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Nerve Growth Factor Receptor TrkA, a New Receptor in Insulin Signaling Pathway in PC12 cells.

Publication Date: 2013 Jun 7 PMID: 23749991
Authors: Geetha, T. - Rege, S. D. - Mathews, S. E. - Meakin, S. O. - White, M. F. - Babu, J. R.
Journal: J Biol Chem

TrkA is a cell surface transmembrane receptor tyrosine kinase for nerve growth factor (NGF). TrkA has an NPXY motif and kinase regulatory loop similar to insulin receptor (INSR) suggesting that NGF-->TrkA signaling might overlap with insulin-->INSR signaling. During insulin or NGF stimulation TrkA, insulin receptor substrate-1 (IRS-1), INSR (and presumably other proteins) forms a complex in PC12 cells. In PC12 cells, tyrosine phosphorylation of INSR and IRS-1 is dependent upon the functional TrkA kinase domain. Moreover, expression of TrkA kinase-inactive mutant blocked the activation of Akt and Erk5 in response to insulin or NGF. Based on these data, we propose that TrkA participates in insulin signaling pathway in PC12 cells.

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Mechanisms of nitrosylation and denitrosylation of cytoplasmic glyceraldehyde-3-phosphate dehydrogenase from Arabidopsis thaliana.

Publication Date: 2013 Jun 7 PMID: 23749990
Authors: Zaffagnini, M. - Morisse, S. - Bedhomme, M. - Marchand, C. H. - Festa, M. - Rouhier, N. - Lemaire, S. D. - Trost, P.
Journal: J Biol Chem

Nitrosylation is a reversible post-translational modification of protein cysteines playing a major role in cellular regulation and signaling in many organisms including plants where it has been implicated in the regulation of immunity and cell death. The extent of nitrosylation of a given cysteine residue is governed by the equilibrium between nitrosylation and denitrosylation reactions. The mechanisms of these reactions remain poorly studied in plants. In the present work, we have employed glycolytic GAPDH from Arabidopsis thaliana as a tool to investigate the molecular mechanisms of nitrosylation and denitrosylation using a combination of approaches including activity assays, the biotin switch technique, site-directed mutagenesis and mass spectrometry. Arabidopsis GAPDH activity was reversibly inhibited by nitrosylation of catalytic Cys-149 mediated either chemically with a strong NO donor or by transnitrosylation with GSNO. GSNO was found to trigger both GAPDH nitrosylation and glutathionylation although nitrosylation was widely prominent. Arabidopsis GAPDH was found to be denitrosylated by GSH but not by plant cytoplasmic thioredoxins. GSH fully converted nitrosylated GAPDH to the reduced, active enzyme, without forming any glutathionylated GAPDH. Thus, we found that nitrosylation of GAPDH is not a step towards formation of the more stable glutathionylated enzyme. GSH-dependent denitrosylation of GapC1 was found to be linked to the [GSH]/[GSNO] ratio and to be independent of the [GSH]/[GSSG] ratio. The possible importance of these biochemical properties for the regulation of Arabidopsis GAPDH functions in vivo is discussed.

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Receptor-type protein tyrosine phosphatase zeta is a functional receptor for interleukin-34.

Publication Date: 2013 Jun 6 PMID: 23744080
Authors: Nandi, S. - Cioce, M. - Yeung, Y. G. - Nieves, E. - Tesfa, L. - Lin, H. - Hsu, A. W. - Halenbeck, R. - Cheng, H. Y. - Gokhan, S. - Mehler, M. F. - Stanley, E. R.
Journal: J Biol Chem

Interleukin-34 (IL-34) is highly expressed in brain. IL-34 signaling via its cognate receptor, colony stimulating factor-1 (CSF-1) receptor (CSF-1R), is required for the development of microglia. However, the differential expression of IL-34 and the CSF-1R in brain suggests that IL-34 may signal via an alternate receptor. By IL-34 affinity chromatography of solubilized mouse brain membrane, followed by mass spectrometric analysis, we identified receptor-type protein tyrosine phosphatase zeta (PTP-zeta), a cell-surface chondroitin sulfate (CS) proteoglycan (CSPG), as a novel IL-34 receptor. PTP-zeta is primarily expressed on neural progenitors and glial cells and is highly expressed in human glioblastomas. IL-34 selectively bound PTP-zeta in CSF-1R-deficient U251 human glioblastoma cell lysates and inhibited the viability, clonogenicity and motility of U251 cells in a PTP-zeta-dependent manner. These effects were correlated with an increase in tyrosine phosphorylation of the previously identified PTP-zeta downstream effectors, focal adhesion kinase and paxillin. IL-34 binding to U251 cells was abrogated by chondroitinase ABC treatment, and IL-34 binding to the extracellular domain of PTP-zeta and to the cells competed for by CS, indicating a dependence of binding on PTP-zeta CS moieties. This study identifies an alternate receptor for IL-34 that may mediate its action on novel cellular targets.

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tBid undergoes multiple conformational changes at the membrane required for Bax activation.

Publication Date: 2013 Jun 7 PMID: 23744079
Authors: Shamas-Din, A. - Bindner, S. - Zhu, W. - Zaltsman, Y. - Campbell, C. - Gross, A. - Leber, B. - Andrews, D. W. - Fradin, C.
Journal: J Biol Chem

Bid is a Bcl-2 family protein that promotes apoptosis by activating Bax and eliciting MOMP2 (Mitochondrial Outer Membrane Permeabilization). Full length Bid is cleaved in response to apoptotic stimuli into two fragments, p7 and tBid (p15) that are held together by strong hydrophobic interactions until the complex binds to membranes. The detailed mechanism(s) of fragment separation including tBid binding to membranes and release of the p7 fragment to the cytoplasm remain unclear. Using liposomes or isolated mitochondria with fluorescently labeled proteins at physiological concentrations as in vitro models, we report that the two components of the complex quickly separate upon interaction with a membrane. Once tBid binds to the membrane, it undergoes slow structural rearrangements that result in an equilibrium between two major tBid conformations on the membrane. The conformational change of tBid is a prerequisite for interaction with Bax and is therefore a novel step that can be modulated to promote or inhibit MOMP. Using automated high-throughput image analysis in cells, we show that downregulation of Mtch2 causes a significant delay between tBid and Bax relocalization in cells. We propose that by promoting insertion of tBid via a conformational change at the mitochondrial outer membrane, Mtch2 accelerates tBid mediated Bax activation and MOMP. Thus the interaction of Mtch2 and tBid is a potential target for therapeutic control of Bid initiated cell death.

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Kdm4b histone demethylase is a DNA damage response protein and confers a survival advantage following gamma-irradiation.

Publication Date: 2013 Jun 6 PMID: 23744078
Authors: Young, L. C. - McDonald, D. C. - Hendzel, M. J.
Journal: J Biol Chem

DNA damage evokes a complex and highly co-ordinated DNA damage response (DDR) that is integral to the suppression of genomic instability. Double strand breaks (DSBs) are considered the most deleterious form damage. Evidence suggests that trimethylation of histone H3 lysine 9 (H3K9me3) presents a barrier to DSB repair. Also, global levels of histone methylation are clinically predictive for several tumor types. Therefore, demethylation of H3K9 may be an important step in the repair of DSBs. The KDM4 subfamily of demethylases removes H3K9 tri- and di-methylation and contributes to the regulation of cellular differentiation and proliferation; mutation or aberrant expression of KDM4 proteins has been identified in several human tumors. We hypothesize that members of the KDM4 subfamily may be components of the DDR. We found that Kdm4b-EGFP and KDM4D-EGFP were recruited rapidly to DNA damage induced by laser micro-irradiation. Focusing on the clinically relevant Kdm4b, we found that recruitment was dependent on poly (ADP-ribose) polymerase 1 (PARP1) activity as well as Kdm4b demethylase activity. The Kdm4 proteins did not measurably accumulate at gamma-irradiation-induced gammaH2AX foci. Nevertheless, increased levels of Kdm4b were associated with decreased numbers of gammaH2AX foci 6 hours following irradiation, as well as increased cell survival. Finally, we found that levels of H3K9me2 and H3K9me3 were decreased at early time points following 2 Gy of gamma-irradiation. Taken together, these data demonstrate that Kdm4b is a DDR protein, and that overexpression of Kdm4b may contribute to the failure of anti-cancer therapy that relies on the induction of DNA damage.

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Anti-HIV Host Factor SAMHD1 Regulates Viral Sensitivity to Nucleoside Reverse Transcriptase Inhibitors via Modulation of Cellular dNTP Levels.

Publication Date: 2013 Jun 5 PMID: 23744077
Authors: Amie, S. M. - Daly, M. B. - Noble, E. - Schinazi, R. F. - Bambara, R. A. - Kim, B.
Journal: J Biol Chem

Newly identified anti-HIV host factor, SAMHD1, restricts replication of lentiviruses such as HIV-1, HIV-2, and SIV in macrophages by enzymatically hydrolyzing and depleting cellular dNTPs, which are the substrates of viral DNA polymerases. HIV-2 and some SIVs express Vpx, which counteracts SAMHD1 and elevates cellular dNTPs, enhancing viral replication in macrophages. Since nucleoside reverse transcriptase inhibitors (NRTIs), the most commonly used anti-HIV drugs, compete against cellular dNTPs for incorporation into proviral DNA, we tested whether SAMHD1 directly affects the efficacy of NRTIs in inhibiting HIV-1. We found that reduction of SAMHD1 levels with the use of virus-like particles (VLPs) expressing Vpx and SAMHD1 specific shRNA, subsequently elevates cellular dNTPs and significantly decreases HIV-1 sensitivity to various NRTIs in macrophages. However, VLP +Vpx treatment of activated CD4+ T cells only minimally reduced NRTI efficacy. Furthermore, our HPLC-based assay could not detect SAMHD1-mediated hydrolysis of various NRTI-triphosphates tested in this study, suggesting that the reduced sensitivity of HIV-1 to NRTIs upon SAMHD1 degradation is most likely caused by the elevation of the cellular dNTP levels.

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Cyclin-dependent kinase 9 links RNA Polymerase II transcription to processing of ribosomal RNA.

Publication Date: 2013 Jun 6 PMID: 23744076
Authors: Burger, K. - Muehl, B. - Rohrmoser, M. - Coordes, B. - Heidemann, M. - Kellner, M. - Gruber-Eber, A. - Heissmeyer, V. - Strasser, K. - Eick, D.
Journal: J Biol Chem

Ribosome biogenesis is a process required for cellular growth and proliferation. Processing of ribosomal (r)RNA is highly sensitive to flavopiridol, a specific inhibitor of cyclin-dependent kinase 9 (Cdk9). Cdk9 has been characterised as the catalytic subunit of the positive transcription elongation factor-b (P-TEFb) of RNA polymerase (RNAP)II. Here we studied the connection between RNAPII transcription and rRNA processing. We show that inhibition of RNAPII activity by alpha-amanitin specifically blocks processing of rRNA. The block is characterised by accumulation of 3' extended unprocessed 47S rRNAs and the entire inhibition of other 47S rRNA specific processing steps. The transcription rate of rRNA is moderately reduced after inhibition of Cdk9, suggesting that defective 3' processing of rRNA negatively feeds back on RNAPI transcription. Knockdown of Cdk9 caused a strong reduction of the levels of RNAPII transcribed U8 small nucleolar (sno)RNA, which is essential for 3' rRNA processing in mammalian cells. Our data demonstrate a pivotal role of Cdk9 activity for coupling of RNAPII transcription with snoRNA production and rRNA processing.

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Pancreatic acinar cell NF-kappaB activation due to bile acid exposure is dependent on calcineurin.

Publication Date: 2013 Jun 6 PMID: 23744075
Authors: Muili, K. A. - Jin, S. - Orabi, A. I. - Eisses, J. F. - Javed, T. A. - Le, T. - Bottino, R. - Jayaraman, T. - Husain, S. Z.
Journal: J Biol Chem

Biliary pancreatitis is the most common etiology of acute pancreatitis, accounting for 30-60% of cases. A dominant theory for the development of biliary pancreatitis is the reflux of bile into the pancreatic duct and subsequent exposure to pancreatic acinar cells. Bile acids are known to induce aberrant Ca2+ signals in acinar cells as well as nuclear translocation of NF-kappaB. In this study, we examined the role of the downstream Ca2+ target calcineurin on NF-kappaB translocation. Freshly isolated mouse acinar cells were infected for 24 hr with an adenovirus expressing an NF-kappaB luciferase reporter. The bile acid taurolithocholic acid-3-sulfate (TLCS) caused NF-kappaB activation at concentrations (500 uM) that were associated with cell injury. We show that the NF-kappaB inhibitor Bay 11-7082 (1 uM) blocked translocation and injury. Pretreatment with the Ca2+ chelator BAPTA, the calcineurin inhibitors, FK506 and cyclosporine A, or use of acinar cells from calcineurin Abeta-deficient mice each led to reduced NF-kappaB activation with TLCS. Importantly, these maniuplations did not affect LPS-induced NF-kappaB activation. A critical upstream regulator of NF-kappaB activation is protein kinase C, which translocates to the membranes of various organelles in the active state. We demonstrate that pharmacologic and genetic inhibition of calcineurin blocks translocation of the PKC-delta isoform. In summary, bile-induced NF-kappaB activation and acinar cell injury are mediated by calcineurin, and a mechanism for this important early inflammatory response appears to be upstream at the level of PKC translocation.

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Precisely Ordered Phosphorylation Reactions in the p38 MAP Kinase Cascade.

Publication Date: 2013 Jun 6 PMID: 23744074
Authors: Humphreys, J. M. - Piala, A. T. - Akella, R. - He, H. - Goldsmith, E. J.
Journal: J Biol Chem

The MAP kinase cascades, comprised of a MAP3K, a MAP2K and a MAPK, control switch responses to extracellular stimuli and stress in eukaryotes. The most important feature of these modules is thought to be the two double phosphorylation reactions catalyzed by MAP3Ks and MAP2Ks. We addressed whether the reactions are sequential or random in the p38 MAP kinase module. Mass spectrometry was used to track the phosphorylation of the MAP2K MEK6 by two MAP3Ks, TAO2 and ASK1, and the subsequent phosphorylation of p38alpha by MEK6/S*T* (where S and T are the two phosphorylation sites and * denotes phos-phorylation). Both double phosphorylation reactions are precisely ordered. MEK6 is phosphorylated first on Thr211 then on Ser207 by both MAP3Ks. This is the first demonstration of a precise reaction order for a MAP2K. p38alpha is phosphorylated first on Tyr182 then on Thr180, the same reaction order observed previously in ERK2. Thus intermediates were MEK6/ST*, and p38alpha/TY*. Similarly, the phosphorylation of the p38alpha transcription factor substrate ATF2 occurs in a precise sequence. Progress curves for the appearance of intermediates were fit to kinetic models. The models confirmed the reaction order, revealed processivity in the phosphorylation of MEK6 by ASK1, and suggested that the order of phos-phorylation is dictated by both binding and catalysis rates.

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Castration induces Parkinson's disease pathologies in young male mice via inducible nitric oxide synthase.

Publication Date: 2013 Jun 6 PMID: 23744073
Authors: Khasnavis, S. - Ghosh, A. - Roy, A. - Pahan, K.
Journal: J Biol Chem

Although Parkinson disease (PD) is a progressive neurodegenerative disorder, available animal models do not exhibit irreversible neurodegeneration and this is a major obstacle in finding out an effective drug against this disease. Here we delineate a new irreversible model to study PD pathogenesis. The model is based on simple castration of young male mice. Levels of inducible nitric oxide synthase (iNOS), glial markers (glial fibrillary acidic protein and CD11b) and a-synuclein were higher in nigra of castrated male mice than normal male mice. On the other hand, after castration, the level of glial-derived neurotrophic factor (GDNF) markedly decreased in the nigra of male mice. Accordingly, castration also induced the loss of tyrosine hydroxylase (TH)-positive neurons in the nigra, decrease in TH-positive fibers and neurotransmitters in the striatum. Reversal of nigrostriatal pathologies in castrated male mice by subcutaneous implantation of 5alpha-dihydrotestosterone (DHT) pellets validates an important role of male sex hormone in castration-induced nigrostriatal pathology. Interestingly, castration was unable to cause glial activation, decrease nigral GDNF, augment the death of nigral dopaminergic neurons, induce the loss of striatal fibers, and impair neurotransmitters in iNOS (-/-) male mice. Furthermore, we demonstrate that iNOS-derived NO is responsible for decreased expression of GDNF in activated astrocytes. Together, our results suggest that castration induces nigrostriatal pathologies via iNOS-mediated decrease in GDNF. These results are important as castrated young male mice may be used as a simple, toxin-free and non-transgenic animal model to study PD-related nigrostriatal pathologies, paving the way for easy drug screening against PD.

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Carbamazepine as a Novel Small Molecule Corrector of Trafficking-Impaired ATP-Sensitive Potassium Channels Identified in Congenital Hyperinsulinism.

Publication Date: 2013 Jun 6 PMID: 23744072
Authors: Chen, P. C. - Olson, E. M. - Zhou, Q. - Kryukova, Y. - Sampson, H. M. - Thomas, D. Y. - Shyng, S. L.
Journal: J Biol Chem

ATP-sensitive potassium (KATP) channels consisting of sulfonylurea receptor 1 (SUR1) and the potassium channel Kir6.2 play a key role in insulin secretion by coupling metabolic signals to beta-cell membrane potential. Mutations in SUR1 and Kir6.2 that impair channel trafficking to the cell surface lead to loss of channel function and congenital hyperinsulinism. We report that carbamazepine, an anticonvulsant, corrects the trafficking defects of mutant KATP channels previously identified in congenital hyperinsulinism. Strikingly, of the nineteen SUR1 mutations examined, only those located in the first transmembrane domain of SUR1 responded to the drug. We show that unlike that reported for several other protein misfolding diseases, carbamazepine does not correct KATP channel trafficking defects by activating autophagy; rather, it directly improves the biogenesis efficiency of mutant channels along the secretory pathway. In addition to its effect on channel trafficking, carbamazepine also inhibits KATP channel activity. Upon subsequent removal of carbamazepine, however, the function of rescued channels is recovered. Importantly, combination of the KATP channel opener diazoxide and carbamazepine leads to enhanced mutant channel function without carbamazepine washout. The corrector effect of carbamazepine on mutant KATP channels is also demonstrated in rat and human beta-cells, with an accompanying increase in channel activity. Our findings identify carbamazepine as a novel small molecule corrector that may be used to restore KATP channel expression and function in a subset of congenital hyperinsulinism patients.

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Alpha-synuclein oligomers impair neuronal microtubule-kinesin interplay.

Publication Date: 2013 Jun 6 PMID: 23744071
Authors: Prots, I. - Veber, V. - Brey, S. - Campioni, S. - Buder, K. - Riek, R. - Bohm, K. J. - Winner, B.
Journal: J Biol Chem

Early alpha-Synuclein (alpha-Syn)-induced alterations are neurite pathologies resulting in Lewy neurites. Alpha-Syn oligomers are a toxic species in synucleinopathies and suspected to cause neuritic pathology. To investigate how alpha-Syn oligomers may be linked to aberrant neurite pathology, we modeled different stages of alpha-Syn aggregation in vitro and investigated their interaction with proteins involved in axonal transport. The interaction of wild type alpha-Syn (WTS) and alpha-Syn variants (E57K, A30P, aSyn30-110) with kinesin, tubulin, and the microtubule (MT) associated proteins, MAP2 and Tau, is stronger for multimers than for monomers. WTS seeds, but not alpha-Syn oligomers significantly and dose-dependently reduced Tau-promoted MT assembly in vitro. In contrast, MT gliding velocity across kinesin-coated surfaces was significantly decreased in the presence of alpha-Syn oligomers but not WTS seeds or fibrils (aSyn30-110 multimers). In a human dopaminergic neuronal cell line, mild overexpression of the oligomerizing E57K alpha-Syn variant significantly impaired neurite network morphology without causing profound cell death. In accordance with these findings, MT stability, neuritic kinesin and neuritic kinesin-dependant cargos were significantly reduced by the presence of alpha-Syn oligomers. In summary, different alpha-Syn species act divergently on the axonal transport machinery. These findings provide new insights into alpha-Syn oligomer-driven neuritic pathology as one of the earliest events in synucleinopathies.

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A Mixed Mirror-image DNA/RNA Aptamer Inhibits Glucagon and Acutely Improves Glucose Tolerance in Models of Type 1 and Type 2 Diabetes.

Publication Date: 2013 Jun 6 PMID: 23744070
Authors: Vater, A. - Sell, S. - Kaczmarek, P. - Maasch, C. - Buchner, K. - Pruszynska-Oszmalek, E. - Kolodziejski, P. - Purschke, W. G. - Nowak, K. W. - Strowski, M. Z. - Klussmann, S.
Journal: J Biol Chem

Excessive secretion of glucagon - a functional insulin antagonist - significantly contributes to hyperglycemia in type 1 and type 2 diabetes. Accordingly, immunoneutralization of glucagon or genetic deletion of the glucagon receptor improved glucose homeostasis in animal models of diabetes. Despite this strong evidence, agents that selectively interfere with endogenous glucagon have not been implemented in clinical practice yet. We report the discovery of mirror-image DNA-aptamers (Spiegelmers) that bind and inhibit glucagon. The affinity of the best-binding DNA oligonucleotide was remarkably increased (>25-fold) by the introduction of oxygen atoms at selected 2'-positions through deoxyribo- to ribonucleotide exchanges resulting in a mixed DNA/RNA-Spiegelmer (NOX-G15) that binds glucagon with a Kd of 3 nM. NOX-G15 shows no cross-reactivity with related peptides such as glucagon-like peptide 1 (GLP-1), GLP-2, gastric-inhibitory peptide, and prepro-vasoactive intestinal peptide (prepro-VIP). In vitro, NOX-G15 inhibits glucagon-stimulated cAMP-production in CHO-cells overexpressing the human glucagon receptor with an IC50 of 3.4 nM. A single injection of NOX-G15 ameliorated glucose excursions in intraperitoneal glucose tolerance tests (ipGTT) in mice with streptozotocin-induced (type 1) diabetes and in a non-genetic mouse model of type 2 diabetes. In conclusion, the data suggest NOX-G15 as a therapeutic candidate with the potential to acutely attenuate hyperglycemia in type 1 and type 2 diabetes.

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Altered Cortical GABAA Receptor Composition, Physiology, and Endocytosis in a Mouse Model of a Human Genetic Absence Epilepsy Syndrome.

Publication Date: 2013 Jun 6 PMID: 23744069
Authors: Zhou, C. - Huang, Z. - Ding, L. - Deel, M. E. - Arain, F. M. - Murray, C. R. - Patel, R. S. - Flanagan, C. D. - Gallagher, M. J.
Journal: J Biol Chem

Patients with generalized epilepsy exhibit cerebral cortical disinhibition. Likewise, mutations in the inhibitory ligand-gated ion channels, GABAA receptors (GABAARs), cause generalized epilepsy syndromes in humans. Recently, we demonstrated that heterozygous knockout (Hetalpha1KO) of the human epilepsy gene, the GABAAR alpha1 subunit, produced generalized absence epilepsy in mice. Here, we determined the effects of Hetalpha1KO on the expression and physiology of GABAARs in mouse cortex. We found that Hetalpha1KO caused modest reductions in the total and surface expression of the beta2 subunit, but did not alter beta1 or beta3 subunit expression, results consistent with a small reduction of GABAARs. Cortices partially compensated for Hetalpha1KO by increasing the fraction of residual alpha1 subunit on the cell surface and by increasing total and surface expression of alpha3, but not alpha2, subunits. Co-immunoprecipitation experiments revealed that Hetalpha1KO increased the fraction of alpha1 subunits, and decreased the fraction of alpha3 subunits, that associated in hybrid alpha1alpha3betagamma receptors. Patch clamp electrophysiology studies showed that Hetalpha1KO layer VI cortical neurons exhibited reduced inhibitory postsynaptic current peak amplitudes, prolonged current rise and decay times, and altered responses to benzodiazepine agonists. Finally, application of inhibitors of dynamin-mediated endocytosis revealed that Hetalpha1KO reduced baseline GABAAR endocytosis, an effect that likely contributes to the observed changes in GABAAR expression. These findings demonstrate that Hetalpha1KO exerts two principle disinhibitory effects on cortical GABAAR-mediated inhibitory neurotransmission 1.) a modest reduction of GABAAR number and 2.) a partial compensation with GABAAR isoforms that possess different physiological properties than the otherwise predominant alpha1betagamma GABAARs.

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Differential contribution of insulin and amino acids to the mTORC1-autophagy pathway in the liver and muscle.

Publication Date: 2013 Jun 6 PMID: 23744068
Authors: Naito, T. - Kuma, A. - Mizushima, N.
Journal: J Biol Chem

Autophagy is a highly inducible intracellular degradation process. It is generally induced by nutrient starvation and suppressed by food intake. Mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) is considered to be the major regulator of autophagy, but the precise mechanism of in vivo regulation remains to be fully characterized. Here, we examined the autophagy-suppressive effect of glucose, insulin, and amino acids in the liver and muscle in mice starved for 1 day. Refeeding after starvation with a standard mouse chow rapidly suppressed autophagy in both tissues, and this suppression was inhibited by rapamycin administration almost completely in the liver and partially in muscle, confirming that mTORC1 is indeed an crucial regulator in vivo. As glucose administration showed no major suppressive effect on autophagy, we examined the role of insulin and amino acids using hyperinsulinemic-euglycemic clamp and intravenous amino acid infusion techniques. Insulin administration showed a clear effect on the mTORC1-autophagy pathway in muscle, but had only a very weak effect in the liver. By contrast, amino acids were able to regulate the mTORC1-autophagy pathway in the liver, but less effectively in muscle. These results suggest that autophagy is differentially regulated by insulin and amino acids in a tissue-dependent manner.

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c-Cbl, a ubiquitin E3 ligase that targets active beta-catenin - A novel layer of Wnt regulation.

Publication Date: 2013 Jun 6 PMID: 23744067
Authors: Chitalia, V. - Shivanna, S. - Martorell, J. - Meyer, R. - Edelman, E. - Rahimi, N.
Journal: J Biol Chem

Regulation of transcriptionally active nuclear beta-catenin during Wnt-on phase is crucial to ensure controlled induction of Wnt target genes. Several ubiquitin E3 ligases are known to regulate cytosolic beta-catenin during Wnt-off phase, but little is known about the fate of active nuclear beta-catenin in Wnt-on phase. We now describe ubiquitination of active beta-catenin in Wnt-on phase by a RING finger ubiquitin E3 ligase, Casitas B-lineage lymphoma (c-Cbl) in endothelial cells (EC). c-Cbl binds preferentially to nuclear active beta-catenin in Wnt-on phase via armadillo repeat region. Wild-type c-Cbl suppresses and E3 ligase-deficient c-Cbl-70Z increases Wnt signaling. Wnt induces nuclear translocation of c-Cbl where it ubiquitinates nuclear beta-catenin. Deletion of c-Cbl UBA domain abrogates its dimerization, binding to beta-catenin, Wnt-induced c-Cbl nuclear translocation and ubiquitination of nuclear beta-catenin. c-Cbl activity inhibits pro-angiogenic Wnt targets IL-8 and VEGF levels and angiogenesis in beta-catenin-dependent manner. This study defines for the first time c-Cbl as an ubiquitin E3 ligase that targets nuclear active beta-catenin in Wnt-on phase and uncovers a novel layer of regulation of Wnt signaling.

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Identification of Serpin Determinants of Specificity and Selectivity for Furin Inhibition through Studies of alpha1PDX (alpha1-Protease Inhibitor Portland)-Serpin B8 and Furin Active-site Loop Chimeras.

Publication Date: 2013 Jun 6 PMID: 23744066
Authors: Izaguirre, G. - Qi, L. - Lima, M. - Olson, S. T.
Journal: J Biol Chem

alpha1PDX is an engineered serpin family inhibitor of the proprotein convertase (PC), furin, that exhibits high specificity but limited selectivity for inhibiting furin over other PC family proteases. Here, we characterize serpin B8, a natural inhibitor of furin, together with alpha1PDX-serpin B8 and furin-PC chimeras to identify determinants of serpin specificity and selectivity for furin inhibition. Replacing reactive center loop (RCL) sequences of alpha1PDX with those of serpin B8 demonstrated that both the P1-P4 RXXR recognition sequence as well as the P1'-P5' sequence are critical determinants of serpin specificity for furin. Alignments of PC catalytic domains revealed four variable active-site loops whose role in furin reactivity with serpin B8 was tested by engineering furin-PC loop chimeras. The furin 298-300 loop but not the other loops differentially affected furin reactivity with serpin B8 and alpha1PDX in a manner that depended on the serpin RCL primed sequence. Modeling of the serpin B8-furin Michaelis complex identified serpin exosites in strand 3C close to the 298-300 loop whose substitution in alpha1PDX differentially affected furin reactivity depending on the furin loop and serpin RCL primed sequences. These studies demonstrate that RCL primed residues, strand 3C exosites and the furin 298-300 loop are critical determinants of serpin reactivity with furin which may be exploited in the design of specific and selective alpha1PDX inhibitors of PCs.

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Enhanced Fasting Glucose Turnover in Mice with Disrupted Action of TUG Protein in Skeletal Muscle.

Publication Date: 2013 Jun 6 PMID: 23744065
Authors: Loffler, M. G. - Birkenfeld, A. L. - Philbrick, K. M. - Belman, J. P. - Habtemichael, E. N. - Booth, C. J. - Castorena, C. M. - Choi, C. S. - Jornayvaz, F. R. - Gassaway, B. M. - Lee, H. Y. - Cartee, G. D. - Philbrick, W. - Shulman, G. I. - Samuel, V. T. - Bogan, J. S.
Journal: J Biol Chem

Insulin stimulates glucose uptake in 3T3-L1 adipocytes in part by causing endoproteolytic cleavage of TUG proteins. Cleavage liberates intracellularly sequestered GLUT4 glucose transporters for translocation to the cell surface. To test the role of this regulation in muscle, we used mice with muscle-specific transgenic expression of a truncated TUG fragment, UBX-Cter. This fragment causes GLUT4 translocation in unstimulated 3T3-L1 adipocytes. We predicted that transgenic mice would have GLUT4 translocation in muscle during fasting. UBX-Cter expression caused depletion of PIST proteins, which transmit an insulin signal to TUG. Whereas insulin stimulated TUG proteolysis in control muscles, proteolysis was constitutive in transgenic muscles. Fasting transgenic mice had decreased plasma glucose and insulin concentrations, compared to controls. Whole-body glucose turnover was increased during fasting, but not during hyperinsulinemic clamp studies. In muscles with greatest UBX-Cter expression, 2-deoxyglucose uptake during fasting was similar to that in control muscles during hyperinsulinemic clamps. Fasting transgenic mice had increased muscle glycogen, and GLUT4 targeting to T-tubule fractions was increased 5.7-fold. Whole-body VO2, VCO2, and energy expenditure were increased by 12-13%. After 3 weeks on a high-fat diet, the decreased fasting plasma glucose in transgenic mice, compared to controls, was more marked, and increased glucose turnover was not observed; the transgenic mice continued to have an increased metabolic rate. We conclude that insulin stimulates TUG proteolysis to translocate GLUT4 in muscle, that this pathway impacts systemic glucose homeostasis and energy metabolism, and that effects of activating this pathway are maintained during high fat diet-induced insulin resistance in mice.

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Lipid, detergent and Coomassie dye affect the migration of small membrane proteins in blue native gels: mitochondrial carriers migrate as monomers not dimers.

Publication Date: 2013 Jun 6 PMID: 23744064
Authors: Crichton, P. G. - Harding, M. - Ruprecht, J. J. - Lee, Y. - Kunji, E. R.
Journal: J Biol Chem

Blue native gel electrophoresis is a popular method for the determination of the oligomeric state of membrane proteins. Studies using this technique have reported that mitochondrial carriers are dimeric (composed of two ~32 kDa monomers) and, in some cases, can form physiologically relevant associations with other proteins. Here, we have scrutinised the behaviour of the yeast mitochondrial ADP/ATP carrier AAC3 in blue native gels. We find that the apparent mass of AAC3 varies in a detergent and lipid dependent manner (~60 to ~130 kDa) that is not related to changes in oligomeric state of the protein, but reflects differences in the associated detergent-lipid micelle and Coomassie dye used in this technique. Higher oligomeric state species are only observed under less favourable solubilisation conditions, consistent with aggregation of the protein. Calibration with an artificial covalent AAC3 dimer indicates that the mass observed for solubilised AAC3 and other mitochondrial carriers corresponds to a monomer. Size exclusion chromatography of purified AAC3 in decyl maltoside under blue native gel-like conditions shows that the mass of the monomer is ~120 kDa, but appears smaller on gels (~60 kDa) due to the unusually high amount of bound negatively charged dye, which increases the electrophoretic mobility of the protein-detergent-dye micelle complex. Our results show that bound lipid, detergent and Coomassie stain alter the behaviour of mitochondrial carriers on gels, which is likely to be true for other small membrane proteins where the associated lipid-detergent micelle is large compared to the mass of the protein.

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In the beginning Escherichia coli assembled the proto-ring: an initial phase of division.

Publication Date: 2013 Jun 5 PMID: 23740256
Authors: Rico, A. I. - Krupka, M. - Vicente, M.
Journal: J Biol Chem

Cell division in Escherichia coli begins by assembling three proteins, FtsZ, FtsA and ZipA to form a proto-ring at midcell. These proteins nucleate an assembly of at least 35 components, the divisome. The structuring of FtsZ to form a ring and the processes that effect constrictions have been explained by alternative but not mutually exclusive mechanisms. We discuss how FtsA and ZipA provide anchoring of the cytoplasmic FtsZ to the membrane and how a temporal sequence of alternative protein interactions may operate in the maturation and stability of the proto-ring. How the force needed for constriction is generated and how the proto-ring proteins relate to peptidoglycan synthesis remain as the main challenges for future research.

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Thermal and spectroscopic characterization of a proton pumping rhodopsin from an extreme thermophile.

Publication Date: 2013 Jun 5 PMID: 23740255
Authors: Tsukamoto, T. - Inoue, K. - Kandori, H. - Sudo, Y.
Journal: J Biol Chem

So far, retinylidene proteins (~ rhodopsin) have only been discovered in organisms living at moderate temperatures (ca. 10-30 degree). In this study, we investigated and characterized a microbial rhodopsin derived from the extreme thermophilic bacterium Thermus thermophilus which lives in a hot spring at around 75 degree. The gene for the retinylidene protein named thermophilic rhodopsin, TR, was chemically synthesized with codon optimization. The codon optimized TR protein was functionally expressed in the cell membranes of E. coli cells and showed active proton transport upon photoillumination. Spectroscopic measurements revealed that the purified TR bound only all-trans retinal as a chromophore and showed an absorption maximum at 530 nm. In addition, TR exhibited both photocycle kinetics and pH-dependent absorption changes, which are characteristic of rhodopsins. Of note, time-dependent thermal denaturation experiments revealed that TR maintained its absorption even at 75 degree and the denaturation rate constant of TR was much lower than those of other proton pumping rhodopsins such as archaerhodopsin-3 (by 200-times), Haloquadratum walsbyi bacteriorhodopsin (by 10-times) and Gloeobacter rhodopsin (by 100-times). Thus, these results suggest that microbial rhodopsins are also distributed among thermophilic organisms and have extremely high stability. TR should allow not only the investigation of the molecular mechanisms of ion transport and protein folding, but also can be utilized as an optogenetic tool.

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Structural Analysis of the N-terminal Domain of Subunit a of the Yeast Vacuolar ATPase (V-ATPase) Using Accessibility of Single Cysteine Substitutions to Chemical Modification.

Publication Date: 2013 Jun 5 PMID: 23740254
Authors: Liberman, R. - Cotter, K. - Baleja, J. D. - Forgac, M.
Journal: J Biol Chem

The vacuolar ATPase (V-ATPase) is a multisubunit complex that carries out ATP-driven proton transport. It is composed of a peripheral V1 domain that hydrolyzes ATP and an integral V0 domain that translocates protons. Subunit a is a 100 kDa integral membrane protein (part of V0) that possesses an N-terminal cytoplasmic domain and a C-terminal hydrophobic domain. While the C-terminal domain functions in proton transport, the N-terminal domain is critical for intracellular targeting and regulation of V-ATPase assembly. Despite its importance, there is currently no high resolution structure for subunit a of the V-ATPase. Recently, the crystal structure of the N-terminal domain of the related subunit I from the archaebacterium Meiothermus ruber was reported. We have used homology modeling to construct a model of the N-terminal domain of Vph1p, one of two isoforms of subunit a expressed in yeast. To test this model, unique cysteine residues were introduced into a Cys-less form of Vph1p and their accessibility to modification by the sulfhydryl reagent 3-(N-maleimido-propionyl) biocytin (MPB) was determined. In addition, accessibility of introduced cysteine residues to MPB modification was compared in the V1V0 complex and the free V0 domain to identify residues protected from modification by the presence of V1. The results provide an experimental test of the proposed model and have identified regions of the N-terminal domain of subunit a that likely serve as interfacial contact sites with the peripheral V1 domain. The possible significance of these results for in vivo regulation of V-ATPase assembly is discussed.

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alpha-synuclein senses lipid packing defects and induces lateral expansion of lipids leading to membrane remodeling.

Publication Date: 2013 Jun 5 PMID: 23740253
Authors: Ouberai, M. M. - Wang, J. - Swann, M. J. - Galvagnion, C. - Guilliams, T. - Dobson, C. M. - Welland, M. E.
Journal: J Biol Chem

There is increasing evidence for the involvement of lipid membranes in both the functional and pathological properties of alpha-synuclein (alpha-Syn). Despite many investigations to characterize the binding of alpha-Syn to membranes, there is still a lack of understanding of the binding mode linking the properties of lipid membranes to alpha-Syn insertion into these dynamic structures. Using a combination of an optical biosensing technique and in situ atomic force microscopy, we show that the binding strength of alpha-Syn is related to the specificity of the lipid environment (the lipid chemistry and steric properties within a bilayer structure) and to the ability of the membranes to accommodate and remodel upon the interaction of alpha-Syn with lipid membranes. We show that this interaction results in the insertion of alpha-Syn into the region of the head groups, inducing a lateral expansion of lipid molecules that can progress to further bilayer remodeling such as membrane thinning and expansion of lipids out of the membrane plane. We provide new insights into the affinity of alpha-Syn for lipid packing defects found in vesicles of high curvature and in planar membranes with cone-shaped lipids and suggest a comprehensive model of the interaction between alpha-Syn and lipid bilayers. The ability of alpha-Syn to sense lipid packing defects and to remodel membrane structure supports its proposed role in vesicle trafficking.

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Functional properties of five Dictyostelium discoideum P2X receptors.

Publication Date: 2013 Jun 5 PMID: 23740252
Authors: Baines, A. - Parkinson, K. - Sim, J. A. - Bragg, L. - Thompson, C. R. - North, R. A.
Journal: J Biol Chem

The Dictyostelium discoideum genome encodes five proteins that share weak sequence similarity with vertebrate P2X receptors. Unlike vertebrate P2X receptors, these proteins are not expressed on the surface of cells, but populate the tubules and bladders of the contractile vacuole. In this study, we expressed humanized cDNAs of P2XA, P2XB, P2XC, P2XD and P2XE in human embryonic kidney cells and altered the ionic and proton environment in an attempt to reflect the situation in amoeba. Recording of whole-cell membrane currents showed that four receptors operated as ATP-gated channels (P2XA, P2XB, P2XD, P2XE). At P2XA receptors, ATP was the only effective agonist of seventeen structurally related putative ligands that were tested. Extracellular sodium, as compared to potassium, strongly inhibited ATP responses in P2XB, P2XD and P2XE receptors. Increasing the proton concentration (pH 6.2) accelerated desensitization at P2XA receptors and decreased currents at P2XD receptors, but increased the currents at P2XB and P2XE receptors. Dictyostelium lacking P2XA receptors showed impaired regulatory volume decrease in hypotonic solution. This phenotype was readily rescued by over-expression of P2XA and P2XD receptors, partially rescued by P2XB and P2XE receptors, and not rescued by P2XC receptors. The failure of the non-functional receptor P2XC to restore the regulatory volume decrease highlights the importance of ATP activation of P2X receptors for a normal response to hypo-osmotic shock, and the weak rescue by P2XB and P2XE receptors, indicates that there is limited functional redundancy among Dictyostelium P2X receptors.

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P2X receptor chimeras highlight roles of the N terminus to partial agonist efficacy, the C terminus to recovery from desensitization, and independent regulation of channel transitions.

Publication Date: 2013 Jun 5 PMID: 23740251
Authors: Allsopp, R. C. - Farmer, L. K. - Fryatt, A. G. - Evans, R. J.
Journal: J Biol Chem

P2X receptor subtypes can be distinguished by their sensitivity to ATP analogues and selective antagonists. We have used chimeras between human P2X1 and P2X2 receptors to address the contribution of the extracellular ligand binding loop, transmembrane segments (TM1- and TM2) and intracellular amino and carboxyl termini to the action of partial agonists (higher potency and efficacy of BzATP and Ap5A at P2X1 receptors) and antagonists. Sensitivity to the antagonists NF449, suramin and PPADS was conferred by the nature of the extracellular loop (e.g. nanomolar for NF449 at P2X1 and P2X2-1EXT and micromolar at P2X2 and P2X1-2EXT). In contrast the effectiveness of partial agonists was similar to P2X1 levels for both the loop transfers suggesting that interactions with the rest of the receptor played an important role. Swapping TM2 had reciprocal effects on partial agonist efficacy. However TM1 swaps increased partial agonist efficacy at both chimeras, and this was similar for swaps of both TM1&2. Changing the amino terminus had no effect on agonist potency but increased partial agonist efficacy at P2X2-1N and decreased it at P2X1-2N chimeras demonstrating that potency and efficacy can be independently regulated. Chimeras and point mutations also identified residues in the carboxyl terminus that regulated recovery from channel desensitization. These results show that interactions between the intracellular, transmembrane and extracellular portions of the receptor regulate channel properties and suggest that transitions to channel opening, the behaviour of the open channel, and recovery from the desensitized state can be controlled independently.

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The triplicated gene APP impairs neuronal precursor differentiation and neurite development through two different domains in the Ts65Dn mouse model for Down syndrome.

Publication Date: 2013 Jun 5 PMID: 23740250
Authors: Trazzi, S. - Fuchs, C. - Valli, E. - Perini, G. - Bartesaghi, R. - Ciani, E.
Journal: J Biol Chem

Intellectual disability in Down syndrome (DS) appears to be related to severe proliferation impairment during brain development. Recent evidence shows that it is not only cellular proliferation that is heavily compromised in DS, but also cell fate specification and dendritic maturation. The amyloid precursor protein (APP), a gene that is triplicated in DS, plays a key role in normal brain development by influencing neural precursor cell proliferation, cell fate specification and neuronal maturation. APP influences these processes via two separate domains, the APP intracellular domain (AICD) and the soluble secreted APP (sAPP). We recently found that the proliferation impairment of neuronal precursors (NPCs) from the Ts65Dn mouse model for DS was caused by derangement of the Shh pathway, due to over-expression of Patched1 (Ptch1), its inhibitory regulator. Ptch1 over-expression was related to increased levels within the APP/AICD system. The overall goal of this study was to determine whether APP contributes to neurogenesis impairment in DS by influencing, in addition to proliferation, cell fate specification and neurite development. We found that normalization of APP expression restored the reduced neuronogenesis, the increased astrogliogenesis and the reduced neurite length of trisomic NPCs, indicating that APP over-expression underpins all aspects of neurogenesis impairment. Moreover, we found that two different domains of APP impair neuronal differentiation and maturation in trisomic NPCs. The APP/AICD system regulates neuronogenesis and neurite length through the Shh pathway, while the APP/sAPP system promotes astrogliogenesis through an IL-6-associated signaling cascade. These results provide novel insight into the mechanisms underlying brain development alterations in DS.

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The minimum M3-M4 loop length of neurotransmitter-activated pentameric receptors is critical for the structural integrity of cytoplasmic portals.

Publication Date: 2013 Jun 5 PMID: 23740249
Authors: Baptista-Hon, D. T. - Deeb, T. Z. - Lambert, J. J. - Peters, J. A. - Hales, T. G.
Journal: J Biol Chem

The 5-HT3A receptor (5-HT3R) homology model, based on the partial structure of the nicotinic acetylcholine receptor (nAChR) from T. marmorata, reveals an asymmetric ion channel with five portals framed by adjacent helical amphipathic (HA) stretches within the 114 residue loop between the M3 and M4 membrane-spanning domains. The positive charge of Arg436, located within the HA stretch is a rate limiting determinant of gamma. Further analysis reveals that positive charge and volume of residue 436 are determinants of 5-HT3R inward rectification, exposing an additional role for portals. A structurally unresolved stretch of 85 residues constitutes the bulk of the M3-M4 loop, leaving a >45-A gap in the model between M3 and the HA stretch. There are no additional structural data for this loop, which is vestigial in bacterial pentameric ligand-gated ion channels (pLGICs) and was largely removed for crystallization of the C. elegans glutamate-activated pLGIC. We created 5-HT3A subunit loop truncation mutants, in which sequences framing the putative portals were retained, to determine the minimum number of residues required to maintain their functional integrity. Truncation to between 90 and 75 amino acids produced 5-HT3Rs with unaltered rectification. Truncation to 70 residues abolished rectification and increased gamma. These findings reveal a critical M3-M4 loop length required for functions attributable to cytoplasmic portals. Examination of all 44 subunits of the human neurotransmitter-activated Cys-loop receptors reveals that, despite considerable variability in their sequences and lengths, all M3-M4 loops exceed 70 residues suggesting a fundamental requirement for portal integrity.

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SLLISWD Sequence in the 10FNIII Domain Initiates Fibronectin Fibrillogenesis.

Publication Date: 2013 Jun 5 PMID: 23740248
Authors: Gee, E. P. - Yuksel, D. - Stultz, C. M. - Ingber, D. E.
Journal: J Biol Chem

Fibronectin (FN) assembly into extracellular matrix is tightly regulated and essential to embryogenesis and wound healing. FN fibrillogenesis is initiated by cytoskeleton-derived tensional forces transmitted across transmembrane integrins onto RGD binding sequences within the tenth FN type III (10FNIII) domains. These forces unfold 10FNIII to expose cryptic FN assembly sites; however, a specific sequence has not been identified in 10FNIII. Our past steered molecular dynamics simulations modeling 10FNIII unfolding by force at its RGD loop predicted a mechanical intermediate with a solvent-exposed N-terminus spanning the A and B beta-strands. Here, we experimentally confirm that the predicted twenty-three residue cryptic peptide 1 (CP1) initiates FN multimerization, which is mediated by interactions with 10FNIII that expose hydrophobic surfaces that support 8-anilino-1-napthalenesulfonic acid (ANS) binding. Localization of multimerization activity to the C-terminus led to the discovery of a minimal seven amino acid 'multimerization sequence' (SLLISWD), which induces polymerization of FN and the clotting protein fibrinogen, in addition to enhancing FN fibrillogenesis in fibroblasts. A point mutation at Trp6 that reduces exposure of hydrophobic sites for ANS binding and beta-structure formation inhibits FN multimerization and prevents physiological cell-based FN assembly in culture. We propose a model for cell-mediated fibrillogenesis whereby cell traction force initiates a cascade of intermolecular exchange starting with the unfolding of 10FNIII to expose the multimerization sequence, which interacts with strand B of another 10FNIII domain via a Trp-mediated beta-strand exchange to stabilize a partially unfolded intermediate that propagates FN self-assembly.

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The MID1 E3 ligase catalyzes the polyubiquitination of Alpha4, a regulatory subunit of Protein Phosphatase 2A (PP2A): Novel insights into MID1-mediated regulation of PP2A.

Publication Date: 2013 Jun 5 PMID: 23740247
Authors: Du, H. - Huang, Y. - Zaghlula, M. - Walters, E. - Cox, T. C. - Massiah, M. A.
Journal: J Biol Chem

Alpha4 is a key regulator of PP2A and mTOR in steps essential for cell-cycle progression. Alpha4 forms a complex with PP2A and MID1, a microtubule-associated ubiquitin E3 ligase, which facilitates MID1-dependent regulation of PP2A and the dephosphorylation of MID1 by PP2A. Ectopic overexpression of alpha4 is associated with hepatocellular carcinomas, breast cancer, and invasive adenocarcincoma. Here, we provide data suggesting that alpha4 is regulated by ubiquitin-dependent degradation mediated by MID1. In cells stably expressing a dominant-negative form of MID1 significantly elevated levels of alpha4 were observed. Treatment of cells with the specific proteasome inhibitor, lactacystin, resulted in a three-fold increase in alpha4 in control cells and a similar level in mutant cells. Using in vitro assays, individual MID1 E3 domains facilitated monoubiquitination of alpha4, while full-length MID1 as well as RING-Bbox1 and RING-Bbox1-Bbox2 constructs catalyzed its polyubiquitination. In a novel non-biased functional screen, we identified a leucine to glutamine substitution at position 146 within Bbox1 that abolished MID1-alpha4 interaction and the subsequent polyubiquitination of alpha4, indicating that direct binding to Bbox1 was necessary for the polyubiquitination of alpha4. The mutant had little impact on the RING E3 ligase functionality of MID1. Mass spectrometry data confirmed Western Blot analysis that ubiquitination of alpha4 occurs only within the last 105 amino acids. These novel findings identify a new role for MID1, and a mechanism of regulation of alpha4 that is likely to impact the stability and activity level of PP2Ac.

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Role of the adapter protein Abi1 in actin-associated signaling and smooth muscle contraction.

Publication Date: 2013 Jun 5 PMID: 23740246
Authors: Wang, T. - Cleary, R. A. - Wang, R. - Tang, D. D.
Journal: J Biol Chem

Actin filament polymerization plays a critical role in the regulation of smooth muscle contraction. However, our knowledge regarding modulation of the actin cytoskeleton in smooth muscle just begins to accumulate. In this study, stimulation with acetylcholine (ACh) induced an increase in the association of the adapter protein c-Abl interactor 1 (Abi1) with neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) (an actin-regulatory protein) in smooth muscle cells/tissues. Furthermore, contractile stimulation activated N-WASP in live smooth muscle cells as evidenced by changes in fluorescence resonance energy transfer efficiency of an N-WASP sensor. Abi1 knockdown by lentivirus-mediated RNAi inhibited N-WASP activation, actin polymerization and contraction in smooth muscle. However, Abi1 silencing did not affect myosin regulatory light chain phosphorylation at Ser-19 in smooth muscle. In addition, c-Abl tyrosine kinase and Crk-associated substrate (CAS) have been shown to regulate smooth muscle contraction. The interaction of Abi1 with c-Abl and CAS has not been investigated. Here, contractile activation induced formation of a multiprotein complex including c-Abl, CAS and Abi1. Knockdown of c-Abl and CAS attenuated the activation of Abi1 during contractile activation. More importantly, Abi1 knockdown inhibited c-Abl phosphorylation at Tyr-412 and the interaction of c-Abl with CAS. These results suggest that Abi1 is an important component of the cellular process that regulates N-WASP activation, actin dynamics and contraction in smooth muscle. Abi1 is activated by the c-Abl-CAS pathway, and Abi1 reciprocally controls the activation of its upstream regulator c-Abl.

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EB1 levels are elevated in ascorbic acid (AA)-stimulated osteoblasts and mediate cell-cell adhesion induced osteoblast differentiation.

Publication Date: 2013 Jun 5 PMID: 23740245
Authors: Pustylnik, S. - Fiorino, C. - Nabavi, N. - Zappitelli, T. - da Silva, R. - Aubin, J. E. - Harrison, R. E.
Journal: J Biol Chem

Osteoblasts are differentiated mesenchymal cells that function as the major bone producing cells of the body. Differentiation cues including ascorbic acid (AA) stimulation provoke intracellular changes in osteoblasts leading to the synthesis of the organic portion of the bone which includes collagen type I, proteoglycans and matrix proteins such as osteocalcin. During our microarray analysis of AA-stimulated osteoblasts, we observed a significant upregulation of the microtubule (MT) plus end binding protein, EB1, compared to undifferentiated osteoblasts. EB1 knockdown significantly impaired AA-induced osteoblast differentiation, as detected by reduced expression of osteoblast differentiation marker genes. Intracellular examination of AA-stimulated osteoblasts treated with EB1 siRNA revealed a reduction in MT stability with a concomitant loss of beta-catenin distribution at the cell cortex and within the nucleus. Diminished beta-catenin levels in EB1 siRNA-treated osteoblasts paralleled an increase in phospho beta-catenin and active GSK-3beta, a kinase known to target beta-catenin to the proteasome. EB1 siRNA treatment also reduced the expression of the beta-catenin gene targets, cyclin D1 and Runx2. Live immunofluorescent imaging of differentiated osteoblasts revealed a cortical association of EB1-mcherry with beta-catenin-GFP. Immunoprecipitation analysis confirmed an interaction between EB1 and beta-catenin. We also determined that cell:cell contacts and cortically associated EB1/beta-catenin interactions are necessary for osteoblast differentiation. Finally, using functional blocking antibodies, we identified E-cadherin as a major contributor to the cell:cell contact-induced osteoblast differentiation.

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An undesired effect of chemotherapy: gemcitabine promotes pancreatic cancer cell invasiveness through ROS-dependent, NF-kappaB- and HIF-1alpha-mediated upregulation of CXCR4.

Publication Date: 2013 Jun 5 PMID: 23740244
Authors: Arora, S. - Bhardwaj, A. - Singh, S. - Srivastava, S. K. - McClellan, S. - Nirodi, C. S. - Piazza, G. A. - Grizzle, W. E. - Owen, L. B. - Singh, A. P.
Journal: J Biol Chem

Recently, we have shown that CXCL12/CXCR4 signaling plays an important role in gemcitabine-resistance of pancreatic cancer (PC) cells. Here, we explored the effect of gemcitabine on this resistance mechanism. Our data demonstrate that gemcitabine induces CXCR4 expression in two PC cell lines (MiaPaCa and Colo357) in a dose- and time- dependent manner. Gemcitabine-induced CXCR4 expression is dependent on reactive oxygen species (ROS) generation as it is abrogated by pre-treatment of PC cells with the free radical scavenger N-acetyl-L-cysteine. CXCR4 upregulation by gemcitabine correlates with time-dependent accumulation of NF-kappaB and HIF-1alpha in the nucleus. Enhanced binding of NF-kappaB and HIF-1alpha to the CXCR4 promoter is observed in gemcitabine-treated PC cells, while their silencing by RNA interference causes suppression of gemcitabine-induced CXCR4 expression. ROS induction upon gemcitabine treatment precedes the nuclear accumulation of NF-kappaB and HIF-1alpha, and suppression of ROS diminishes these effects. The effect of ROS on NF-kappaB and HIF-1alpha is mediated through activation of ERK1/2 and Akt and their pharmacological inhibition also suppresses gemcitabine-induced CXCR4 upregulation. Interestingly, our data demonstrate that nuclear accumulation of NF-kappaB results from phosphorylation-induced degradation of IkappaBalpha, while HIF-1alpha upregulation is NF-kappaB-dependent. Lastly, our data demonstrate that gemcitabine-treated PC cells are more motile and exhibit significantly greater invasiveness against a CXCL12 gradient. Together, these findings reinforce the role of CXCL12/CXCR4 signaling in gemcitabine resistance and point toward an unintended and undesired effect of chemotherapy.

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Activation of the Transcription Factor GLI1 by WNT Signaling Underlies the Role of SULFATASE 2 as a Regulator of Tissue Regeneration.

Publication Date: 2013 Jun 5 PMID: 23740243
Authors: Nakamura, I. - Fernandez-Barrena, M. G. - Ortiz-Ruiz, M. C. - Almada, L. L. - Hu, C. - Elsawa, S. F. - Mills, L. D. - Romecin, P. A. - Gulaid, K. H. - Moser, C. D. - Han, J. J. - Vrabel, A. - Hanse, E. A. - Akogyeram, N. A. - Albrecht, J. H. - Monga, S. P. - Sanderson, S. O. - Prieto, J. - Roberts, L. R. - Fernandez-Zapico, M. E.
Journal: J Biol Chem

Tissue regeneration requires the activation of a set of specific growth signaling pathways. The identity of these cascades and their biological roles are known, however, the molecular mechanisms regulating the interplay between these pathways remain poorly understood. Here, we define a new role for SULFATASE 2 (SULF2) in regulating tissue regeneration and define the WNT-GLI1 axis as a novel downstream effector for this sulfatase in a liver model of tissue regeneration. SULF2 is a heparan sulfate 6-O-endosulfatase, which releases growth factors from extracellular storage sites turning activate multiple signaling pathways. We demonstrate that SULF2-KO mice display delayed regeneration after partial hepatectomy (PH). Mechanistic analysis of the SULF2-KO phenotype showed a decrease in WNT signaling pathway activity in vivo. In isolated hepatocytes, SULF2 deficiency blocked WNT-induced beta-CATENIN nuclear translocation, TCF activation, and proliferation. Furthermore, we identified the transcription factor GLI1 as a novel target of the SULF2-WNT cascade. WNT induces GLI1 expression in a SULF2- and beta-CATENIN-dependent manner. GLI1-KO mice phenocopied the SULF2-KO, showing delayed regeneration and decreased hepatocyte proliferation. Moreover, we identified CYCLIN D1, a key mediator of cell growth during tissue regeneration, as a GLI1 transcriptional target. GLI1 binds to the cyclin d1 promoter and regulates its activity and expression. Finally, restoring GLI1 expression in the liver of SULF2-KO mice after PH rescues CYCLIN D1 expression and hepatocyte proliferation to wild-type levels. Thus, together these findings define a novel pathway in which SULF2 regulates tissue regeneration in part via the activation of a novel WNT-GLI1-CYCLIN D1 pathway.

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Orm proteins integrate multiple signals to maintain sphingolipid homeostasis.

Publication Date: 2013 Jun 4 PMID: 23737533
Authors: Gururaj, C. - Federman, R. - Chang, A.
Journal: J Biol Chem

Sphingolipids are structural components of membranes, and sphingolipid metabolites serve as signaling molecules. The first and rate-limiting step in sphingolipid synthesis is catalyzed by serine palmitoyltransferase (SPT). The recently discovered SPT-associated proteins, Orm1 and Orm2, are critical regulators of sphingolipids. Orm protein phosphorylation mediating feedback regulation of SPT activity occurs in response to multiple sphingolipid intermediates, including long chain base and complex sphingolipids. Both branches of the TOR signaling network, TORC1 and TORC2, participate in regulating sphingolipid synthesis via Orm phosphorylation in response to the status of the sphingolipids as well as nutritional conditions. Moreover, a calcium and calcineurin-dependent pathway is activated in response to ER stress to regulate sphingolipid synthesis, including transcriptional induction of ORM2. Conversely, the calcium, calcineurin-dependent pathway signals the unfolded stress response upon lipid dysregulation in the absence of the Orm proteins to restore ER homeostasis.

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Hsp110 is a bona fide chaperone using ATP to unfold stable misfolded polypeptides and reciprocally collaborate with Hsp70 to solubilize protein aggregates.

Publication Date: 2013 Jun 4 PMID: 23737532
Authors: Mattoo, R. U. - Sharma, S. K. - Priya, S. - Finka, A. - Goloubinoff, P.
Journal: J Biol Chem

Structurally and sequence-wise, the Hsp110s belong to a subfamily of the Hsp70 chaperones. Like the classical Hsp70s, members of the Hsp110 subfamily can bind misfolding polypeptides and hydrolyse ATP. Yet they apparently act as a mere subordinate nucleotide exchange factors, regulating the ability of Hsp70 to hydrolyse ATP and convert stable protein aggregates into native proteins. Using stably misfolded and aggregated polypeptides as substrates in optimised in vitro chaperone assays, we show that the human cytosolic Hsp110s (HSPH1, HSPH2) are bona fide chaperones on its own that collaborates with Hsp40 (DNAJA1, DNAJB1) to hydrolyse ATP and unfold and convert stable misfolded polypeptides into natively refolded proteins. Moreover, equimolar Hsp70 (HSPA1A) and Hsp110 (HSPH1) formed a powerful molecular machinery that optimally reactivated stable luciferase aggregates in an ATP- and DNAJA1-dependent manner, in a disaggregation mechanism whereby the two paralogous chaperones alternatively activate the release of bound unfolded polypeptide substrates from one another, leading to native protein refolding.

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Interaction of aryl hydrocarbon receptor-interacting protein-like 1 with the farnesyl moiety.

Publication Date: 2013 Jun 4 PMID: 23737531
Authors: Majumder, A. - Gopalakrishna, K. N. - Cheguru, P. - Gakhar, L. - Artemyev, N. O.
Journal: J Biol Chem

Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a photoreceptor specific chaperone of the visual effector enzyme phosphodiesterase-6 (PDE6). AIPL1 has been shown to bind the farnesylated PDE6A subunit. Mutations in AIPL1 are thought to destabilize PDE6 and thereby cause Leber congenital amaurosis type 4 (LCA4), a severe form of childhood blindness. Here, we examined the solution structure of AIPL1 by Small Angle X-ray Scattering (SAXS). A structural model of AIPL1 with the best fit to the scattering data features two independent FK506-binding protein (FKBP)-like and tetratricopeptide repeat (TPR) domains. Guided by the model, we tested the hypothesis that AIPL1 directly binds the farnesyl moiety. Our studies revealed high-affinity binding of the farnesylated-Cys probe to the FKBP-like domain of AIPL1, thus uncovering a novel function of this domain. Mutational analysis of the potential farnesyl-binding sites on AIPL1 identified two critical residues, Cys89 and Leu147, located in close proximity in the structure model. The L147A mutation and the LCA-linked C89R mutation prevented the binding of the farnesyl-Cys probe to AIPL1. Furthermore, Cys89 and Leu147 flank the unique insert region of AIPL1, deletion of which also abolished the farnesyl interaction. Our results suggest that the binding of PDE6A farnesyl is essential to normal function of AIPL1 and its disruption is one of the mechanisms underlying LCA.

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Nuclear factor of activated T cells c1 mediates p21-activated kinase 1 activation in the modulation of chemokine-induced human aortic smooth muscle cell F-actin stress fiber formation, migration and proliferation and injury-induced vascular wall remodeling.

Publication Date: 2013 Jun 4 PMID: 23737530
Authors: Kundumani-Sridharan, V. - Singh, N. K. - Kumar, S. - Gadepalli, R. - Rao, G. N.
Journal: J Biol Chem

Recent literature suggests that cyclin-dependent kinases (CDKs) mediate cell migration. However, the mechanisms were not known. Therefore, the objective of this study is to test whether cyclin-CDKs activate Pak1, an effector of Rac1, in the modulation of monocyte chemotactic protein 1 (MCP1)-induced human aortic smooth muscle cell (HASMC) migration and proliferation. MCP1 stimulated Pak1 phosphorylation and F-actin stress fiber formation in a delayed manner in HASMCs. Inhibition of Pak1 suppressed MCP1-induced HASMC F-actin stress fiber formation, migration and proliferation. Down regulation of cyclin D1, CDK6 or CDK4 levels blocked MCP1-induced Pak1 phosphorylation and its activity leading to decreased HASMC F-actin stress fiber formation, migration and proliferation. Depletion of NFATc1 levels also attenuated MCP1-induced cyclin D1 expression, CDK6 and CDK4 activities as well as Pak1 phosphorylation and its activity, resulting in decreased HASMC F-actin stress fiber formation, migration and proliferation. CDK4 forms a complex with Pak1 in response to MCP1 and immunocomplex kinase assay showed that Pak1 is a substrate for CDK4. Furthermore, MCP1-induced NFATc1-cyclin D1-CDK6-CDK4-Pak1 activation requires Rac1. SM-specific deletion of NFATc1 led to decreased cyclin D1 expression, CDK6, CDK4 and Pak1 activities resulting in reduced neointima formation in response to guidewire injury. Thus, the present observations uncover a new signaling that shows that Pak1 is a substrate for CDK4 and this event is mediated by Rac1-dependent NFATc1-mediated cyclin D1 expression and CDK6 activity. In addition, SM NFATc1 activation mediates balloon injury-induced cyclin D1-CDK6-CDK4-Pak1 signaling and neointima formation.

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An uncharacterized member of the ribokinase family in Thermococcus kodakarensis exhibits myo-inositol kinase activity.

Publication Date: 2013 Jun 4 PMID: 23737529
Authors: Sato, T. - Fujihashi, M. - Miyamoto, Y. - Kuwata, K. - Kusaka, E. - Fujita, H. - Miki, K. - Atomi, H.
Journal: J Biol Chem

Here we performed structural and biochemical analyses on the TK2285 gene product from the hyperthermophilic archaeon Thermococcus kodakarensis, an uncharacterized protein annotated as a member of the ribokinase family. The three-dimensional structure of the TK2285 protein resembled those of previously characterized members of the ribokinase family, including ribokinase, adenosine kinase and phosphofructokinase. Conserved residues characteristic of this protein family were located in a cleft of the TK2285 protein, as in other members whose structures have been determined. We thus examined the kinase activity of the TK2285 protein towards various sugars recognized by well-characterized ribokinase family members. Although activity with sugar phosphates and nucleosides were not detected, kinase activity was observed toward D-allose, D-lyxose, D-tagatose, D-talose, D-xylose, and D-xylulose. Kinetic analyses with the six sugar substrates revealed high Km values, suggesting that they were not the true physiological substrates. By examining activity toward amino sugars, sugar alcohols, and disaccharides, we found that the TK2285 protein exhibited prominent kinase activity toward myo-inositol. Kinetic analyses with myo-inositol revealed a greater kcat and much lower Km value than those obtained with the monosaccharides, resulting in over a 2000-fold increase in kcat/Km values. TK2285 homologs are distributed among members of Thermococcales, and in most species, the gene is positioned close to a myo-inositol monophosphate synthase gene. Our results suggest the presence of a novel subfamily of the ribokinase family whose members are present in the archaea and recognize myo-inositol as a substrate.

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The NAD+ synthesis enzyme NMNAT1 regulates rRNA transcription.

Publication Date: 2013 Jun 4 PMID: 23737528
Authors: Song, T. - Yang, L. - Kabra, N. - Chen, L. - Koomen, J. - Haura, E. B. - Chen, J.
Journal: J Biol Chem

The chromosomal region encoding the nuclear NAD+ synthesis enzyme NMNAT1 is frequently deleted in human cancer. We describe evidence that NMNAT1 interacts with the nucleolar repressor protein nucleomethylin (NML) and is involved in regulating rRNA transcription. NMNAT1 binds to NML and is recruited into a ternary complex containing the NAD+-dependent deacetylase SirT1. NMNAT1 expression stimulates the deacetylase function of SirT1. Knockdown of NMNAT1 enhances rRNA transcription and promotes cell death after nutrient deprivation. Furthermore, NMNAT1 expression is induced by DNA damage and plays a role in preventing cell death after damage. Heterozygous deletion of NMNAT1 in lung tumor cell lines correlates with low expression level and increased sensitivity to DNA damage. These results suggest that NMNAT1 deletion in tumors may contribute to transformation by increasing rRNA synthesis, but may also increase sensitivity to nutrient stress and DNA damage.

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Transforming Growth Factor-beta Induces transcription factors MafK and Bach1 to Suppress Expression of the Heme Oxygenase-1 Gene.

Publication Date: 2013 Jun 4 PMID: 23737527
Authors: Okita, Y. - Kamoshida, A. - Suzuki, H. - Itoh, K. - Motohashi, H. - Igarashi, K. - Yamamoto, M. - Ogami, T. - Koinuma, D. - Kato, M.
Journal: J Biol Chem

Transforming growth factor-beta (TGF-beta) has multiple functions in embryogenesis, adult homeostasis, tissue repair, and development of cancer. Here, we report that TGF-beta suppresses the transcriptional activation of the heme oxygenase-1 gene (HO-1), which is implicated in protection against oxidative injury and lung carcinogenesis. HO-1 is a target of the oxidative stress-responsive transcription factor, Nrf2. TGF-beta did not affect the stabilization or nuclear accumulation of Nrf2 after stimulation with electrophiles. Instead, TGF-beta induced expression of transcription factors MafK and Bach1. Enhanced expression of either MafK or Bach1 was enough to suppress the electrophile-inducible expression of HO-1 even in the presence of accumulated Nrf2 in the nucleus. Knockdown of MafK and Bach1 by siRNA abolished TGF-beta-dependent suppression of HO-1. Furthermore, chromatin immunoprecipitation assays revealed that Nrf2 substitutes for Bach1 at the antioxidant response elements (AREs; E1 and E2), which are responsible for the induction of HO-1 in response to oxidative stress. On the other hand, pretreatment with TGF-beta suppressed binding of Nrf2 to both E1 and E2 but marginally increased the binding of MafK to E2 together with Smads. As TGF-beta is activated after injury and in the process of cancer development, these findings suggest a novel mechanism by which damaged tissue becomes vulnerable to oxidative stress and xenobiotics.

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Streptococcus pneumoniae serotype 11D has a bi-specific glycosyltransferase and expresses two different capsular polysaccharide repeating units.

Publication Date: 2013 Jun 4 PMID: 23737526
Authors: Oliver, M. B. - Jones, C. - Larson, T. R. - Calix, J. J. - Zartler, E. R. - Yother, J. - Nahm, M. H.
Journal: J Biol Chem

Streptococcus pneumoniae (pneumococci) express a capsular polysaccharide (CPS) that protects against host immunity and is synthesized by enzymes in the capsular polysaccharide synthesis (cps ) locus. Serogroup 11 has 6 members (11A-E) and the CPS structure of all members has been solved, except for serotype 11D. The cps loci of 11A and 11D differ by one codon (Asn112Ser) in wcrL, which putatively encodes a glycosyltransferase that adds the fourth sugar of the CPS repeating unit (RU). Gas chromatography and nuclear magnetic resonance analysis revealed that 11A and 11D PS contain identical CPS RUs that contain alphaGlc as the fourth sugar. However, approximately 25% of 11D CPS RUs contain instead alphaGlcNAc as the fourth sugar, suggesting that 11D wcrL encodes a bi-specific glycosyl-transferase. To test the hypothesis that codon 112 of WcrL determines enzyme specificity, and therefore the fourth sugar in the RU, we generated three isogenic pneumococcal strains with 11A cps loci containing wcrL encoding Ser112 (MBO128) or Ala112 (MBO130). MBO128 was serologically and biochemically identical to serotype 11D. MBO130 had a unique serologic profile, has as much alphaGlcNAc as 11F, 11B and 11C CPS does, and may represent a new serotype. These findings demonstrate how pneumococci alter their CPS structure and their immunologic properties with a minimal genetic change.

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Mammalian CAP1 (adenylyl Cyclase-Associated Protein 1) Regulates Cofilin Function, the Actin Cytoskeleton and Cell Adhesion.

Publication Date: 2013 Jun 4 PMID: 23737525
Authors: Zhang, H. - Ghai, P. - Wu, H. - Wang, C. - Field, J. - Zhou, G. L.
Journal: J Biol Chem

CAP (adenylyl Cyclase-Associated Protein) was first identified in yeast as a protein that regulates both the actin cytoskeleton and the Ras/cAMP pathway. Whereas the role in Ras signaling does not extend beyond yeast, evidence supports that CAP regulates the actin cytoskeleton in all eukaryotes including mammals. In vitro actin polymerization assays show that both mammalian and yeast CAP homologues facilitate cofilin-driven actin filament turnover. We generated HeLa cells with stable CAP1 knockdown using RNA Interference. Depletion of CAP1 led to larger cell size, remarkably developed lamellipodia as well as accumulation of filamentous actin (F-actin). Moreover, we found that CAP1 depletion also led to changes in cofilin phosphorylation and localization as well as activation of FAK (Focal Adhesion Kinase) and enhanced cell spreading. CAP1 forms complexes with the adhesion molecules FAK and Talin, which likely underlie the cell adhesion phenotypes through inside-out activation of integrin signaling. CAP1-depleted HeLa cells also had substantially elevated cell motility as well as invasion through Matrigel. In summary, in addition to generating in vitro and in vivo evidence further establishing the role of mammalian CAP1 in actin dynamics, we identified a novel cellular function for CAP1 in regulating cell adhesion.

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Roles of Long-range Electrostatic Domain Interactions and K+ in Phosphoenzyme Transition of Ca2+-ATPase.

Publication Date: 2013 Jun 4 PMID: 23737524
Authors: Yamasaki, K. - Daiho, T. - Danko, S. - Suzuki, H.
Journal: J Biol Chem

Sarcoplasmic reticulum Ca2+-ATPase couples the motions and rearrangements of three cytoplasmic domains (A, P, N) with Ca2+ transport. We explored the role of electrostatic force in the domain dynamics in a rate-limiting phosphoenzyme (EP) transition by a systematic approach combining electrostatic screening with salts, computer analysis of electric fields in crystal structures, and mutations. Low KCl concentration activated and increasing salt above 0.1 M inhibited the EP transition. A plot of the logarithm of the transition rate versus the square of the mean activity coefficient of the protein gave a linear relationship allowing division of the activation energy into an electrostatic component and a non-electrostatic component in which the screenable electrostatic forces are shielded by salt. Results show that the structural change in the transition is sterically restricted, but that strong electrostatic forces, when K+ is specifically bound at the P domain, come into play to accelerate the reaction. Electric fields analysis revealed long-range electrostatic interactions between the N and P domains around their hinge. Mutations of the residues directly involved and other charged residues at the hinge disrupted in parallel the electric field and the structural transition. Favorable electrostatics evidently provides a low energy path for the critical N-domain motion towards the P domain, overcoming steric restriction. The systematic approach employed here is, in general, a powerful tool for understanding structural mechanisms of enzymes.

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The Chlorite Dismutase (HemQ) from Staphylococcus aureus Has a Redox-Sensitive Heme and is Associated with the Small Colony Variant Phenotype.

Publication Date: 2013 Jun 4 PMID: 23737523
Authors: Mayfield, J. A. - Hammer, N. D. - Kurker, R. C. - Chen, T. K. - Ojha, S. - Skaar, E. P. - Dubois, J. L.
Journal: J Biol Chem

The chlorite dismutases (Clds or C-family proteins) are a widespread family of heme-binding proteins for which chemical and biological roles remain unclear. An association of the gene with heme biosynthesis in Gram-positive bacteria was previously demonstrated by experiments involving introduction of genes from two Gram positive species into heme biosynthesis mutant strains of E. coli, leading to the gene being renamed hemQ. To assess the biological role of the gene product more directly, a Staphylococcus aureus strain with an inactivated hemQ gene was generated and shown to be a slow-growing small colony variant (SCV) under aerobic but not anaerobic conditions. The SCV phenotype is rescued by the addition of exogenous heme despite an otherwise wild type heme biosynthetic pathway. The DeltahemQ mutant accumulates coproporphyrin specifically under aerobic conditions. Though its sequence is highly similar to functional chlorite dismutases, the HemQ protein has no steady state reactivity with chlorite, very modest reactivity with H2O2 or peracetic acid, and no observable transient intermediates. The HemQ equilibrium affinity for heme is in the low micromolar range. Holo-HemQ reconstituted with heme exhibits heme lysis after <50 turnovers with peroxide <10 turnovers with chlorite. The heme-free apo protein aggregates or unfolds over time. IsdG-like proteins and antibiotic biosynthesis monooxygenases are close sequence and structural relatives of HemQ that use heme or porphyrin-like organic molecules as substrates. The genetic and biochemical data suggest a similar substrate role for heme or porphyrin, with possible sensor-regulator functions for the protein.

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Divergent sequence tunes ligand sensitivity in phospholipid-regulated hormone receptors.

Publication Date: 2013 Jun 4 PMID: 23737522
Authors: Musille, P. M. - Pathak, M. - Lauer, J. L. - Griffin, P. R. - Ortlund, E. A.
Journal: J Biol Chem

The NR5A subfamily family of nuclear receptors (NRs) are important regulators of pluripotency, lipid and glucose homeostasis, and steroidogenesis. Liver receptor homologue 1 (LRH-1; NR5A2) and steroidogenic factor 1 (SF-1; NR5A1), have therapeutic potential for the treatment of metabolic and neoplastic disease; however, a poor understanding of their ligand regulation has hampered the pursuit of these proteins as pharmaceutical targets. In this study, we dissect how sequence variation among LRH-1 orthologs affects phospholipid (PL) binding and regulation. Both human and mouse LRH-1 (mLRH-1) respond to newly discovered medium chain PL agonists to modulate lipid and glucose homeostasis. These PLs activate human LRH-1 (hLRH-1) by altering receptor dynamics in a newly identified alternate activation function region. Mouse and Drosophila orthologs contain divergent sequence in this region potentially altering PL-driven activation. Structural evidence suggests that these sequence differences in mouse LRH-1 (mLRH-1) and Drosophila FTZ-f1 (dmFTZ-f1), confer at least partial ligand independence, making them poor models for hLRH-1 studies; however, the mechanisms of ligand independence remain untested. We show using structural and biochemical methods that the recent evolutionary divergence of the mLRH-1 stabilizes the active conformation in the absence of ligand, yet does not abrogate PL-dependent activation. We also show by mass spectrometry and biochemical assays that FTZ-f1 is incapable of PL binding. This work provides a structural mechanism for the differential tuning of PL-sensitivity in NR5A orthologs and supports the use of mice as viable therapeutic models for LRH-1-dependent diseases.

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Identification of a novel Endoplasmic Reticulum Stress Response Element regulated by XBP1.

Publication Date: 2013 Jun 4 PMID: 23737521
Authors: Misiewicz, M. - Dery, M. A. - Foveau, B. - Jodoin, J. - Ruths, D. - Leblanc, A. C.
Journal: J Biol Chem

Understanding regulatory mechanisms mediating PRNP gene expression is highly relevant to elucidating normal cellular prion protein (PrP) function(s) and the transmissibility of prion protein neurodegenerative diseases. Here, luciferase reporter assays show that an endoplasmic reticulum stress element (ERSE)-like element, CCAAT-N26-CCACG in the human PRNP promoter, is regulated by ER stress and the X-Box protein 1 (XBP1), but not by the activating transcription factor alpha (ATF6alpha). Bioinformatics identify the ERSE-26 motif in 37 other human genes in the absence of canonical ERSE sites, except for three genes. Several of these genes are associated with a synaptic function or are involved in oxidative stress. Brefeldin A, tunicamycin, and thapsigargin ER stressors induce gene expression of PRNP and four randomly chosen ERSE-26-containing genes, ERLEC1, GADD45B, SESN2, and SLC38A5, in primary human neuron cultures or in the breast carcinoma MCF-7 cell line, although the level of the response depends on the gene analyzed, the genetic background of the cells, the cell type and the ER stressor. Overexpression of XBP1 increases, whereas siRNA knock down of XBP1 considerably reduces, PRNP and ERLEC1 mRNA levels in MCF-7 cells. Taken together, these results identify a novel ER stress regulator implicating the ER stress response in previously unrecognized cellular functions.

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The Drosophila PDGF and VEGF-Receptor Related (Pvr) Ligands Pvf2 and Pvf3 Control Hemocyte Viability and Invasive Migration.

Publication Date: 2013 Jun 4 PMID: 23737520
Authors: Parsons, B. - Foley, E.
Journal: J Biol Chem

Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) family members are essential and evolutionary conserved determinants of blood cell development and dispersal. In addition, VEGFs are integral to vascular growth and permeability with detrimental contributions to ischemic diseases and metastatic cancers. The PDGF/VEGF-receptor related (Pvr) protein is implicated in the migration and trophic maintenance of macrophage-like hemocytes in Drosophila melanogaster embryos. pvr mutants have a depleted hemocyte population and a breakdown in hemocyte distribution. Previous studies suggested redundant functions for the Pvr ligands, Pvf2 and Pvf3 in the regulation of hemocyte migration, proliferation and size. However, the precise roles that Pvf2 and Pvf3 play in hematopoiesis remain unclear due to the lack of available mutants. To determine Pvf2 and Pvf3 functions in vivo, we generated a genomic deletion that simultaneously ablates Pvf2 and Pvf3. From our studies, we identified redundant contributions of Pvf2 and Pvf3 to the Pvr trophic maintenance of hemocytes. Furthermore, we uncovered a novel role for Pvfs in invasive migrations. We showed that Pvf2 and Pvf3 are not required for the directed migration of hemocytes, but act locally in epithelial cells to coordinate trans-epithelial migration of hemocytes. Our findings redefine Pvf roles in hemocyte migration and highlight novel Pvf roles in hemocyte invasive migration. These new parallels between the Pvr and PDGF/VEGF pathways extend the utility of the Drosophila embryonic system to dissect physiological and pathological roles of PDGF/VEGF-like growth factors.

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Anti-antimicrobial peptides: folding-mediated host defense antagonists.

Publication Date: 2013 Jun 4 PMID: 23737519
Authors: Ryan, L. - Lamarre, B. - Diu, T. - Ravi, J. - Judge, P. J. - Temple, A. - Carr, M. - Cerasoli, E. - Su, B. - Jenkinson, H. F. - Martyna, G. - Crain, J. - Watts, A. - Ryadnov, M. G.
Journal: J Biol Chem

Antimicrobial or host defense peptides are innate immune regulators found in all multicellular organisms. Many of them fold into membrane-bound alpha-helices and function by causing cell wall disruption in microorganisms. Herein we probe the possibility and functional implications of antimicrobial antagonism mediated by complementary coiled coil interactions between antimicrobial peptides and de novo designed antagonists, anti-antimicrobial peptides. Using sequences from native helical families such as cathelicidins, cecropins and magainins we demonstrate that designed antagonists can co-fold with antimicrobial peptides into functionally inert helical oligomers. The properties and function of the resulting assemblies were studied in solution, membrane environments and in bacterial culture by a combination of chiroptical and solid state NMR spectroscopies, microscopy, bioassays and molecular dynamics simulations. The findings offer a molecular rationale for anti-antimicrobial responses with potential implications for antimicrobial resistance.

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Diaminothiazoles Modify Tau Phosphorylation and Improve the Tauopathy in Mouse Models.

Publication Date: 2013 Jun 4 PMID: 23737518
Authors: Zhang, X. - Hermandez, I. - Rei, D. - Mair, W. - Laha, J. K. - Cornwell, M. E. - Cuny, G. D. - Tsai, L. H. - Steen, J. A. - Kosik, K. S.
Journal: J Biol Chem

Although tau accumulation is a feature of several neurodegenerative conditions treatment options for these conditions are non-existent. Targeting tau kinases represents a potential therapeutic approach. Small molecules in the diaminothiazole class are potent tau kinase inhibitors that target CDK5 and GSK3beta. Lead compounds from the series have IC50 values toward CDK5/p25 and GSK3beta in the low nanomolar range and no observed toxicity in the therapeutic dose range. Neuronal protective effects and decreased PHF-1 immunoreactivity were observed in two animal models, 3xTg-AD and CK-p25. Treatment nearly eliminated sarkosyl-insoluble tau with the most prominent effect on the phosphorylation at Ser404. Treatment also induced the recovery of memory in a fear conditioning assay. Given the contribution of both CDK5/p25 and GSK3beta to tau phosphorylation, effective treatment of tauopathies may require dual kinase targeting.

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Rab Family Proteins Regulate the Endosomal Trafficking and Function of RGS4.

Publication Date: 2013 Jun 3 PMID: 23733193
Authors: Bastin, G. - Heximer, S. P.
Journal: J Biol Chem

RGS4, a heterotrimeric G-protein inhibitor, localizes to plasma membrane (PM) and endosomal compartments. We here examined Rab-mediated control of RGS4 internalization and recycling. Wild type (WT) and constitutively active Rab5 decreased RGS4 PM levels while increasing its endosomal targeting. Rab5, however, did not appreciably affect the PM localization or function of the M1 muscarinic receptor (M1R)/Gq signaling cascade. RGS4-containing endosomes colocalized with subsets of Rab5-, transferrin receptor- and Lamp1/Lysotracker-marked compartments suggesting RGS4 traffics through PM recycling or acidified endosome pathways. Rab7 activity promoted TGN association whereas Rab7(DN) trapped RGS4 in late endosomes. Furthermore, RGS4 was found to colocalize with an endosomal pool marked by Rab11, the protein that mediates recycling/sorting of proteins to the PM. The Cys12 residue in RGS4 appeared important for its Rab11-mediated trafficking to the PM. Rab11(DN) decreased RGS4 PM levels and increased the number of RGS4-containing endosomes. Inhibition of Rab11 activity decreased RGS4 function as an inhibitor of M1R activity without affecting localization and function of the M1R/Gq signaling complex. Thus, both Rab5 activation and Rab11 inhibition decrease RGS4 function in a manner that is independent from their effects on the localization and function of the M1R/Gq signaling complex. This is the first study to implicate Rab GTPases in the intracellular trafficking of an RGS protein. Thus, Rab GTPases may be novel molecular targets for the selective regulation of M1R-mediated signaling via their specific effects on RGS4 trafficking and function.

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Human P-glycoprotein Contains a Greasy Ball-and-Socket Joint at the Second Transmission Interface.

Publication Date: 2013 Jun 3 PMID: 23733192
Authors: Loo, T. W. - Bartlett, M. C. - Clarke, D. M.
Journal: J Biol Chem

The P-glycoprotein (P-gp) drug pump protects us from toxins. Drug-binding sites in the transmembrane (TM) domains (TMDs) are connected to the nucleotide-binding domains (NBDs) by intracellular helices (IHs). TMD-NBD crosstalk is a key step in the transport mechanism since drug binding stimulates ATP hydrolysis followed by drug efflux. Here, we tested whether the IHs are critical for maturation and TMD-NBD coupling by characterizing the effects of mutations to the IH1 and IH2 interfaces. While IH1 mutations had little effect, most mutations at the IH2-NBD2 interface inhibited maturation or activity. For example, the F1086A mutation at the IH2-NBD2 interface abolished drug-stimulated ATPase activity. The mutant F1086A however, retained the ability to bind ATP and drug substrates. The mutant was defective in mediating ATP-dependent conformational changes in the TMDs because binding of ATP no longer promoted cross-linking between cysteines located at the extracellular ends of TM segments 6 and 12. Replacement of Phe1086 (in NBD2) with hydrophobic but not charged residues yielded active mutants. The activity of the F1086A mutant could be restored when the nearby residue Ala266 (in IH2) was replaced with aromatic residues. These results suggest that Phe1086/Ala266 lies in a hydrophobic IH2-NBD2 ball-and-socket joint.

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Non-destructive and selective imaging of the functionally active, pro-invasive membrane type-1 matrix metalloproteinase (MT1-MMP) enzyme in cancer cells.

Publication Date: 2013 Jun 3 PMID: 23733191
Authors: Remacle, A. G. - Shiryaev, S. A. - Golubkov, V. S. - Freskos, J. N. - Brown, M. A. - Karwa, A. S. - Naik, A. D. - Howard, C. P. - Sympson, C. J. - Strongin, A. Y.
Journal: J Biol Chem

Proteolytic activity of cell surface-associated MT1-MMP (MMP-14) is directly related to cell migration, invasion and metastasis. MT1-MMP is regulated as a proteinase by activation and conversion of the latent proenzyme into the active enzyme, and also via inhibition by tissue inhibitors of MMPs (TIMPs) and self-proteolysis. MT1-MMP is also regulated as a membrane protein through its internalization and recycling. Routine immunohistochemistry, flow cytometry, reverse transcription-PCR and immunoblotting methodologies do not allow quantitative imaging and assessment of the cell-surface levels of the active, TIMP-free MT1-MMP enzyme. Here, we developed a fluorescent reporter prototype that targets the cellular active MT1-MMP enzyme alone. The reporter (MP-3653) represents a liposome tagged with a fluorochrome and functionalized with a PEG chain spacer linked to an inhibitory hydroxamate warhead. Our studies using the MP-3653 reporter and its inactive derivative demonstrated that MP-3653 can be efficiently used not only to visualize the trafficking of MT1-MMP through the cell compartment, but also to quantify the femtomolar range amounts of the cell surface-associated active MT1-MMP enzyme in multiple cancer cell types, including breast carcinoma, fibrosarcoma and melanoma. Thus, the levels of the naturally expressed, fully functional, active cellular MT1-MMP enzyme are roughly equal to 1x105 molecules/cell, while these levels are in a 1x106 range in the cells with the enforced MT1-MMP expression. We suggest that the reporter we developed will contribute to the laboratory studies of MT1-MMP and then, ultimately, to the design of novel, more efficient prognostic approaches and personalized cancer therapies.

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NO and KLF4 epigenetically modify CIITA to repress MHC-II expression during M. bovis BCG infection.

Publication Date: 2013 Jun 3 PMID: 23733190
Authors: Ghorpade, D. S. - Holla, S. - Sinha, A. Y. - Alagesan, S. K. - Balaji, K. N.
Journal: J Biol Chem

Pathogenic mycobacteria employ several immune evasion strategies such as inhibition of CIITA and MHC-II expression, in order to survive and persist in host macrophages. However, precise roles for specific signaling components executing downregulation of CIITA/MHC-II have not been adequately addressed. Here, we demonstrate that M. bovis BCG mediated, TLR2 signaling induced iNOS/NO expression is obligatory for the suppression of IFN-gamma induced CIITA/MHC-II functions. Significantly, NOTCH/PKC/MAPK-triggered signaling crosstalk was found critical for iNOS/NO production. NO responsive recruitment of a bifunctional transcription factor, KLF4 to the promoter of CIITA during M. bovis BCG infection of macrophages was essential to orchestrate the epigenetic modifications mediated by histone methyltransferase EZH2 or miR-150 and thus calibrate CIITA/MHC-II expression. NO dependent KLF4 regulated processing and presentation of ovalbumin by infected macrophages to reactive T cells. Altogether, our study delineates a novel role for iNOS/NO/KLF4 in dictating mycobacterial capacity to inhibit CIITA/MHC-II mediated antigen presentation by infected macrophages and thereby elude immune surveillance.

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Signal transducer and activator of transcription 3 (STAT3) degradation by proteasome controls a developmental switch in neurotrophin-dependence.

Publication Date: 2013 Jun 3 PMID: 23733189
Authors: Murase, S.
Journal: J Biol Chem

Neonatal brains develop through a program that eliminates about half of neurons. During this period, neurons depend on neurotrophins for their survival. Recently, we reported that, at the conclusion of the naturally occurring death period, neurons become neurotrophin independent, and further, that this developmental switch is achieved by the emergence of a second survival pathway mediated by signal transducer and activator of transcription 3 (STAT3). Here I show that calcineurin plays a key role in controlling the developmental switch in mouse hippocampal neurons. Calcineurin promotes the degradation of STAT3 via the ubiquitin-proteasome pathway. Inhibition of calcineurin acutely increases total levels of STAT3 as well as its activated forms, resulting in decreased levels of the tumor suppressor p53 and its pro-apoptotic target, Bax. In vivo and in vitro, calcineurin regulates levels of STAT3 and neurotrophin-dependence. TMF/ARA 160, the key mediator of STAT3 ubiquitination, is required for calcineurin-dependent STAT3 degradation. Thus, these results show the ubiquitin-proteasome pathway controls the critical developmental switch of neurotrophin-dependence in the newborn hippocampus.

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Estrogen receptor alpha L543A, L544A mutation changes antagonists to agonists which correlates with the ligand binding domain dimerization associated with DNA binding activity.

Publication Date: 2013 Jun 3 PMID: 23733188
Authors: Arao, Y. - Hamilton, K. J. - Coons, L. A. - Korach, K. S.
Journal: J Biol Chem

A ligand-dependent nuclear transcription factor, ERalpha has two transactivating functional domains (AF), AF-1 and AF-2. AF-1 is localized in the N-terminal region and AF-2 is distributed in the C-terminal ligand-binding domain (LBD) of the ERalpha protein. Helix 12 (H12) in the LBD is a component of the AF-2 and the configuration of H12 is ligand inducible to an active or inactive form. Previously, we demonstrated that the ERalpha mutant (AF2ER) possessing L543A, L544A mutations in H12 disrupts AF-2 function and reverses antagonists such as fulvestrant/ICI182780 (ICI) or 4-hydoxytamoxifen (OHT) into agonists, in the AF2ER knock-in mouse. Our previous in vitro studies suggested that the mode of AF2ER activation is similar to the partial agonist activity of OHT for WT-ERalpha. However it is still unclear how antagonists activate ERalpha. To understand the molecular mechanism of antagonist reversal activity, we analyzed the correlation between the ICI dependent ERE-mediated transcription activity of AF2ER and AF2ER-LBD dimerization activity. We report here that ICI dependent AF2ER activation correlated with the activity of AF2ER-LBD homodimerization. Prevention of dimerization impaired the ICI dependent ERE binding and transcription activity of AF2ER. The dislocation of H12 caused ICI dependent LBD homodimerization involving the F-domain, the adjoining region of H12. Furthermore, F-domain truncation also strongly depressed the dimerization of WT-ERalpha-LBD with antagonists but not with E2. AF2ER activation levels with ICI, OHT and raloxifene were parallel with the degree of AF2ER-LBD homodimerization; supporting a mechanism that antagonist-dependent LBD homodimerization involving the F-domain results in antagonist reversal activity of ERalpha.

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A novel family of soluble minimal scaffolds provides structural insight into the catalytic domains of integral-membrane metallopeptidases.

Publication Date: 2013 Jun 3 PMID: 23733187
Authors: Lopez-Pelegrin, M. - Cerda-Costa, N. - Martinez-Jimenez, F. - Cintas-Pedrola, A. - Canals, A. - Peinado, J. R. - Marti-Renom, M. A. - Lopez-Otin, C. - Arolas, J. L. - Gomis-Ruth, F. X.
Journal: J Biol Chem

In the search for structural models of integral-membrane metallopeptidases (MPs), we discovered three related proteins from thermophilic prokaryotes, which we grouped into a novel family called minigluzincins. We determined the crystal structures of the zymogens of two of these (Pyrococcus abyssi proabylysin and Methanocaldococcus jannaschii projannalysin) which are soluble and, with ~100 residues, constitute the shortest structurally characterized MPs to date. Despite relevant sequence and structural similarity, the structures revealed two unique mechanisms of latency maintenance through the C-terminal segments hitherto unseen in MPs: intra-molecular, through an extended tail, in proabylysin, and crosswise inter-molecular, through a helix swap, in projannalysin. In addition, structural and sequence comparisons, as well as phylogenetic and bioinformatics analyses, revealed large similarity with MPs of the gluzincin tribe such as thermolysin, leukotriene A4 hydrolase relatives, and cowrins. Interestingly, gluzincins mostly have a glutamate as third characteristic zinc ligand while minigluzincins have a histidine. Sequence and structure similarity further allowed us to ascertain that minigluzincins are very similar to the catalytic domains of integral-membrane MPs of MEROPS database families M48 and M56, such as FACE1, HtpX, Oma1, and BlaR1/MecR1, which are provided with trans-membrane helices flanking or inserted into a minigluzincin-like catalytic domain. In a time where structural biochemistry of integral-membrane proteins in general still faces formidable challenges, the minigluzincin soluble minimal scaffold may contribute to our understanding of the working mechanisms of these membrane MPs and to the design of novel inhibitors through structure-aided rational drug design approaches.

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Protein signatures that promote operator selectivity among paralog MerR monovalent metal-ion regulators.

Publication Date: 2013 Jun 3 PMID: 23733186
Authors: Humbert, M. V. - Rasia, R. M. - Checa, S. K. - Soncini, F. C.
Journal: J Biol Chem

Two paralog transcriptional regulators of the MerR family, CueR and GolS, are responsible for monovalent metal ion sensing and resistance in Salmonella enterica. Although similar in sequence and also in their target binding sites these proteins differ in signal detection, and in the set of target genes they control. Recently, we demonstrated that selective promoter recognition depends on the presence of specific bases located at positions 3' and 3 within the operators they interact with. Here, we identify the amino acid residues within the N-terminal DNA-binding domain of these sensor proteins that are directly involved in operator discrimination. We demonstrate that a methionine residue at position 16 of GolS, absolutely conserved among GolS-like proteins, but absent in all CueR-like xenologs, is key to selectively recognize operators that harbor the distinctive GolS-operator signature, while residue at position 19 finely tunes the regulator/operator interaction. Furthermore, swapping these residues switches the set of genes recognized by these transcription factors. These results indicate that co-evolution of a regulator and its cognate operators within the bacterial cell provides the conditions to avoid cross-recognition and guarantees the proper response to metal injury.

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4-Hydroxynonenal induces G2/M phase cell cycle arrest by the activation of ataxia telangiectasia mutated and Rad3-related protein (ATR)/checkpoint kinase 1 (Chk1) signaling pathway.

Publication Date: 2013 Jun 3 PMID: 23733185
Authors: Chaudhary, P. - Sharma, R. - Sahu, M. - Vishwanatha, J. K. - Awasthi, S. - Awasthi, Y. C.
Journal: J Biol Chem

4-Hydroxynonenal (HNE) has been widely implicated in the mechanisms of oxidant- induced toxicity but the detrimental effects of HNE associated with DNA damage or cell cycle arrest have not been thoroughly studied. Here we demonstrate for the first time that HNE caused G2/M cell cycle arrest of hepatocellular carcinoma HepG2 (p53 wild type) and Hep3B (p53 null) cells that was accompanied with decreased expression of CDK1 and cyclin B1 and activation of p21 in a p53 independent manner. HNE treatment suppressed Cdc25C level which led to the inactivation of CDK1. HNE-induced phosphorylation of Cdc25C at Ser-216 resulted in its translocation from nucleus to cytoplasm, thereby facilitating its degradation via ubiquitin-mediated proteasomal pathway. This phosphorylation of Cdc25C was regulated by the activation of ATR/Chk1 pathway. Role of HNE in DNA double strand break was strongly suggested by a remarkable increase in comet tail formation and H2A.X phosphorylation in HNE treated cells in vitro. This was supported by increased in vivo phosphorylation of H2A.X in mGsta4 null mice that have impaired HNE metabolism and increased HNE levels in tissues. HNE-mediated ATR/Chk1 signaling was inhibited by ATR kinase inhibitor (caffeine). Additionally, most of the signaling effects of HNE on cell cycle arrest were attenuated in hGSTA4 transfected cells, thereby indicating the involvement of HNE in these events. A novel role of GSTA4-4 in the maintenance of genomic integrity is also suggested.

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Structure-function studies of the staphylococcal methicillin resistance anti-repressor, MecR2.

Publication Date: 2013 Jun 3 PMID: 23733184
Authors: Arede, P. - Botelho, T. - Guevara, T. - Uson, I. - Oliveira, D. C. - Gomis-Ruth, F. X.
Journal: J Biol Chem

Methicillin resistance in Staphylococcus aureus is elicited by the MecI-MecR1-MecA axis encoded by the mec locus. Recently, MecR2 was also identified as the third regulator of mec through binding of the methicillin repressor, MecI. Here we show that plasmid-encoded full-length MecR2 restores resistance in a sensitive S. aureus mecR2 deletion mutant of the resistant strain N315. The crystal structure of MecR2 reveals an N-terminal DNA-binding domain (NDD), an intermediate scaffold domain (ISD), and a C-terminal dimerization domain (CDD) that contributes to oligomerization. The protein shows structural similarity to ROK (from repressors, open-reading frames and kinases) family proteins, which bind DNA and/or sugar molecules. We found that functional cell-based assays of three point mutants affecting residues participating in sugar binding in ROK proteins had no effect on the resistance phenotype. By contrast, MecR2 bound short double-stranded DNA oligonucleotides non-specifically and a deletion mutant affecting the NDD showed a certain effect on activity, thus contributing to resistance less than the wild-type protein. Similarly, a deletion mutant, in which a flexible segment of ISD had been replaced by four glycines, significantly reduced MecR2 function, thus indicating that this domain may likewise be required for activity. Taken together, these results provide the structural basis for the activity of methicillin anti-repressor, MecR2, which would sequester MecI away from its cognate promoter region and facilitate its degradation.

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The PI(3,5)P2-dependent Tup1 conversion regulates metabolic reprogramming from glycolysis to gluconeogenesis.

Publication Date: 2013 Jun 3 PMID: 23733183
Authors: Han, B. K. - Emr, S. D.
Journal: J Biol Chem

Glucose/carbon metabolism is a fundamental cellular process in living cells. In response to varying environments, eukaryotic cells reprogram their glucose/carbon metabolism between aerobic or anaerobic glycolysis, oxidative phosphorylation, and/or gluconeogenesis. The distinct type of glucose/carbon metabolism that a cell carries out has significant effects on the cell's proliferation and differentiation. However, it is poorly understood how the reprogramming of glucose/carbon metabolism is regulated. Here, we report a novel endosomal PI(3,5)P2 lipid-dependent regulatory mechanism that is required for metabolic reprogramming from glycolysis to gluconeogenesis in Saccharomyces cerevisiae. Certain gluconeogenesis genes such as FBP1 (encoding fructose-1,6-bisphosphatase 1) and ICL1 (encoding isocitrate lyase 1) are under control of the Mig1 repressor and Cyc8-Tup1 corepressor complex. We previously identified the PI(3,5)P2-dependent Tup1 conversion (PIPTC), a mechanism to convert Cyc8-Tup1 corepressor to Cti6-Cyc8-Tup1 coactivator. We demonstrate the PIPTC plays a critical role for transcriptional activation of FBP1 and ICL1. Furthermore, without the PIPTC, the Cat8 and Sip4 transcriptional activators cannot be efficiently recruited to the promoters of FBP1 and ICL1, suggesting a key role for the PIPTC in remodulating the chromatin architecture at the promoters. Our findings expand our understanding of the regulatory mechanisms for metabolic reprogramming in eukaryotes to include key regulation steps outside the nucleus. Given that Tup1 and the metabolic enzymes that control PI(3,5)P2 are highly conserved among eukaryotes, our findings may provide important insights toward understanding glucose/carbon metabolic reprogramming in other eukaryotes including humans.

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A novel type of peptidoglycan-binding domain highly specific for amidated D-Asp cross-bridge, identified in Lactobacillus casei bacteriophage endolysins.

Publication Date: 2013 Jun 3 PMID: 23733182
Authors: Regulski, K. - Courtin, P. - Kulakauskas, S. - Chapot-Chartier, M. P.
Journal: J Biol Chem

Peptidoglycan-hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell-wall binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-L-alanine amidase whereas Lc-Lys2 is a gamma-D-glutamyl-L-lysyl-endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains which possess PG with D-Ala4-->D-Asx-L-Lys3 in their cross-bridge, such as L. casei, Lactococcus lactis and Enterococcus faecium. By testing a panel of L. lactis cell-wall mutants, we observed that Lc-Lys and Lc-Lys2 were not able to lyse mutants with a modified PG cross-bridge, constituted of D-Ala4-->L-Ala-(L-Ala/L-Ser)-L-Lys3; moreover they do not lyse L. lactis mutant containing only non-amidated D-Asp cross-bridge i.e. D-Ala4-->D-Asp-L-Lys3. In contrast, Lc-Lys could lyse ampicillin-resistant E. faecium mutant with 3-->3 L-Lys3-D-Asn-L-Lys3 bridges replacing the wild-type 4-->3 D-Ala4-D-Asn-L-Lys3 bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly D-Asn but not PG with only non-amidated D-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically D-Asn interpeptide bridge of PG.

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The Crystal Structure of Six Transmembrane Antigen of the Prostate 4 (Steap4), a Ferri/Cuprireductase, Suggests a Novel Inter-Domain Flavin Binding Site.

Publication Date: 2013 Jun 3 PMID: 23733181
Authors: Gauss, G. H. - Kleven, M. D. - Sendamarai, A. K. - Fleming, M. D. - Lawrence, C. M.
Journal: J Biol Chem

Steap4 is a cell surface metalloreductase linked to obesity associated insulin resistance. Initial characterization of its cell surface metalloreductase activity has been reported, but thorough biochemical characterization of this activity is lacking. Here we report detailed kinetic analysis of the Steap4 cell surface metalloreductase activities. Steap4 shows physiologically relevant Km values for both Fe3+ and Cu2+ and retains activity at acidic pH, suggesting it may also function within intracellular organelles to reduce these metals. Flavin dependent NADPH oxidase activity that was much greater than the equivalent Steap3 construct was observed for the isolated N-terminal oxidoreductase domain. The crystal structure of the Steap4 oxidoreductase domain was determined, providing a structural explanation for these differing activities. Structure-function work also suggested Steap4 utilizes an inter-domain flavin binding site to shuttle electrons between the oxidoreductase and transmembrane domains, and showed that the disordered N-terminal residues do not contribute to enzymatic activity.

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STAT5 and Prolactin Participate in a Positive Autocrine Feedback Loop that Promotes Angiogenesis.

Publication Date: 2013 Jun 2 PMID: 23729680
Authors: Yang, X. - Meyer, K. - Friedl, A.
Journal: J Biol Chem

We have shown previously that the murine prolactin/growth hormone family member proliferin (PLF) plays a pivotal role in angiogenesis induced by the FGF2/STAT5 signaling cascade. To delineate the signaling pathway downstream of STAT5 in the human system, where PLF does not exist, we expressed constitutively active (CA) or dominant-negative (DN) mutant STAT5A in hCMEC/D3 human brain endothelial cells. We found that conditioned medium from CA-STAT5A but not from DN-STAT5A overexpressing EC is sufficient to induce EC migration and tube formation but not proliferation, indicating that STAT5A regulates the secretion of autocrine proangiogenic factors. We identified prolactin (PRL) as a candidate autocrine factor. CA-STAT5A expression stimulates PRL production at the RNA and protein level and STAT5A binds to the PRL promoter region, suggesting direct transcriptional regulation. Medium conditioned by CA-STAT5 overexpressing EC induces phosphorylation of the PRL receptor and activates MAPK. Knockdown of PRL expression by sh-RNA or blocking of PRL activity with neutralizing antibodies removed the CA-STAT5A-dependent proangiogenic activity from the conditioned medium of EC. The addition of recombinant PRL restores this activity. STAT5-induced PRL in the conditioned medium can activate STAT5, STAT1 and to a lesser extent STAT3 in hCMEC/D3 cells, suggesting the existence of a positive feedback loop between STAT5 and PRL that promotes angiogenesis. Furthermore, we find that VEGF, a potent proangiogenic factor is induced by activation of STAT5A and VEGF induction depends on PRL expression. These observations demonstrate a STAT5/PRL/VEGF signaling cascade in human brain EC and implicate PRL and VEGF as autocrine regulators of EC migration, invasion and tube formation.

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Parathyroid Hormone (PTH)/PTH-related Peptide Type 1 Receptor (PPR) Signaling in Osteocytes Regulates Anabolic and Catabolic Skeletal Responses to PTH.

Publication Date: 2013 Jun 2 PMID: 23729679
Authors: Saini, V. - Marengi, D. J. - Barry, K. J. - Fulzele, K. S. - Heiden, E. - Liu, X. - Dedic, C. - Maeda, A. - Lotinun, S. - Baron, R. - Pajevic, P. D.
Journal: J Biol Chem

Parathyroid hormone (PTH) is the only FDA approved anabolic agent to treat osteoporosis; however cellular targets of PTH action in bone remain controversial. PTH modulates bone turnover by binding to the PTH/PTH- related Peptide (PTHrP) type 1 receptor (PPR), a G-protein-coupled-receptor highly expressed in bone and kidneys. Osteocytes, the most abundant cells in adult bone, also express PPR. However, physiological relevance of PPR signaling in osteocytes remains to be elucidated. Towards this goal, we generated mice with PPR deletion in osteocytes (Ocy-PPRKO). Skeletal analysis of these mice revealed a significant increase in bone mineral density, and trabecular and cortical bone parameters. Osteoblast activities were reduced in these animals, as demonstrated by decrease Col1alpha1 mRNA and RANKL expression. Importantly, when subjected to anabolic or catabolic PTH regimen, Ocy-PPRKO animals demonstrated blunted skeletal responses. PTH failed to suppress SOST/Sclerostin or induce RANKL expression in Ocy-PPRKO animals compared to controls. In vitro osteoclastogenesis was significantly impaired in Ocy-PPRKO upon PTH administration, indicating that osteocytes control osteoclast formation through a PPR-mediated mechanism. Taken together, these data indicate that PPR signaling in osteocytes is required for bone remodeling and receptor signaling in osteocytes is needed for anabolic and catabolic skeletal responses.

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Regulation of Deinococcus radiodurans RecA protein function via modulation of active and inactive nucleoprotein filament states.

Publication Date: 2013 May 31 PMID: 23729671
Authors: Ngo, K. V. - Molzberger, E. T. - Chitteni-Pattu, S. - Cox, M. M.
Journal: J Biol Chem

The RecA protein of Deinococcus radiodurans (DrRecA) has a central role in genome reconstitution after exposure to extreme levels of ionizing radiation. When bound to DNA, filaments of DrRecA protein exhibit active and inactive states that are readily interconverted in response to several sets of stimuli and conditions. At 30 C, the optimal growth temperature, and at physiological pH 7.5, DrRecA protein binds to double-stranded DNA (dsDNA) and forms extended helical filaments in the presence of ATP. However, the ATP is not hydrolyzed. ATP hydrolysis of the DrRecA-dsDNA filament is activated by addition of single-stranded DNA, with or without the single-stranded DNA binding protein (SSB). The ATPase function of DrRecA nucleoprotein filaments thus exists in a inactive default state under some conditions. ATPase activity is thus not a reliable indicator of DNA binding for all bacterial RecA proteins. Activation is effected by situations in which the DNA substrates needed to initiate recombinational DNA repair are present. The inactive state can also be activated by decreasing the pH (protonation of multiple ionizable groups is required), or by addition of volume exclusion agents. SSB plays a much more central role in DNA pairing and strand exchange catalyzed by DrRecA than is the case for the cognate proteins in E. coli. The data suggests a mechanism to enhance the efficiency of recombinational DNA repair in the context of severe genomic degradation in D. radiodurans.

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Insights into Okazaki Fragment Synthesis by the T4 Replisome: The Fate of Lagging-Strand Holoenzyme Components and Their Influence on Okazaki Fragment Size.

Publication Date: 2013 May 31 PMID: 23729670
Authors: Chen, D. - Yue, H. - Spiering, M. M. - Benkovic, S. J.
Journal: J Biol Chem

In this study, we employed a circular replication substrate with a low priming site frequency (1 site per 1.1 Kb) to quantitatively examine size distribution and formation pattern of Okazaki fragments. Replication reactions by the T4 replisome on this substrate yielded a patterned series of Okazaki fragments whose size distribution shifted through collision and signaling mechanisms as the gp44/62 clamp loader levels changed, but was insensitive to changes in the gp43 polymerase concentration as expected for a processive, recycled laggingstrand polymerase. In addition, we showed that only one gp45 clamp is continuously associated with the replisome and no additional clamps accumulate on the DNA, providing further evidence that the clamp departs whereas the polymerase is recycled upon completion of an Okazaki fragment synthesis cycle. We found no support for the participation of a third polymerase in Okazaki fragment synthesis.

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Inducible IL-32 exerts extensive antiviral function via selective stimulation of IFN-lambda1.

Publication Date: 2013 May 31 PMID: 23729669
Authors: Li, Y. - Xie, J. - Xu, X. - Liu, L. - Wan, Y. - Liu, Y. - Zhu, C. - Zhu, Y.
Journal: J Biol Chem

Interleukin (IL)-32 has been recognized as a proinflammatory cytokine that participates in responses to viral infection. However, little is known about how IL-32 is induced in response to viral infection and the mechanisms of IL-32-mediated antiviral activities. We discovered that IL-32 is elevated by the infection of Hepatitis B virus (HBV) both in vitro and in vivo, and that HBV-induced IL-32 expression at the level of both transcription and post-transcription. Furthermore, microRNA(miR)-29b was found to be a key factor in HBV-regulated IL-32 expression by directly targeting the mRNA 3'-untranslated region of IL-32. Antiviral analysis showed that IL-32 was not sufficient to alter HBV replication in HepG2.2.15 cells. To mimic the viremic phase of viral infection, freshly isolated peripheral blood mononuclear cells (PBMCs) were treated with IL-32gamma, the secretory isoform, and the supernatants were used for antiviral assays. Surprisingly, these supernatants exhibited extensive antiviral activity against multiplex viruses beside HBV. Thus, we speculated that the IL-32gamma-treated PBMCs produced and secreted an unknown antiviral factor. Using antibody neutralization assays, we identified the factor as interferon (IFN)-lambda1 and not IFN-alpha. Further studies indicated that IL-32gamma effectively inhibited HBV replication in hydrodynamic injection mouse model. Clinical data showed that elevated levels of IFN-lambda1 both in serum and liver tissue of HBV patients were positively correlated to the increased levels of IL-32. Our results demonstrate that elevated IL-32 levels during viral infection mediate antiviral effects by stimulating the expression of IFN-lambda1.

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Circadian regulation of intestinal lipid absorption by apolipoprotein AIV involves forkhead transcription factors A2/O1 and microsomal triglyceride transfer protein.

Publication Date: 2013 May 31 PMID: 23729668
Authors: Pan, X. - Munshi, M. K. - Iqbal, J. - Queiroz, J. - Sirwi, A. A. - Shah, S. - Younus, A. - Hussain, M. M.
Journal: J Biol Chem

We have previously shown that Clock, microsomal triglyceride transfer protein (MTP) and nocturnin are involved in the circadian regulation of intestinal lipid absorption. Here, we clarified the role of apolipoprotein AIV (apoAIV) in the diurnal regulation of plasma lipids and intestinal lipid absorption in mice. Plasma triglyceride in ApoAIV-/- mice showed diurnal variations similar to ApoAIV+/+ mice; however, increases at night were significantly lower in these mice. ApoAIV-/- mice absorbed fewer lipids at night and showed blunted response to daytime feeding. To explain reasons for these lower responses, we measured MTP expression; intestinal MTP was low at night and its induction after food entrainment was less in ApoAIV-/- mice. Conversely, apoAIV overexpression increased MTP mRNA in hepatoma cells indicating transcriptional regulation. Mechanistic studies revealed that sequences between -204 and -775 bp in the MTP promoter respond to apoAIV and that apoAIV enhances expression of FoxA2 andFoxO1 transcription factors and their binding to the identified cis-elements in the MTP promoter at night. Knockdown of FoxA2 and FoxO1 abolished apoAIV mediated MTP induction. Similarly, knockdown of apoAIV in differentiated Caco-2 cells, reduced MTP, FoxA2 and FoxO1 mRNA levels, cellular MTP activity and media apoB. Moreover, FoxA2 and FoxO1 expression showed diurnal variations and their expressions were significantly lower in ApoAIV-/- mice. These data indicate that apoAIV modulates diurnal changes in lipid absorption by regulating forkhead transcription factors and MTP, and that inhibition of apoAIV expression might reduce plasma lipids.

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Ten-eleven translocation 1 (Tet1) Is Regulated by O-GlcNAc Transferase (Ogt) for Target Gene Repression in Mouse Embryonic Stem Cells.

Publication Date: 2013 May 31 PMID: 23729667
Authors: Shi, F. - Kim, H. - Lu, W. - He, Q. - Liu, D. - Goodell, M. A. - Wan, M. - Songyang, Z.
Journal: J Biol Chem

As a member of the Tet (Ten-eleven translocation) family proteins that can convert 5-methylcytosine (5mC) to 5-hydroxyl methylcytosine (5hmC), Tet1 has been implicated in regulating global DNA de-methylation and gene expression. Tet1 is highly expressed in embryonic stem (ES) cells and appears to mainly repress developmental genes for maintaining pluripotency. To understand how Tet1 may regulate gene expression, we conducted large-scale immunoprecipitation (IP) followed by mass spectrometry of endogenous Tet1 in mouse ES cells. We found that Tet1 could interact with multiple chromatin regulators, including Sin3A and NuRD complexes. In addition, we showed that Tet1 could also interact with the O-GlcNAc transferase (Ogt) and be O-GlcNAcylated. Depletion of Ogt led to reduced Tet1 and 5hmC levels on Tet1-target genes, while ectopic expression of wild-type but not enzymatically inactive Ogt increased Tet1 levels. Mutation of the putative O-GlcNAcylation site on Tet1 led to decreased O-GlcNAcylation and level of the Tet1 protein. Our results suggest that O-GlcNAcylation can positively regulate Tet1 protein concentration and indicate that Tet1-mediated 5hmC modification and target repression is controlled by Ogt.

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Structural Requirements for Sterol Regulatory Element-Binding Protein (SREBP) Cleavage in Fission Yeast.

Publication Date: 2013 May 31 PMID: 23729666
Authors: Cheung, R. - Espenshade, P. J.
Journal: J Biol Chem

Sterol regulatory element-binding proteins (SREBP) are central regulators of cellular lipid synthesis and homeostasis. Mammalian SREBPs are proteolytically activated and liberated from the membrane by Golgi Site-1 and Site-2 proteases. Fission yeast SREBPs, Sre1 and Sre2, employ a different mechanism that genetically requires the Golgi Dsc E3 ligase complex for cleavage activation. Here, we established Sre2 as a model to define structural requirements for SREBP cleavage. We showed that Sre2 cleavage does not require the N-terminal basic helix-loop-helix zipper transcription factor domain (bHLH-zip), thus separating cleavage of Sre2 from its transcription factor function. From a mutagenesis screen of 94 C-terminal residues of Sre2, we isolated 15 residues required for cleavage and further identified a glycine-leucine sequence required for Sre2 cleavage. Importantly, the glycine-leucine sequence is located at a conserved distance before the first transmembrane segment of both Sre1 and Sre2, and cleavage occurs in between this sequence and the membrane. Bioinformatic analysis revealed a broad conservation of this novel glycine-leucine motif in SREBP homologs of ascomycete fungi, including the opportunistic human pathogen Aspergillus fumigatus where SREBP is required for virulence. Consistent with this, the sequence was also required for cleavage of the oxygen-responsive transcription factor Sre1 and adaptation to hypoxia, demonstrating functional conservation of this cleavage recognition motif. These cleavage mutants will aid identification of the fungal SREBP protease and facilitate functional dissection of the Dsc E3 ligase required for SREBP activation and fungal pathogenesis.

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Identification and Functional Analysis of the Primary Pantothenate Transporter, PfPAT, of the Human Malaria Parasite Plasmodium falciparum.

Publication Date: 2013 May 31 PMID: 23729665
Authors: Augagneur, Y. - Jaubert, L. - Schiovani, M. - Pachikara, N. - Garg, A. - Usmani-Brown, S. - Wesolowski, D. - Zeller, S. - Ghosal, A. - Cornillot, E. - Said, H. M. - Kumar, P. - Altman, S. - Ben Mamoun, C.
Journal: J Biol Chem

The human malaria parasite Plasmodium falciparum is absolutely dependent on the acquisition of host pantothenate for its development within human erythrocytes. While the biochemical properties of this transport have been characterized, the molecular identity of the parasite encoded pantothenate transporter remains unknown. Here we report the identification and functional characterization of the first protozoan pantothenate transporter, PfPAT, from P. falciparum. We show using cell biological, biochemical and genetic analyses that this transporter is localized to the parasite plasma membrane and plays an essential role in parasite intraerythrocytic development. We have targeted PfPAT to the yeast plasma membrane and showed that the transporter complements the growth defect of the yeast fen2Delta pantothenate transporter-deficient mutant and mediates the entry of the fungicide drug, fenpropimorph. Our studies in P. falciparum revealed that fenpropimorph inhibits the intraerythrocytic development of both chloroquine and pyrimethamine resistant P. falciparum strains with potency equal or better than that of currently available pantothenate analogs. The essential function of PfPAT and its ability to deliver both pantothenate and fenpropimorph makes it an attractive target for the development and delivery of new classes of antimalarial drugs.

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Domain Requirements of the JIL-1 Tandem Kinase for Histone H3 Serine10 Phosphorylation and Chromatin Remodeling in Vivo.

Publication Date: 2013 May 30 PMID: 23723094
Authors: Li, Y. - Cai, W. - Wang, C. - Yao, C. - Bao, X. - Deng, H. - Girton, J. - Johansen, J. - Johansen, K. M.
Journal: J Biol Chem

The JIL-1 kinase localizes to Drosophila polytene chromosome interbands and phosphorylates histone H3 at interphase counteracting histone H3 lysine9 (H3K9) dimethylation and gene silencing. JIL-1 can be divided into four main domains including an NH2-terminal domain, two separate kinase domains, and a COOH-terminal domain. In this study we characterize the domain requirements of the JIL-1 kinase for H3S10 phosphorylation and chromatin remodeling in vivo. We show that a JIL-1 construct without the amino-terminal domain is without H3S10 phosphorylation activity despite that it localizes properly to polytene interband regions and that it contains both kinase domains. JIL-1 is a double kinase and we demonstrate that both kinase domains of JIL-1 are required to be catalytically active for H3S10 phosphorylation to occur. Furthermore, we provide evidence that JIL-1 is phosphorylated at serine424 and that this phosphorylation is necessary for JIL-1 H3S10 phosphorylation activity. Thus, these data are compatible with a model where the amino-terminal domain of JIL-1 is required for chromatin complex interactions that position the kinase domain(s) for catalytic activity in the context of the state of higher order nucleosome packaging and chromatin structure, and where catalytic H3S10 phosphorylation activity mediated by the first kinase domain is dependent on autophosphorylation of serine424 by the second kinase domain. Furthermore, using a lacO repeat tethering system to target mutated JIL-1 constructs with or without catalytic activity we show that the epigenetic histone H3S10 phosphorylation mark itself functions as a causative regulator of chromatin structure independently of any structural contributions from the JIL-1 protein.

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The structure of two ferryl-oxo intermediates at the same oxidation level in the heme-copper binuclear center of cytochrome c oxidase: The protein effect.

Publication Date: 2013 May 30 PMID: 23723073
Authors: Pinakoulaki, E. - Daskalakis, V. - Ohta, T. - Richter, O. M. - Budiman, K. - Kitagawa, T. - Ludwig, B. - Varotsis, C.
Journal: J Biol Chem

Identification of the intermediates and determination of their structures in the reduction of dioxygen to water by cytochrome c oxidase (CcO) are particularly important to understanding both O2-activation and proton pumping by the enzyme. In this work, we report the products of the rapid reaction of O2 with the mixed valence form (CuA2+, heme a3+, heme a32+-CuB1+) of the enzyme. The resonance Raman results show the formation of two ferryl-oxo species with characteristic Fe(IV)=O stretching modes at 790 and 804 cm-1 at the peroxy oxidation level (PM). Density functional theory calculations show that the protein environment of the proximal H-bonded His411 determines the strength of the distal Fe(IV)=O bond. In contrast to previous proposals, the PM intermediate is also formed in the reaction of Y167F with O2. These results suggest that in the fully reduced enzyme, the proton pumping nuFe(IV)=O = 804 cm-1 to nuFe(IV)=O =790 cm-1 transition (P-->F), is triggered not only by electron transfer from heme a to heme a3 but also by the formation of the H-bonded form of the His411-Fe(IV)=O conformer in the proximal site of heme a3. The implications of these results with respect to the role of an O=Fe(IV)-His411-H-bonded form to the ring A propionate of heme a3-Asp399-H2O site and, thus, to the exit/output proton channel (H2O) pool during the proton pumping P-->F transition are discussed. We propose that the proximal to the heme a3 environment controls the spectroscopic properties of the ferryl intermediates in cytochrome oxidases.

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Modulation of lipid kinase PI4KIIalpha activity and lipid raft association of presenilin 1 underlies gamma-secretase inhibition by ginsenoside(20S)Rg3.

Publication Date: 2013 May 30 PMID: 23723072
Authors: Kang, M. S. - Baek, S. H. - Chun, Y. S. - Moore, A. Z. - Landman, N. - Berman, D. - Yang, H. O. - Morishima-Kawashima, M. - Osawa, S. - Funamoto, S. - Ihara, Y. - Di Paolo, G. - Park, J. H. - Chung, S. - Kim, T. W.
Journal: J Biol Chem

Amyloid beta-peptide (Abeta) pathology is an invariant feature of Alzheimer's disease (AD), preceding any detectable clinical symptoms by more than a decade. To this end, we seek to iidentifyagents that can reduce Abeta levels in the brain via novel mechanisms. We found that (20S)Rg3, a triterpene natural compound known as ginsenoside, reduced Abeta levels in cultured primary neurons and in the brains of a mouse model of AD. The (20S)Rg3 treatment induced a decrease in the association of presenilin 1 (PS1) fragments with lipid rafts, where catalytic components of the gamma-secretase complex are enriched. The Abeta-lowering activity of (20S)Rg3 directly correlated with increased activity of phosphatidylinositol 4-kinase IIalpha (PI4KIIalpha), a lipid kinase that mediates the rate-limiting step in phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) synthesis. PI4KIIalpha overexpression recapitulated the effects of (20S)Rg3 while reduced expression of PI4KIIalpha abolished the Abeta-reducing activity of (20S)Rg3 in neurons. Our results substantiate an important role for PI4KIIalpha and phosphoinositide modulation in gamma-secretase activity and Abeta biogenesis.

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Association/dissociation of the nucleotide-binding domains of the ATP-binding cassette protein MsbA measured during continuous hydrolysis.

Publication Date: 2013 May 30 PMID: 23723071
Authors: Cooper, R. S. - Altenberg, G. A.
Journal: J Biol Chem

In ATP binding cassette (ABC) proteins the two nucleotide-binding domains (NBDs) work as dimers to bind and hydrolyze ATP, but the molecular mechanism of nucleotide hydrolysis is controversial. It is still unresolved whether hydrolysis leads to dissociation of the ATP-induced dimers, or partial opening of the dimers such that the NBDs remain in contact during the hydrolysis cycle. We studied the bacterial lipid flippase MsbA by luminescence resonance energy transfer (LRET). The LRET signal between optical probes reacted with single-cysteine mutants was employed to follow NBD association/dissociation in real time. The inter-monomer distances calculated from LRET data indicate that the NBDs separate completely following ATP hydrolysis, even in the presence of mM MgATP, and that the dissociation occurs following each hydrolysis cycle. The results support association/dissociation, as opposed to constant-contact models, for the mode of operation of ABC proteins.

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AMPK inhibits oxidative stress induced caveolin-1 phosphorylation and endocytosis by suppressing the dissociation between c-Abl and prdx1 in endothelial cells.

Publication Date: 2013 May 30 PMID: 23723070
Authors: Takeuchi, K. - Morizane, Y. - Kamami-Levy, C. - Suzuki, J. - Kayama, M. - Cai, W. - Miller, J. W. - Vavvas, D. G.
Journal: J Biol Chem

Caveolin-1 is the primary structural component of endothelial caveolae, which is essential for transcellular trafficking of albumin, and also a critical scaffolding protein, which regulates the activity of signaling molecules in caveolae. Phosphorylation of caveolin-1 plays a fundamental role in the mechanism of oxidant-induced vascular hyper permeability. However, the regulatory mechanism of caveolin-1 phosphorylation remains unclear. Here, we identify a previously unexpected role for AMPK in inhibition of caveolin-1 phosphorylation under oxidative stress. A pharmacological activator of AMPK, AICAR, inhibited oxidative stress induced phosphorylation of both caveolin-1 and c-Abl, which is the major kinase of caveolin-1, and endocytosis of albumin in HUVEC. These effects were abolished by treatment with two specific inhibitors of AICAR, dypiridamole and 5-iodotubericidin. Consistently, knock down of catalytic AMPK! subunit by siRNA abolished the inhibitory effect of AICAR on oxidant-induced phosphorylation of both caveolin-1 and c-Abl. Pretreatment with specific c-Abl inhibitor, imatinib mesylate, and knock down of c-Abl significantly decreased the caveolin-1 phosphorylation after H2O2 exposure and abolished the inhibitory effect of AICAR on the caveolin-1 phosphorylation. Interestingly, knock down of prdx-1, an antioxidant enzyme associated with c-Abl, increased phosphorylation of both caveolin-1 and c-Abl, and abolished the inhibitory effect of AICAR on the caveolin-1 phosphorylation. Furthermore, co-immunoprecipitation experiment showed that AICAR suppressed the oxidant-induced dissociation between c-Abl and prdx1. Overall, our results suggest that activation of AMPK inhibits oxidative stress induced caveolin-1 phosphorylation and endocytosis and this effect is mediated in part by stabilizing the interaction between c-Abl and prdx-1.

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Leucine-rich repeat and immunoglobulin domain-containing protein-1 negative (Lrig1) regulatory action towards ErbB receptor tyrosine kinases is opposed by Leucine-rich repeat and immunoglobulin domain-containing protein-3 (Lrig3).

Publication Date: 2013 May 30 PMID: 23723069
Authors: Rafidi, H. - Mercado, F. - Astudillo, M. - Fry, W. H. - Saldana, M. - Carraway, K. L. - Sweeney, C.
Journal: J Biol Chem

Lrig1 is the founding member of the Lrig family of transmembrane leucine rich repeat proteins, which also includes Lrig2 and Lrig3. Lrig1 is a negative regulator of oncogenic receptor tyrosine kinases, including ErbB and Met receptors, and promotes receptor degradation. Lrig1 has recently emerged as both a tumor suppressor and a key regulator of epidermal and epithelial stem cell quiescence. Despite this, little is known of mechanisms by which Lrig1 is regulated. Lrig3 was recently reported to increase ErbB receptor expression suggesting that it may function in a manner opposite to Lrig1. In this study, we explore the interaction between Lrig1 and Lrig3 and demonstrate that Lrig1 and Lrig3 functionally oppose one another. Lrig3 opposes Lrig1 negative regulatory activity and stabilizes ErbB receptors. Conversely, Lrig1 destabilizes Lrig3, limiting Lrig3s positive effects on receptors and identifying Lrig3 as a new target of Lrig1. These studies provide new insight into the regulation of Lrig1 and uncover a complex cross-talk between Lrig1 and Lrig3.

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Regulation of Phospholipase D Activity and Phosphatidic Acid Production Following Purinergic (P2Y6) Receptor Stimulation.

Publication Date: 2013 May 30 PMID: 23723068
Authors: Scott, S. A. - Xiang, Y. - Mathews, T. P. - Plumley, H. C. - Myers, D. S. - Armstrong, M. D. - Tallman, K. A. - O'Reilly, M. C. - Lindsley, C. W. - Brown, H. A.
Journal: J Biol Chem

Phosphatidic acid (PA) is a lipid second messenger located at the intersection of several lipid metabolism and cell signaling events including membrane trafficking, survival, and proliferation. Generation of signaling PA has long been primarily attributed to the activation of phospholipase D (PLD). PLD catalyzes the hydrolysis of phosphatidylcholine into PA. A variety of both receptor tyrosine kinase and G-protein coupled receptor stimulations have been shown to lead to PLD activation and PA generation. This study focuses on profiling the PA pool upon P2Y6 receptor signaling manipulation to determine the major PA producing enzymes. Here we show that PLD, although highly active, is not responsible for the majority of stable PA being produced upon UDP stimulation of the P2Y6 receptor and that PA levels are tightly regulated. By following PA flux in the cell we show that PLD is involved in an initial increase in PA upon receptor stimulation, however when PLD is blocked the cell compensates by increasing PA production from other sources. We further delineate the P2Y6 signaling pathway showing that PLCbeta3, PLCdelta1, DGKzeta and PLD are all downstream of receptor activation. We also show that DGKzeta is a novel negative regulator of PLD activity in this system which occurs through an inhibitory mechanism with PKCalpha. These results further define the downstream events resulting in PA production in the P2Y6 receptor signaling pathway.

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Nuclear BAG6-UBL4A-GET4 complex mediates DNA damage signaling and cell death.

Publication Date: 2013 May 30 PMID: 23723067
Authors: Krenciute, G. - Liu, S. - Yi, S. - Yucer, N. - Ortiz, P. - Liu, Q. - Kim, B. J. - Odejimi, O. A. - Leng, M. - Qin, J. - Wang, Y.
Journal: J Biol Chem

BAG6 is a member of the BAG protein family, which is implicated in diverse cellular processes including apoptosis, co-chaperone and DNA damage response. Recently, it has been shown that BAG6 forms a stable complex with UBL4A and GET4 and functions in membrane protein targeting and protein quality control. The BAG6 sequence contains a canonical nuclear localization signal (NLS) and is localized predominantly in the nucleus. However, GET4 and UBL4A are found mainly in cytoplasm. Whether GET4 and UBL4A are also involved in DNA damage response (DDR) in the context of the BAG6 complex remains unknown. Here, we provide evidence that the nuclear BAG6-UBL4A-GET4 complex mediates DDR signaling and damage-induced cell death. BAG6 appears to be the central component for the process, as depletion of BAG6 leads to the loss of both UBL4A and GET4 proteins and resistance to cell killing by DNA damage reagents. In addition, nuclear localization of BAG6 and phosphorylation of BAG6 by checkpoint kinase ATM are all required for cell killing and BAG6 appears to regulate UBL4A and GET4 to translocate to the nucleus upon DNA damage. GET4 and UBL4A play redundant roles in cell killing, as depletion of either one has no effect but co-depletion leads resistance. All 3 components of the BAG6 complex are required for optimal DDR signaling, as BAG6, and to a lesser extent, GET4 and UBL4A, regulate the recruitment of BRCA1 complex to sites of DNA damage. Together our results suggest that the nuclear BUG complex is an effector in DNA damage response pathway and its phosphorylation and nuclear localization are important determinants for its function.

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TLR-TRIF pathway enhances the expression of KSHV replication and transcription activator.

Publication Date: 2013 May 30 PMID: 23723066
Authors: Meyer, F. - Ehlers, E. - Steadman, A. - Waterbury, T. - Cao, M. - Zhang, L.
Journal: J Biol Chem

Kaposis sarcoma-associated herpesvirus (KSHV) is a human gamma herpesvirus. KSHV replication and transcription activator (RTA) is necessary and sufficient for KSHV reactivation from latency. Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns, act through adaptors, and initiate innate and adaptive immune responses against pathogens. Toll/interleukin-1-receptor-domain containing adaptor protein inducing interferon-beta (TRIF) is an adaptor associated with TLR3 and TLR4 signaling, and is closely related to antiviral signaling to activate type I interferon (IFN) production. We previously found that KSHV RTA degrades TRIF indirectly and blocks TLR3 pathways. In this report, we find that TRIF, as well as TLR3 activation, enhances KSHV RTA protein expression. The C-terminal region of the RTA is involved in the responding TRIF-mediated enhancement. The degradation of TRIF and the enhancement of RTA expression are using two different pathways. The enhancement by TLR-TRIF is at least partially via promoting translational efficiency of RTA mRNA. Finally, the receptor interacting protein 1 (RIP1) may be involved in TRIF-mediated enhancement of RTA expression, but not in the RTA-mediated degradation of TRIF. Therefore, Therefore, the activation of TLR-TRIF pathway enhances KSHV RTA protein expression, and KSHV RTA in turn degrades TRIF to block innate immunity. The putative KSHV-TLR-adaptor interacting loop may be a critical element to evade and usurp host innate immunity in KSHV life-cycle.

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Collagen-binding MSCRAMMs of Gram-positive bacteria inhibit complement activation via the classical pathway.

Publication Date: 2013 May 29 PMID: 23720782
Authors: Kang, M. - Ko, Y. P. - Liang, X. - Ross, C. L. - Liu, Q. - Murray, B. E. - Hook, M.
Journal: J Biol Chem

Members of a family of collagen-binding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) from Gram-positive bacteria are established virulence factors in several infectious diseases models. Here we report that these adhesins also can bind C1q and act as potent inhibitors of the classical complement pathway. Molecular analyses of Cna from Staphylococcus aureus (S. aureus) suggested that this prototype MSCRAMM bound to the collagenous domain of C1q and interfered with the interactions of C1r with C1q. As a result, C1r2C1s2 was displaced from C1q and the C1 complex deactivated. This novel function of the Cna-like MSCRAMMs represents an potential immune evasion strategy that could be used by numerous Gram-positive pathogens.

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Epidermal Growth Factor Receptor (EGFR) signaling promotes proliferation and survival in osteoprogenitors by increasing Early Growth Response Protein (Egr2) expression.

Publication Date: 2013 May 29 PMID: 23720781
Authors: Chandra, A. - Lan, S. - Zhu, J. - Siclari, V. - Qin, L.
Journal: J Biol Chem

Maintaining bone architecture requires continuous generation of osteoblasts from osteoprogenitor pool. Our previous study of mice with epidermal growth factor receptor (EGFR) specifically inactivated in osteoblast lineage cells revealed that EGFR stimulates bone formation by expanding the population of mesenchymal progenitors. EGFR ligands are potent regulators for the osteoprogenitor pool but the underlying mechanisms are largely unknown. Here we demonstrate that activation of EGFR increases the number of osteoprogenitors by promoting cell proliferation and suppressing either serum- or TNFalpha-induced apoptosis mainly through MAPK/Erk pathway. Mouse calvarial organ culture revealed that EGF elevated the number of proliferative cells and decreased the number of apoptotic cells, which lead to increased osteoblasts. Microarray analysis of MC3T3 cells, an osteoprogenitor cell line, revealed that EGFR signaling stimulates the expression of Mcl1, an anti-apoptotic protein, and a family of Egr transcription factors (Egr1, 2, and 3). The up-regulation of Mcl1 and Egr2 by EGF were further confirmed in osteoprogenitors close to the calvarial bone surface. Overexpression of Nab2, a co-repressor for Egrs, attenuated the EGF-induced increase in osteoprogenitor number. Interestingly, knocking down the expression of Egr2, but not Egr1 or 3, resulted in a similar effect. Using inhibitor, adenovirus overexpression, and siRNA approaches, we demonstrate that EGFR signaling activates MAPK/Erk pathway to stimulate the expression of Egr2, which in turn leads to cell growth and Mcl1-mediated cell survival. Taken together, our data clearly demonstrate that EGFR-induced Egr2 expression is critical for osteoprogenitor maintenance and new bone formation.

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Anthrax SET protein: a potential virulence determinant that epigenetically represses NF-kappaB activation in infected macrophages.

Publication Date: 2013 May 29 PMID: 23720780
Authors: Mujtaba, S. - Winer, B. Y. - Jaganathan, A. - Patel, J. - Sgobba, M. - Schuch, R. - Gupta, Y. K. - Haider, S. - Wang, R. - Fischetti, V. A.
Journal: J Biol Chem

Toxins play a major role in the pathogenesis of Bacillus anthracis (B. anthracis) by subverting the host defenses. However, besides toxins, B. anthracis expresses effector proteins, whose role in pathogenesis are yet to be investigated. Here, we present that Suppressor-of-variegation, Enhancer-of-zeste, Trithorax protein from B. anthracis (BaSET) methylates human histone H1, resulting in repression of NF-kappaB functions. Notably, BaSET is secreted and undergoes nuclear translocation to enhance H1 methylation in B. anthracis-infected macrophages. Compared to wild type Sterne, delayed growth kinetics and altered septum formation were observed in the BaSET knockout (BaSET) bacilli. Uncontrolled BaSET expression during complementation of the BaSET gene in BaSET, partially restored growth during stationary phase, however, resulted in substantially shorter bacilli throughout the growth cycle. Importantly, in contrast to Sterne, the BaSET B. anthracis is avirulent in a lethal murine bacteremia model of infection. Collectively, BaSET is required for repression of host's transcription as well as proper B. anthracis growth, making it a potentially unique virulence determinant.

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XIAP regulation of cyclin D1 expression and cancer cell anchorage-independent growth via Its E3 ligase-mediated protein phosphatase 2A/C-Jun axis.

Publication Date: 2013 May 29 PMID: 23720779
Authors: Cao, Z. - Zhang, R. - Li, J. - Huang, H. - Zhang, D. - Zhang, J. - Gao, J. - Chen, J. - Huang, C.
Journal: J Biol Chem

The X-linked inhibitor of apoptosis protein (XIAP) is a well-known potent inhibitor of apoptosis, however, it is also involved in other cancer cell biological behavior. In the current study, we discovered that XIAP and its E3 ligase played a crucial role in regulation of cyclin D1 expression in cancer cells. We found that deficiency of XIAP expression resulted in a marked reduction in cyclin D1 expression. Consistently, cell cycle transition and anchorage-independent cell growth were also attenuated in XIAP-deficient cancer cells compared to those of the parental wild-type cells. Subsequent studies demonstrated that E3 ligase activity within the RING domain of XIAP is crucial for its regulation ability of cyclin D1 transcription, cell cycle transition and anchorage- independent cell growth by upregulating transactivation of C-Jun/AP-1. Moreover, we found that E3 ligase within RING domain was required for XIAP inhibiting phosphatase PP2A activity by upregulation of PP2A phosphorylation at Tyr307 in its catalytic subunit. Such PP2A phosphorylation and inactivation resulted in its downstream target C-Jun phosphorylation and activation, in turn leading to cyclin D1 expression. Collectively, our studies uncovered a novel function of E3 ligase activity of XIAP in the upregulation of cyclin D1 expression, providing significant insight into the understanding of the biomedical significance of overexpressed XIAP in cancer development, further offering a new molecular basis for utilizing XIAP E3 ligase as a cancer therapeutic target.

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Squeezing at Entrance of Proton Transport Pathway in Proton-translocating Pyrophosphatase upon Substrate Binding.

Publication Date: 2013 May 29 PMID: 23720778
Authors: Huang, Y. T. - Liu, T. H. - Lin, S. M. - Chen, Y. W. - Pan, Y. J. - Lee, C. H. - Sun, Y. J. - Tseng, F. G. - Pan, R. L.
Journal: J Biol Chem

Homodimeric proton-translocating pyrophosphatase (H+-PPase; EC 3.6.1.1) is indispensable for many organisms in maintaining organellar pH homeostasis. This unique proton pump couples the hydrolysis of PPi to proton translocation across the membrane. H+-PPase consists of 14-16 relatively hydrophobic transmembrane domains (TMs) presumably for proton translocation and hydrophilic loops primarily embedding a catalytic site. Several highly conserved polar residues located at/near the entrance of the transport pathway in H+-PPase are essential for proton pumping activity. In this investigation, single molecule FRET (smFRET) was employed to dissect the action at the pathway entrance in homodimeric Clostridium tetani H+-PPase (CtH+-PPase) upon ligands binding. The presence of the substrate analogue, imidodiphosphate (IDP), mediated two sites at the pathway entrance moving towards each other. Moreover, smFRET analyses following the mutation at the first proton-carrying residue (Arg-169) demonstrated that conformational changes at the entrance are conceivably essential for the initial step of H+-PPase proton translocation. A working model is accordingly to illustrate the squeeze at the entrance of the transport pathway in H+-PPase upon substrate binding.

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Accelerated Development of Pulmonary Fibrosis via Cu,Zn-SOD-induced Alternative Activation of Macrophages.

Publication Date: 2013 May 29 PMID: 23720777
Authors: He, C. - Ryan, A. J. - Murthy, S. - Carter, A. B.
Journal: J Biol Chem

Macrophages not only initiate and accentuate inflammation after tissue injury, but they are also involved in resolution and repair. This difference in macrophage activity is the result of a differentiation process to either M1 or M2 phenotypes. M1 macrophages are pro-inflammatory and have microbicidal and tumoricidal activity, whereas the M2 macrophages are involved in tumor progression and tissue remodeling, and can be pro-fibrotic in certain conditions. Because mitochondrial Cu,Zn-SOD-mediated H2O2 is crucial for development of pulmonary fibrosis, we hypothesized that Cu,Zn-SOD modulated the macrophage phenotype. In this study, we demonstrate that Cu,Zn-SOD polarized macrophages to an M2 phenotype, and Cu,Zn-SOD-mediated H2O2 levels modulated M2 gene expression at the transcriptional level by redox regulation of a critical cysteine in STAT6. Furthermore, overexpression of Cu,Zn-SOD in mice resulted in a pro-fibrotic environment and accelerated the development of pulmonary fibrosis, while polarization of macrophages to the M1 phenotype attenuated pulmonary fibrosis. Taken together, these observations provide a novel mechanism of Cu,Zn-SOD-mediated and Th2-independent M2 polarization and provide a potential therapeutic target for attenuating the accelerated development of pulmonary fibrosis.

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Sequence Determinants of GLUT1 Oligomerization - Analysis by Homology-Scanning Mutagenesis.

Publication Date: 2013 May 29 PMID: 23720776
Authors: De Zutter, J. K. - Levine, K. B. - Deng, D. - Carruthers, A.
Journal: J Biol Chem

The human blood brain barrier glucose transport protein (GLUT1) forms homodimers and homotetramers in detergent micelles and in cell membranes where GLUT1 oligomeric state determines GLUT1 transport behavior. GLUT1 and the neuronal glucose transporter GLUT3 do not form heterocomplexes in HEK 293 cells as judged by co-immunoprecipitation assays. Using homology-scanning mutagenesis in which GLUT1 domains are substituted with equivalent GLUT3 domains and vice versa, we show that GLUT1 transmembrane helix 9 (TM9) is necessary for optimal association of GLUT1-GLUT3 chimeras with parental GLUT1 in HEK cells. GLUT1 TMs 2, 5, 8 and 11 also contribute to a less abundant hetero-complex. Cell surface GLUT1 and GLUT3 containing GLUT1 TM9 are 4-fold more catalytically active than GLUT3 and GLUT1 containing GLUT3 TM9. GLUT1 and GLUT3 display allosteric transport behavior. Size exclusion chromatography of detergent solubilized, purified GLUT1 resolves GLUT1/lipid/detergent micelles as 6 and 10 nm Stokes radius particles which correspond to GLUT1 dimers and tetramers respectively. Studies with GLUTs expressed in and solubilized from HEK cells show that HEK cell GLUT1 resolves as 6 and 10 nm Stokes radius particles whereas GLUT3 resolves as a 6 nm particle. Substitution of GLUT3 TM9 with GLUT1 TM9 causes chimeric GLUT3 to resolve as 6 and 10 nm Stokes radius particles. Substitution of GLUT1 TM9 with GLUT3 TM9 causes chimeric GLUT1 to resolve as a mixture of 6 and 4 nm particles. We discuss these findings in the context of determinants of GLUT oligomeric structure and transport function.

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Small Oligomers of Ribulose-bisphosphate carboxylase/oxygenase (Rubisco) Activase are required for Biological Activity.

Publication Date: 2013 May 29 PMID: 23720775
Authors: Keown, J. R. - Griffin, M. D. - Mertens, H. D. - Pearce, F. G.
Journal: J Biol Chem

Rubisco activase uses the energy from ATP hydrolysis to remove tight binding inhibitors from Rubisco, thus playing a key role in regulating photosynthesis in plants. While several structures have recently added much needed structural information for different Rubisco activase enzymes, the arrangement of these subunits in solution remains unclear. In this study, we use a variety of techniques to show that Rubisco activase forms a wide range of structures in solution, ranging from monomers to much higher order species, and that the distribution of these species is highly dependent on protein concentration. The data supports a model in which Rubisco activase forms an open spiralling structure rather than a closed hexameric structure. At protein concentrations of mu1 M, corresponding to the maximal activity of the enzyme, Rubisco activase has an oligomeric state of 2-4 subunits. We propose a model in which Rubisco activase requires at least one neighbouring subunit for hydrolysis of ATP.

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A Brucella virulence factor targets macrophages to trigger B-cell proliferation.

Publication Date: 2013 May 29 PMID: 23720774
Authors: Spera, J. M. - Herrmann, C. K. - Roset, M. S. - Comerci, D. J. - Ugalde, J. E.
Journal: J Biol Chem

Brucella spp. and Trypanosoma cruzi are two intracellular pathogens that have no evolutionary common origins but share a similar lifestyle as they establish chronic infections for which they have to circumvent the host immune response. Both pathogens have a virulence factor (prpA in Brucella and tcPrac in T. cruzi) that induces B-cell proliferation and promotes the establishment of the chronic phase of the infectious process. We show here that, even though PrpA promotes B-cell proliferation, it targets macrophages in vitro and is translocated to the cytoplasm during the intracellular replication phase. We observed that PrpA treated macrophages induce the secretion of a soluble factor responsible for B-cell proliferation and identified non-muscular myosin IIA (NMM-IIA) as a receptor required for binding and function of this virulence factor. Finally, we show that the Trypanosoma cruzi homolog of PrpA also targets macrophages to induce B-cell proliferation through the same receptor, indicating that this virulence strategy is conserved between a bacterial and a protozoan pathogen.

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An Engineered GluCl Channel for Sensitive, Consistent Neuronal Silencing by Ivermectin.

Publication Date: 2013 May 29 PMID: 23720773
Authors: Frazier, S. J. - Cohen, B. N. - Lester, H. A.
Journal: J Biol Chem

A modified invertebrate glutamate-gated Cl- channel (GluCl alphabeta) was previously employed to allow pharmacologically induced silencing of electrical activity in CNS neurons upon exposure to the anthelmintic drug ivermectin (IVM). Usefulness of the previous receptor was limited by (1) the high concentration of IVM necessary to elicit a consistent silencing phenotype, raising concern about potential side effects, and (2) the variable extent of neuronal spike suppression, due to variations in the co-expression levels of the fluorescent protein-tagged alpha and beta subunits. To address these issues, mutant receptors generated via rational protein engineering strategies were examined for improvement. Introduction of a gain-of-function mutation (L9'F) in the second transmembrane domain of the alpha subunit appears to facilitate beta subunit incorporation and substantially increase heteromeric GluCl alphabeta sensitivity to IVM without increasing background conductance (see Frazier et al., JBC Submitted). Removal of an arginine-based endoplasmic reticulum retention motif (RSR mutated to AAA) from the intracellular loop of the beta subunit further promotes heteromeric expression at the plasma membrane possibly by preventing endoplasmic reticulum (ER)-associated degradation of the beta subunit rather than simply reducing ER retention. A monomeric XFP (mXFP) mutation that prevents fluorescent protein dimerization complements the mutant channel effects. Expression of the newly engineered GluCl opt alpha-mXFP L9'F + opt beta-mXFP Y182F RSR_AAA receptor in dissociated neuronal cultures markedly increases conductance and reduces variability in spike suppression at 1 nM IVM. This receptor, named GluClv2.0, is an improved tool for IVM-induced silencing.

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Metformin Increases Mitochondrial Energy Formation in L6 Muscle Cell Cultures.

Publication Date: 2013 May 29 PMID: 23720772
Authors: Vytla, V. S. - Ochs, R. S.
Journal: J Biol Chem

A popular hypothesis for the action of metformin, the widely used anti-diabetes drug, is the inhibition of mitochondrial respiration, specifically at complex I. This is consistent with metformin stimulation of glucose uptake by muscle and inhibition of gluconeogenesis by liver. Yet, mitochondrial inhibition is inconsistent with metformin stimulation of fatty acid oxidation in both tissues. In the present study we measured mitochondrial energy production in intact cells adapting an in vivo technique of phosphocreatine (PCr) formation following energy interruption (PCr recovery) to cell cultures. Metformin increased PCr recovery from either dinitrophenol (DNP) or azide in L6 cells. We found that metformin alone had no effect on cell viability as measured by total ATP concentration, trypan blue exclusion, or MTT reduction. However, treatments with low concentrations of DNP or azide reversibly decreased ATP concentration. Metformin increased MTT reduction during recovery from either agent. Viability measured by trypan blue exclusion indicated that cells were intact under these conditions. We also found that metformin increased free AMP, and to a smaller extent, free ADP concentrations in cells, an action that was duplicated by a structurally unrelated AMP deaminase inhibitor. We conclude that, in intact cells, metformin can lead to a stimulation of energy formation, rather than an inhibition.

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Par14 associates with IRS-1, thereby enhancing insulin-induced IRS-1 phosphorylation and metabolic actions.

Publication Date: 2013 May 29 PMID: 23720771
Authors: Zhang, J. - Nakatsu, Y. - Sinjo, T. - Guo, Y. - Sakoda, H. - Yamamotoya, T. - Otani, Y. - Okubo, H. - Kushiyama, A. - Fujishiro, M. - Fukushima, T. - Tsuchiya, Y. - Kamata, H. - Nishimura, F. - Katagiri, H. - Takahashi, S. I. - Kurihara, H. - Uchida, T. - Asano, T.
Journal: J Biol Chem

Pin1 and Par14 are parvulin-type peptidyl prolyl cis/trans isomerases. While numerous proteins have been identified as Pin1 substrates, the target proteins of Par14 remain largely unknown. Par14 expression levels are increased in the livers and embryonic fibroblasts of Pin1 KO mice, suggesting a compensatory relationship between the functions of Pin1 and Par14. In this study, first, the association of Par14 with IRS-1 was demonstrated in HepG2 cells overexpressing both as well as endogenously in the mouse liver. The analysis using deletion-mutated Par14 and IRS-1 constructs revealed the N-terminal portion containing the basic domain of Par14 and the two relatively C-terminal portions of IRS-1 to be involved in these associations, in contrast to the WW domain of Pin1 and the SAIN domain of IRS-1. Par14 overexpression in HepG2 markedly enhanced insulin-induced IRS-1 phosphorylation and its downstream events; PI 3-kinase binding with IRS-1 and Akt phosphorylation. In contrast, treating HepG2 cells with Par14 siRNA suppressed these events. In addition, overexpression of Par14 in the insulin-resistant ob/ob mouse liver by adenoviral transfer significantly improved hyperglycemia with normalization of hepatic PEPCK and G6Pase mRNA levels, and gene suppression of Par14 using shRNA adenovirus significantly exacerbated the glucose intolerance in Pin1 KO mice. Therefore, although Pin1 and Par14 associate with different portions of IRS-1, the prolyl cis-trans isomerization in multiple sites of IRS-1 by these isomerases appears to be critical for efficient insulin receptor-induced IRS-1 phosphorylation. This process is likely to be one of the major mechanisms regulating insulin sensitivity and also constitutes a potential therapeutic target for novel insulin-sensitizing agents.

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mRNA on the move: the road to its biological destiny.

Publication Date: 2013 May 28 PMID: 23720759
Authors: Eliscovich, C. - Buxbaum, A. R. - Katz, Z. B. - Singer, R. H.
Journal: J Biol Chem

Cells have evolved to regulate the asymmetric distribution of specific mRNA targets to institute spatial and temporal control over gene expression. Over the last few decades evidence has mounted as to the importance of localizing elements in the mRNA sequence and their respective RNA-binding proteins. Live imaging methodologies have shown mechanistic details of this phenomenon. In this minireview we will focus on the advanced biochemical and cell imaging techniques used to tweeze out the finer aspects of mechanisms of mRNA movement.

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Glycoprotein biosynthesis in a eukaryote lacking the membrane protein Rft1.

Publication Date: 2013 May 28 PMID: 23720757
Authors: Jelk, J. - Gao, N. - Serricchio, M. - Signorell, A. - Schmidt, R. S. - Bangs, J. D. - Acosta-Serrano, A. - Lehrman, M. A. - Butikofer, P. - Menon, A. K.
Journal: J Biol Chem

Mature dolichol-linked oligosaccharides (mDLOs) needed for eukaryotic protein N-glycosylation are synthesized by a multi-step pathway in which the biosynthetic lipid intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) flips from the cytoplasmic to the luminal face of the endoplasmic reticulum (ER). The ER membrane protein Rft1 is intimately involved in mDLO biosynthesis. Yeast genetic analyses implicated Rft1 as the M5-DLO flippase, but since biochemical tests challenged this assignment, the function of Rft1 remains obscure. To understand the role of Rft1 we sought to analyze mDLO biosynthesis in vivo in the complete absence of the protein. Rft1 is essential for yeast viability, and no Rft1-null organisms are currently available. Here, we exploited Trypanosoma brucei (Tb), an early diverging eukaryote whose Rft1 homologue functions in yeast. We report that TbRft1-null procyclic trypanosomes grow nearly normally. They have normal steady-state levels of mDLO and significant N-glycosylation, indicating robust M5-DLO flippase activity. Remarkably, the mutant cells have 30-100-fold greater steady-state levels of M5-DLO than wild-type cells. All N-glycans in the TbRft1-null cells originate from mDLO indicating that the M5-DLO excess is not available for glycosylation. These results suggest that rather than facilitating M5-DLO flipping, Rft1 facilitates conversion of M5-DLO to mDLO by another mechanism, possibly by acting as an M5-DLO chaperone.

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Differential virulence of Candida glabrata glycosylation mutants.

Publication Date: 2013 May 28 PMID: 23720756
Authors: West, L. - Lowman, D. W. - Mora-Montes, H. M. - Grubb, S. - Murdoch, C. - Thornhill, M. H. - Gow, N. A. - Williams, D. - Haynes, K.
Journal: J Biol Chem

The fungus Candida glabrata is an important and increasingly common pathogen of humans, particularly in immunocompromised hosts. Despite this little is known about the attributes that allow this organism to cause disease, or its interaction with the host immune system. However, in common with other fungi the cell wall of C. glabrata is the initial point of contact between the host and pathogen, and as such is likely to play an important role in mediating interactions and hence virulence. Here we show both through genetic complementation and polysaccharide structural analyses that C. glabrata ANP1, MNN2 and MNN11 encode functional orthologues of the respective Saccharomyces cerevisiae mannosyl-transferases. Furthermore we show that deletion of the C. glabrata Anp1, Mnn2 & Mnn11 mannosyltransferases, directly affects the structure of the fungal N-linked mannan, in-line with their predicted functions and this has implications for cell wall integrity and consequently virulence. C. glabrata anp1 and mnn2 mutants showed increased virulence, compared to wild-type (and mnn11) cells. This is in contrast to Candida albicans where inactivation of genes involved in mannan biosynthesis has usually been linked to an attenuation of virulence. In the longer-term better understanding of the attributes that allow C. glabrata to cause disease will provide insights that can be adopted for the development of novel therapeutic and diagnostic approaches.

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A blue-shifted light-driven proton pump for neural silencing.

Publication Date: 2013 May 28 PMID: 23720753
Authors: Sudo, Y. - Okazaki, A. - Ono, H. - Yagasaki, J. - Sugo, S. - Kamiya, M. - Reissig, L. - Inoue, K. - Ihara, K. - Kandori, H. - Takagi, S. - Hayashi, S.
Journal: J Biol Chem

Ion-transporting rhodopsins are widely utilized as optogenetic tools both for light-induced neural activation and silencing. The most studied representative is Bacteriorhodopsin (BR) which absorbs green/red light (~ 570 nm) and functions as a proton pump. Upon photoexcitation, BR induces a hyperpolarization across the membrane, which, if incorporated into a nerve cell, results in its neural silencing. In this study, we show that several residues around the retinal chromophore, which are completely conserved among BR homologs from the archaea, are involved in the spectral tuning in a BR homolog (HwBR), and that the combination mutation causes a large spectral blue-shift (lambdamax = 498 nm) whilst preserving the robust pumping activity. QM/MM calculations revealed that, compared with the wild-type, the beta-ionone ring of the chromophore in the mutant is rotated about 130 degrees caused by the lack of steric hindrance between the methyl groups of the retinal and the mutated residues, resulting in the breakage of the pi conjugation system on the polyene chain of the retinal. By the same mutations, similar spectral blue-shifts are also observed in another BR homolog, archearhodopsin-3 (AR3, also called Arch). The color variant of AR3 could be successfully expressed in the neural cells of C. elegans, and illumination with blue light (500 nm) lead to the effective locomotory paralysis of the worms. Thus, we successfully produced a blue-shifted proton pump for neural silencing.

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Molecular mechanisms, thermodynamics, and dissociation kinetics of knob-hole interactions in fibrin.

Publication Date: 2013 May 31 PMID: 23720752
Authors: Kononova, O. - Litvinov, R. I. - Zhmurov, A. - Alekseenko, A. - Cheng, C. H. - Agarwal, S. - Marx, K. A. - Weisel, J. W. - Barsegov, V.
Journal: J Biol Chem

Polymerization of fibrin, the primary structural protein of blood clots and thrombi, occurs through binding of knobs `A' and `B' in the central nodule of fibrin monomer to complementary holes `a' and `b' in the gamma- and beta-nodules, respectively, of another monomer. We characterized the A:a and B:b knob-hole interactions under varying solution conditions using Molecular Dynamics simulations of the structural models of fibrin(ogen) fragment D complexed with synthetic peptides GPRP (knob `A' mimetic) and GHRP (knob `B' mimetic). The strength of A:a and B:b knob-hole complexes was roughly equal, decreasing with pulling force; yet, the dissociation kinetics were sensitive to variations in acidity (pH=5-7) and temperature (T=25-37 degrees C). There were similar structural changes in holes `a' and `b' during forced dissociation of the knob-hole complexes: elongation of loop I, stretching of interior region, and translocation of the moveable flap. The disruption of the knob-hole interactions was not an "all-or-none" transition, as it occurred through distinct two-step or single-step pathways with or without intermediate states. The knob-hole bonds were stronger, tighter, and more brittle at pH=7 than at pH=5. The B:b knob-hole bonds were weaker, looser, and more compliant than the A:a knob-hole bonds at pH=7, but stronger, tighter, and less compliant at pH=5. Surprisingly, the knob-hole bonds were stronger, not weaker, at elevated temperature (T=37 degrees C) compared to T=25 degrees C due to the helix-to-coil transition in loop I, which helps stabilize the bonds. These results provide detailed qualitative and quantitative characteristics underlying the most significant non-covalent interactions involved in fibrin polymerization.

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Pentalysine clusters mediate silica targeting of silaffins in Thalassiosira pseudonana.

Publication Date: 2013 May 28 PMID: 23720751
Authors: Poulsen, N. - Scheffel, A. - Sheppard, V. C. - Chesley, P. M. - Kroger, N.
Journal: J Biol Chem

The biological formation of inorganic materials (biomineralization) often occurs in specialized intracellular vesicles. Prominent examples are diatoms, a group of single-celled eukaryotic microalgae that produce their SiO2 (silica) based cell walls within intracellular silica deposition vesicles (SDVs). SDVs contain protein-based organic matrices that control silica formation resulting in species specifically nanopatterned biosilica, an organic-inorganic composite material. So far no information is available regarding the molecular mechanisms of SDV biogenesis. Here we have investigated by fluorescence microscopy and subcellular membrane fractionation the intracellular transport of silaffin Sil3. Silaffins are a group of phosphoproteins constituting the main components of the organic matrix of diatom biosilica. We demonstrate that the N-terminal signal peptide of Sil3 mediates import into a specific subregion of the endoplasmic reticulum (ER). Additional segments from the mature part of Sil3 are required to reach post-ER compartments. Further transport of Sil3 and incorporation into the biosilica (silica targeting) requires protein segments that contain a high density of modified lysine residues and phosphoserines. Silica targeting of Sil3 is not dependent on a particular peptide sequence, yet a lysine rich 12-14 amino acid peptide motif (pentalysine cluster), which is conserved in all silaffins, strongly promotes silica targeting. The results of the present work provide the first insight into the molecular mechanisms for biogenesis of mineral forming vesicles from an eukaryotic organism.

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An epigenetic antimalarial resistance mechanism involving parasite genes linked to nutrient uptake.

Publication Date: 2013 May 28 PMID: 23720749
Authors: Sharma, P. - Wollenberg, K. - Sellers, M. - Zainabadi, K. - Galinsky, K. - Moss, E. - Nguitragool, W. - Neafsey, D. - Desai, S. A.
Journal: J Biol Chem

Acquired antimalarial drug resistance produces treatment failures and has led to periods of global disease resurgence. In P. falciparum, resistance is known to arise through genome-level changes such as mutations and gene duplications. We now report an epigenetic resistance mechanism involving genes responsible for the plasmodial surface anion channel, a nutrient channel that also transports ions and antimalarial compounds at the host erythrocyte membrane. Two blasticidin S-resistant lines exhibited markedly reduced expression of clag genes linked to channel activity, but had no genome-level changes. Silencing aborted production of the channel protein and was directly responsible for reduced uptake. Silencing affected clag paralogs on two chromosomes and was mediated by specific histone modifications, allowing a rapidly reversible drug resistance phenotype advantageous to the parasite. These findings implicate a novel epigenetic resistance mechanism that involves reduced host cell uptake and is a worrisome liability for water-soluble antimalarial drugs.

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Cytoplasmic Degradation of the Arabidopsis Transcription Factor ABSCISIC ACID INSENSITIVE 5 is Mediated by the RING-type E3 Ligase KEEP ON GOING.

Publication Date: 2013 May 29 PMID: 23720747
Authors: Liu, H. - Stone, S. L.
Journal: J Biol Chem

To mitigate the effects of environmental stress the ABA-responsive transcription factor ABI5 is required to delay growth of germinated seedlings. In the absence of stress, KEG E3 is required to maintain low levels of ABI5. However, the mechanism underlying KEG-dependent turnover of ABI5 is not known. In addition, localization studies place KEG at the trans-Golgi network, while ABI5 is nuclear. Here we show that KEG interacts directly with ABI5 via its conserved C3 region. Interactions between KEG and ABI5 were observed in the cytoplasm and trans-Golgi network only when the RING domain of KEG was inactivated or when ABI5 was stabilized via mutations. Deletion of the C-terminal region of ABI5 or substituting lysine 344 for alanine (K344A) prohibited protein turnover. Furthermore, ABI5 is observed in the cytoplasm of Arabidopsis thaliana root cells when the K344A mutation is combined with the deletion of a nuclear localization signal (NLS). Other lysine mutations (K353A, K364A and K376A) in conjunction with the NLS deletion did not result in cytoplasmic accumulation of ABI5. Loss of K344 did not affect the ability of ABI5 to promote ABA responses, which demonstrates that the mutant transcription factor is still functional. Based on the results, a model is suggested where KEG targets ABI5 for degradation in the cytoplasm, thus reducing nuclear accumulation of the transcription factor in the absence of ABA.

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Mice lacking alpha-tubulin acetyltransferase 1 are viable but display alpha-tubulin acetylation deficiency and dentate gyrus distortion.

Publication Date: 2013 May 28 PMID: 23720746
Authors: Kim, G. W. - Li, L. - Gorbani, M. - You, L. - Yang, X. J.
Journal: J Biol Chem

alpha-Tubulin acetylation at Lys-40, located on the luminal side of microtubules, has been widely studied and used as a marker for stable microtubules in the cilia and other subcellular structures, but the functional consequences remain perplexing. Recent studies have shown that Mec-17 and its paralog are responsible for alpha-tubulin acetylation in C. elegans. There is one such protein known as Atat1 (alpha tubulin acetyltransferase 1) per higher organism. Zebrafish Atat1 appears to govern embryo development, raising the intriguing possibility that Atat1 is also critical for development in mammals. In addition to Atat1, three other mammalian acetyltransferases, ARD1-NAT1, ELP3 and GCN5, have been shown to acetylate alpha-tubulin in vitro, so an important question is how these four enzymes contribute to the acetylation in vivo. We demonstrate here that Atat1 is a major alpha-tubulin acetyltransferase in mice. It is widely expressed in mouse embryos and tissues. While Atat1-null animals display no overt phenotypes, alpha-tubulin acetylation is lost in sperm flagella and the dentate gyrus is deformed. Furthermore, human ATAT1 colocalizes in bundled microtubules with doublecortin. These results thus suggest that mouse Atat1 may regulate advanced functions such as learning and memory, thereby shedding novel light on physiological roles of alpha-tubulin acetylation in mammals.

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Matrix Metalloproteinase (MMP)-9 transcription in mouse brain induced by fear learning.

Publication Date: 2013 May 28 PMID: 23720741
Authors: Ganguly, K. - Rejmak, E. - Mikosz, M. - Nikolaev, E. - Knapska, E. - Kaczmarek, L.
Journal: J Biol Chem

Memory formation requires learning based molecular and structural changes in neurons, whereas matrix metalloproteinase (MMP)-9 is involved in the synaptic plasticity by cleaving extracellular matrix proteins and thus is associated with learning processes in the mammalian brain. As the mechanisms of MMP-9 transcription in the brain are poorly understood, this study aimed at elucidating regulation of MMP-9 gene expression in the mouse brain after fear learning. We show herein that contextual fear conditioning markedly increases MMP-9 transcription, followed by enhanced enzymatic levels in the three major brain structures implicated in fear learning, i.e., the amygdala, hippocampus and prefrontal cortex. To reveal the role of AP-1 transcription factor in MMP-9 gene expression, we have used reporter gene constructs with specifically mutated AP-1 gene promoter sites. The constructs were introduced into the medial prefrontal cortex of neonatal mouse pups by electroporation, and the regulation of MMP-9 transcription was studied after contextual fear conditioning in the adult animals. Specifically, -42/-50 and -478/-486 bp AP-1 binding motifs of mouse MMP-9 promoter sequence have been found to play a major role in MMP-9 gene activation. Furthermore, increases in MMP-9 gene promoter binding by the AP-1 transcription factor proteins c-Fos and c-Jun have been demonstrated in all three brain structures under investigation. Hence, our results suggest that AP-1 acts as a positive regulator of MMP-9 transcription in the brain following fear learning.

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CaMKII-mediated intramolecular opening of Integrin Cytoplasmic domain Associated Protein-1 (ICAP-1alpha) negatively regulates beta1 integrins.

Publication Date: 2013 May 28 PMID: 23720740
Authors: Millon-Fremillon, A. - Brunner, M. - Abed, N. - Collomb, E. - Ribba, A. S. - Block, M. R. - Albiges-Rizo, C. - Bouvard, D.
Journal: J Biol Chem

Focal adhesion turnover during cell migration is an integrated cyclic process requiring tight regulation of integrin function. Interaction of integrin with its ligand depends on its activation state which is regulated by the direct recruitment of proteins onto the beta integrin chain cytoplasmic domain. We previously reported that ICAP-1alpha, a specific cytoplasmic partner of beta1A integrins, limits both talin and kindlin interaction with beta1 integrin and thereby restraining focal adhesion assembly. Here we provide evidences that the calcium and calmodulin-dependent serine/threonine protein kinase of type II (CaMKII) is an important regulator of ICAP-1alpha for controlling focal adhesion dynamics. CaMKII directly phosphorylates ICAP-1alpha and disrupts an intramolecular interaction between the N- and the C-terminal domains of ICAP-1alpha, unmasking the PTB domain thereby permitting ICAP-1alpha binding onto beta1 integrin tail. ICAP-1alpha direct interaction with the beta1 integrin tail and the modulation of beta1 integrin affinity state are required for down regulating focal adhesion assembly. Our results point to a molecular mechanism for the phosphorylation-dependent control of ICAP-1alpha function by CaMKII allowing the dynamic control of beta1 integrin activation and cell adhesion.

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MicroRNA-34c inversely couples the biological functions of the runt-related transcription factor RUNX2 and the tumor suppressor p53 in osteosarcoma.

Publication Date: 2013 May 29 PMID: 23720736
Authors: van der Deen, M. - Taipaleenmaki, H. - Zhang, Y. - Teplyuk, N. M. - Gupta, A. - Cinghu, S. - Shogren, K. - Maran, A. - Yaszemski, M. J. - Ling, L. - Cool, S. M. - Leong, D. T. - Dierkes, C. - Zustin, J. - Salto-Tellez, M. - Ito, Y. - Bae, S. C. - Zielenska, M. - Squire, J. A. - Lian, J. B. - Stein, J. L. - Zambetti, G. P. - Jones, S. N. - Galindo, M. - Hesse, E. - Stein, G. S. - van Wijnen, A. J.
Journal: J Biol Chem

Osteosarcoma (OS) is a primary bone tumor that is most prevalent during adolescence. RUNX2, which stimulates differentiation and suppresses proliferation of osteoblasts, is deregulated in OS. Here, we define pathological roles of RUNX2 in the etiology of OS and mechanisms by which RUNX2 expression is stimulated. RUNX2 is often highly expressed in human OS biopsies and cell lines. Small interference RNA (siRNA)-mediated depletion of RUNX2 inhibits growth of U2OS OS cells. RUNX2 levels are inversely linked to loss of p53 (which predisposes to OS) in distinct OS cell lines and osteoblasts. RUNX2 protein levels decrease upon stabilization of p53 with the MDM2 inhibitor Nutlin-3. Elevated RUNX2 protein expression is post-transcriptionally regulated and directly linked to diminished expression of several validated RUNX2 targeting microRNAs (miRNAs) in human OS cells compared to mesenchymal progenitor cells. The p53-dependent miR-34c is the most significantly down-regulated RUNX2 targeting miRNA in OS. Exogenous supplementation of miR-34c markedly decreases RUNX2 protein levels, while 3UTR reporter assays establish RUNX2 as a direct target of miR-34c in OS cells. Importantly, Nutlin-3 mediated stabilization of p53 increases expression of miR-34c and decreases RUNX2. Thus, a novel RUNX2-p53-miR34 network controls cell growth of osseous cells and is compromised in OS.

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A model of the membrane-bound cytochrome b5-cytochrome p450 complex from NMR and mutagenesis data.

Publication Date: 2013 May 24 PMID: 23709268
Authors: Ahuja, S. - Jahr, N. - Im, S. C. - Vivekanandan, S. - Popovych, N. - Le Clair, S. V. - Huang, R. - Soong, R. - Xu, J. - Yamamoto, K. - Nanga, R. P. - Bridges, A. - Waskell, L. - Ramamoorthy, A.
Journal: J Biol Chem

Microsomal cytochrome b5 (cytb5) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb5 (16-kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb5 NMR resonances and site-directed mutagenesis data were used to characterize the cytb5 interaction epitope recognized by ferric microsomal cytP450 (56-kDa). Subsequently, a data-driven docking algorithm, HADDOCK, was used to generate the structure of the complex between cytP4502B4 and cytb5 using experimentally derived restraints from NMR, mutagenesis and double-mutant cycle data obtained on the full-length proteins. Our docking and experimental results point to the formation of a dynamic electron-transfer complex between the acidic, convex surface of cytb5 and the concave, basic proximal surface of cytP4502B4. The majority of the binding energy for the complex is provided by interactions between residues on the C-helix and beta-bulge of cytP450 and residues at the end of helix alpha4 of cytb5. The structure of the complex allows us to propose an interprotein electron transfer pathway involving the highly conserved Arg125 on cytP450 serving as a salt bridge between the heme propionates of cytP450 and cytb5. We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb5-cytP450 interaction. This study paves the way to obtaining valuable structural, functional and dynamic information on membrane-bound complexes.

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Structural characterization of carbohydrate binding by LMAN1 provides new insight into the endoplasmic reticulum export of FV and FVIII.

Publication Date: 2013 May 24 PMID: 23709226
Authors: Zheng, C. - Page, R. C. - Das, V. - Nix, J. C. - Wigren, E. - Misra, S. - Zhang, B.
Journal: J Biol Chem

LMAN1 (ERGIC-53) is a key mammalian cargo receptor responsible for the export of a subset of glycoproteins from the endoplasmic reticulum. Together with its soluble coreceptor MCFD2, LMAN1 transports coagulation factors V (FV) and VIII (FVIII). Mutations in LMAN1 or MCFD2 cause the genetic bleeding disorder combined deficiency of FV and FVIII (F5F8D). The LMAN1 carbohydrate recognition domain (CRD) binds to both glycoprotein cargo and to MCFD2 in a Ca2+-dependent manner. To understand the biochemical basis and regulation of LMAN1 binding to glycoprotein cargo, we solved crystal structures of the LMAN1-CRD bound to Man-alpha-1,2-Man, the terminal carbohydrate moiety of high-mannose glycans. Our structural data, combined with mutagenesis and in vitro binding assays, define the central mannose binding site on LMAN1 and pinpoint histidine 178 and glycines 251/252 as critical residues for FV/FVIII binding. We also show that mannobiose binding is relatively independent of pH in the range relevant for ER-to-Golgi traffic, but is sensitive to lowered Ca2+ concentrations. The distinct LMAN1-MCFD2 interaction is maintained at these lowered Ca2+ concentrations. Our results suggest that compartmental changes in Ca2+ concentration regulate glycoprotein cargo binding and release from the LMAN1-MCFD2 complex in the early secretory pathway.

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The molecular basis for species-specific activation of human TRPA1 by protons involves poorly conserved residues within transmembrane domains 5 and 6.

Publication Date: 2013 May 24 PMID: 23709225
Authors: de la Roche, J. - Eberhardt, M. J. - Klinger, A. B. - Stanslowksy, N. - Wegner, F. - Koppert, W. - Reeh, P. W. - Lampert, A. - Fischer, M. J. - Leffler, A.
Journal: J Biol Chem

The surveillance of acid-base homeostasis is concerted by diverse mechanisms, including an activation of sensory afferents. Proton-evoked activation of rodent sensory neurons is mainly mediated by the capsaicin receptor TRPV1 and acid sensing ion channels. In this study we demonstrate that extracellular acidosis activates and sensitizes the human irritant receptor TRPA1 (hTRPA1). Proton-evoked membrane currents and calcium influx through hTRPA1 occurred at physiological acidic pH-values, were concentration-dependent, and blocked by the selective TRPA1 antagonist HC030031. Both rodent and rhesus monkey TRPA1 failed to respond to extracellular acidosis, and protons even inhibited rodent TRPA1. Accordingly, mouse dorsal root ganglion neurons lacking TRPV1 only responded to protons when hTRPA1 was heterologously expressed. This species specific activation of hTRPA1 by protons was reversed in both mouse and rhesus monkey TRPA1 by exchange of distinct residues within transmembrane domains 5 and 6. Furthermore, protons seem to interact with an extracellular interaction site to gate TRPA1 and not via a modification of intracellular N-terminal cysteines known as important interaction sites for electrophilic TRPA1-agonists. Our data suggest that hTRPA1 acts as a sensor for extracellular acidosis in human sensory neurons and should thus be taken into account as a yet unrecognized transduction molecule for proton-evoked pain and inflammation. The species specificity of this property is unique among known endogenous TRPA1-agonists, possibly indicating that evolutionary pressure enforced TRPA1 to inherit the role as an acid-sensor in human sensory neurons.

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Isolation and characterization of two geometric allene oxide isomers synthesized from 9S-hydroperoxy-linoleic acid by P450 CYP74C3: Stereochemical assignment of natural fatty acid allene oxides.

Publication Date: 2013 May 24 PMID: 23709224
Authors: Brash, A. R. - Boeglin, W. E. - Stec, D. F. - Voehler, M. - Schneider, C. - Cha, J. K.
Journal: J Biol Chem

Specialized cytochromes P450 or catalase-related hemoproteins transform fatty acid hydroperoxides to allene oxides, highly reactive epoxides leading to cyclopentenones and other products. The stereochemistry of the natural allene oxides is incompletely defined, as are the structural features required for their cyclization. We investigated the transformation of 9S-hydroperoxy-linoleic acid (9S-HPODE) with the allene oxide synthase CYP74C3, a reported reaction that unexpectedly produces an allene oxide-derived cyclopentenone. Using biphasic reaction conditions at 0 degrees C we isolated the initial products and separated two allene oxide isomers by HPLC at -15 degrees C. One matched previously described allene oxides in its UV spectrum (lambdamax 236 nm) and NMR spectrum (defining a 9,10-epoxy-octadec-10,12Z-dienoate). The second was a novel stereoisomer (UV lambdamax 239 nm) with distinctive NMR chemical shifts. Comparison of NOE interactions of the epoxy proton at C9 in the two allene oxides (and the equivalent NOE experiment in 12,13-epoxy allene oxides) allowed assignment at the isomeric C10 epoxy-ene carbon as Z in the new isomer and the E configuration in all previously characterized allene oxides. The novel 10Z isomer spontaneously formed a cis-cyclopentenone at room temperature in hexane. These results explain the origin of the cyclopentenone, provide insights into the mechanisms of allene oxide cyclization, and define the double bond geometry in naturally occurring allene oxides.

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The nucleic acid binding domain and translational repression activity of a Xenopus terminal uridylyl transferase.

Publication Date: 2013 May 24 PMID: 23709223
Authors: Lapointe, C. P. - Wickens, M.
Journal: J Biol Chem

Terminal uridylyl transferases (TUTs) catalyze the addition of uridines to the 3' ends of RNAs and are implicated in the regulation of both messenger RNAs and microRNAs. To better understand how TUTs add uridines to RNAs, we focused on a putative TUT from Xenopus laevis, XTUT7. We determined that XTUT7 catalyzed the addition of uridines to RNAs. Mutational analysis revealed that a truncated XTUT7 enzyme, which contained solely the nucleotidyl transferase and poly(A) polymerase-associated domains, was sufficient for catalytic activity. XTUT7 activity decreased upon removal of the CCHC zinc finger domains and a short segment of basic amino acids (the basic region). Furthermore, the basic region bound nucleic acids in vitro. We also demonstrated that XTUT7 repressed translation of a polyadenylated RNA, to which it added a distinct number of uridines. We generated a predicted structure of XTUT7's catalytic core that indicated histidine 1269 was likely important for uridine specificity. Indeed, mutation of histidine 1269 broadened XTUT7's nucleotide specificity and abolished XTUT7-dependent translational repression. Our data reveal key aspects of how XTUT7 adds uridines to RNAs, highlight the role of the basic region, illustrate that XTUT7 can repress translation, and identify an amino acid important for uridine specificity.

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Enzyme Action in the Regulation of Plant Hormone Responses.

Publication Date: 2013 May 24 PMID: 23709222
Authors: Westfall, C. S. - Muehler, A. M. - Jez, J. M.
Journal: J Biol Chem

Plants synthesize a chemically diverse range of hormones that regulate growth, development, and responses to environmental stresses. The major classes of plant hormones are specialized metabolites with exquisitely tailored perception and signaling systems, but equally important are the enzymes that control the dose and exposure to the bioactive forms of these molecules. Here we review new insights on the role of enzyme families, including the SABATH methyltransferases, the MES methylesterases, the GH3 acyl acid amido synthetases, and the hormone peptidyl hydrolases, in controlling the biosynthesis and modifications of plant hormones and how these enzymes contribute to the network of chemical signals responsible for plant growth, development, and environmental adaptation.

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Two salt bridges differentially contribute to the maintenance of CFTR channel function.

Publication Date: 2013 May 24 PMID: 23709221
Authors: Cui, G. - Freeman, C. S. - Knotts, T. - Prince, C. Z. - Kuang, C. - McCarty, N. A.
Journal: J Biol Chem

Previous studies have identified two salt bridges in human CFTR chloride ion channels, R352-D993 and R347-D924, that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that R347 not only interacts with D924 but also interacts with D993. The tripartite interaction R347-D924-D993 mainly contributes to maintaining a stable s2 open subconductance state. The R352-D993 salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of R352 and D993, channels bearing cysteines at these sites could be latched into a full open state using the bifunctional crosslinker MTS-2-MTS, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle.

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A new gene involved in coenzyme Q biosynthesis in Escherichia coli: UbiI functions in aerobic C5-hydroxylation.

Publication Date: 2013 May 24 PMID: 23709220
Authors: Hajj Chehade, M. - Loiseau, L. - Lombard, M. - Pecqueur, L. - Ismail, A. - Smadja, M. - Golinelli-Pimpaneau, B. - Mellot-Draznieks, C. - Hamelin, O. - Aussel, L. - Kieffer-Jaquinod, S. - Labessan, N. - Barras, F. - Fontecave, M. - Pierrel, F.
Journal: J Biol Chem

Coenzyme Q (ubiquinone or Q) is a redox active lipid found in organisms ranging from bacteria to mammals in which it plays a crucial role in energy generating processes. Q biosynthesis is a complex pathway which involves multiple proteins. In this work, we show that the uncharacterized conserved visC gene is involved in Q biosynthesis in Escherichia coli and we have renamed it ubiI. Based on genetic and biochemical experiments, we establish that the UbiI protein functions in the C5-hydroxylation reaction. A strain deficient for ubiI has low level of Q and accumulates a compound derived from the Q biosynthetic pathway, which we purified and characterized. We also demonstrate that UbiI is only implicated in aerobic Q biosynthesis and that an alternative enzyme catalyzes the C5-hydroxylation reaction in the absence of oxygen. We have solved the crystal structure of a truncated form of UbiI. This structure shares many features with the canonical FAD-dependent para-hydroxybenzoate hydroxylase (PHBH) and represents the first structural characterization of a monooxygenase involved in Q biosynthesis. Site directed mutagenesis confirms that residues of the flavin binding pocket of UbiI are important for activity. With our identification of UbiI, the three monooxygenases necessary for aerobic Q biosynthesis in E. coli are known.

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Mechanisms of granule membrane recapture following exocytosis in intact mast cells.

Publication Date: 2013 May 24 PMID: 23709219
Authors: Cabeza, J. M. - Acosta, J. - Ales, E.
Journal: J Biol Chem

In secretory cells, several exocytosis-coupled forms of endocytosis have been proposed including clathrin-mediated endocytosis, kiss-and-run endocytosis, cavicapture and bulk endocytosis. These forms of endocytosis can be induced under different conditions, but their detailed molecular mechanisms and functions are largely unknown. We studied exocytosis and endocytosis in mast cells with both perforated-patch and whole-cell configurations of the patch-clamp technique using cell capacitance measurements in combination with amperometric serotonin detection. We found that intact mast cells exhibit an early endocytosis that follows exocytosis induced by compound 48/80. Direct observation of individual exocytic and endocytic events showed a higher percentage of capacitance flickers (27.3%) and off-steps (11.4%) in intact mast cells than in dialysed cells (5.4% and 2.9%, respectively). Moreover, we observed a type of endocytosis of large pieces of membrane that were likely formed by cumulative fusion of several secretory granules with the cell membrane. We also identified large-capacitance flickers that occur after large endocytosis events. Pore conductance analysis indicated that these transient events may represent compound cavicapture, most likely due to the flickering of a dilated fusion pore. Using fluorescence imaging of individual exocytic and endocytic events we observed that granules can fuse to granules already fused with the plasma membrane and then the membranes and dense cores of fused granules are internalized. Altogether, our results suggest that stimulated exocytosis in intact mast cells is followed by several forms of compensatory endocytosis, including kiss-and-run endocytosis and a mechanism for efficient retrieval of the compound membrane of several secretory granules through a single membrane fission event.

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EPR Spectroscopy of MolB2C2-A Reveals Mechanism of Transport for a Bacterial Type II Molybdate Importer.

Publication Date: 2013 May 24 PMID: 23709218
Authors: Rice, A. J. - Alvarez, F. J. - Schultz, K. M. - Klug, C. S. - Davidson, A. L. - Pinkett, H. W.
Journal: J Biol Chem

In bacteria, ABC transporters are vital for the uptake of nutrients and cofactors. Based on differences in structure and activity, ABC importers are divided into two types. Type I transporters have been well studied and employ a tightly regulated alternating access mechanism. Less is known about Type II importers, but much of what we do know has been observed in studies of the vitamin B12 importer, BtuC2D2. MolB2C2 (formally known as HI1470/71) is also a Type II importer, but its substrate, molybdate, is about 10x smaller than vitamin B12. To understand mechanistic differences among Type II importers, we focused our studies on MolBC where alternative conformations may be required to transport its relatively small substrate. To investigate the mechanism of MolBC, we employed disulfide cross-linking and electron paramagnetic resonance (EPR) spectroscopy. From these studies, we found nucleotide binding is coupled to a conformational shift at the periplasmic gate. Unlike the larger conformation changes in BtuCD-F, this shift in MolBC-A is akin to unlocking a swinging door: allowing just enough space for molybdate to slip into the cell. The lower cytoplasmic gate, identified in BtuCD-F as gate I, remains open throughout the MolBC-A mechanism and cytoplasmic gate II closes in the presence of nucleotide. Combining our results, we propose a peristaltic mechanism for MolBC-A, which gives new insight in the transport of small substrates by a Type II importer.

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Type IV P-type ATPases distinguish mono- versus di-acyl phosphatidylserine using a cytofacial exit gate in the membrane domain.

Publication Date: 2013 May 24 PMID: 23709217
Authors: Baldridge, R. D. - Xu, P. - Graham, T. R.
Journal: J Biol Chem

P4-ATPases use the energy from ATP to ''flip'' phospholipid across a lipid bilayer facilitating membrane trafficking events and maintaining the characteristic plasma membrane phospholipid asymmetry. Preferred translocation substrates for the budding yeast P4-ATPases Dnf1 and Dnf2 include lysophosphatidylcholine (lyso-PC), lysophosphatidylethanolamine (lyso-PE), derivatives of PC and PE containing a 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) group on the sn-2 C6 position, and was presumed to include PC and PE species with two intact acyl chains. We previously identified several mutations in Dnf1 transmembrane segments 1 through 4 (TM1-4) that greatly enhance recognition and transport of NBD-phosphatidylserine (NBD-PS). Here we show that most of these Dnf1 mutants cannot flip diacylated PS to the cytosolic leaflet to establish PS asymmetry. However, mutation of a highly conserved asparagine (N550) in TM3 allowed Dnf1 to restore plasma membrane PS asymmetry in a strain deficient for the P4-ATPase Drs2, the primary PS flippase. Moreover, Dnf1 N550 mutants could replace the Drs2 requirement for growth at low temperature. A screen for additional Dnf1 mutants capable of replacing Drs2 function identified substitutions of TM1 and 2 residues, within a region called the exit gate, that permit recognition of dually acylated PS. These TM1, 2 and 3 residues coordinate with the ''P+4'' residue within TM4 to determine substrate preference at the exit gate. Moreover, residues from Atp8a1, a mammalian ortholog of Drs2, in these positions allow PS recognition by Dnf1. These studies indicate Dnf1 poorly recognizes diacylated phospholipid and define key substitutions enabling recognition of endogenous PS.

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Discrete Control of TRPV4 Function in the Distal Nephron by PKA and PKC.

Publication Date: 2013 May 24 PMID: 23709216
Authors: Mamenko, M. - Zaika, O. - Boukelmoune, N. - Berrout, J. - O'Neil, R. G. - Pochynyuk, O.
Journal: J Biol Chem

We have recently documented that Ca2+-permeable TRPV4 channel, which is abundantly expressed in the distal nephron cells, mediates cellular Ca2+ responses to elevated luminal flow. The current study combined Fura 2-based [Ca2+]i imaging with immunofluorescent microscopy in isolated split-opened distal nephrons of C57BL/6 mice to probe the molecular determinants of TRPV4 activity and subcellular distribution. We found that activation of PKC pathway with PMA significantly increases [Ca2+]i responses to flow without affecting the subcellular distribution of TRPV4. Inhibition of PKC with BIM-1 diminishes cellular responses to elevated flow. On the contrary, activation of PKA pathway with forskolin does not affect TRPV4-mediated [Ca2+]i responses to flow, while markedly shifting subcellular distribution of the channel towards the apical membrane. These actions are blocked with a specific PKA inhibitor H89. Concomitant activation of PKA and PKC cascades additively enhances the amplitude of flow-induced [Ca2+]i responses and greatly increases basal [Ca2+]i levels indicating constitutive TRPV4 activation. This effect is precluded by a selective TRPV4 antagonist, HC-067047. Therefore, functional status of TRPV4 channel in the distal nephron is regulated by two distinct signaling pathways. While PKA-dependent cascade promotes TRPV4 trafficking and translocation to the apical membrane, PKC-dependent pathway increases the activity of the channel on the plasma membrane.

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Structural basis for activity regulation and substrate preference of clostridial collagenases G, H, and T.

Publication Date: 2013 May 23 PMID: 23703618
Authors: Eckhard, U. - Schonauer, E. - Brandstetter, H.
Journal: J Biol Chem

Clostridial collagenases are among the most efficient enzymes to degrade by far the most predominant protein in the biosphere. Here we present crystal structures of the peptidases of three clostridial collagenase isoforms (ColG, ColH and ColT). The comparison of unliganded and liganded structures reveal a quaternary subdomain dynamics. In the unliganded ColH structure this globular dynamics is modulated by an aspartate switch motion that binds to the catalytic zinc. We further identified a calcium binding site in proximity to the catalytic zinc. Both ions are required for full activity, explaining why calcium critically affects the enzymatic activity of clostridial collagenases. Our studies further reveal that loops close to the active site thus serve as characteristic substrate selectivity filter. These elements explain the distinct peptidolytic and collagenolytic activities of these enzymes and provide a rational to engineer collagenases with customized substrate specificity as well as for inhibitor design.

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Structure of the Chlamydia trachomatis Immunodominant Antigen Pgp3.

Publication Date: 2013 May 23 PMID: 23703617
Authors: Galaleldeen, A. - Taylor, A. B. - Chen, D. - Schuermann, J. P. - Holloway, S. P. - Hou, S. - Gong, S. - Zhong, G. - Hart, P. J.
Journal: J Biol Chem

Chlamydia trachomatis infection is the most common sexually transmitted bacterial disease. Left untreated, it can lead to ectopic pregnancy, pelvic inflammatory disease, and infertility. Here we present the structure of the secreted C. trachomatis protein Pgp3, an immunodominant antigen and putative virulence factor. The ~84 kDa Pgp3 homotrimer, encoded on a cryptic plasmid, consists of globular N- and C-terminal assemblies connected by a triple helical coiled-coil (THCC). The C-terminal domains (CTDs) possess folds similar to members of the tumor necrosis factor (TNF) family of cytokines. The closest Pgp3 CTD structural homologs include a lectin from Burkholderia cenocepacia, the C1q component of complement, and a portion of the Bacillus anthracis spore surface protein BclA, all of which play roles in bioadhesion. The N-terminal domain (NTD) consists of a concatenation of structural motifs typically found in trimeric viral proteins. The central parallel THCC contains an unusual alternating pattern of apolar and polar residue pairs that generate a rare right-handed superhelical twist. The unique architecture of Pgp3 provides the basis for understanding its role in chlamydial pathogenesis and serves as the platform for its optimization as a potential vaccine antigen candidate.

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The Involvement of Mitochondrial Amidoxime Reducing Components 1 and 2 and Mitochondrial Cytochrome b5 in N-reductive Metabolism in Human Cells.

Publication Date: 2013 May 23 PMID: 23703616
Authors: Plitzko, B. - Ott, G. - Reichmann, D. - Henderson, C. J. - Wolf, C. R. - Mendel, R. - Bittner, F. - Clement, B. - Havemeyer, A.
Journal: J Biol Chem

The mitochondrial amidoxime reducing component mARC is a recently discovered molybdenum enzyme in mammals. mARC is not active as a stand-alone protein but together with the electron transport proteins NADH-cytochrome b5 reductase (CYB5R) and cytochrome b5 (CYB5) it catalyzes the reduction of N-hydroxylated compounds such as amidoximes. The mARC-containing enzyme system is therefore considered to be responsible for the activation of amidoxime-prodrugs. All hitherto analyzed mammalian genomes code for two mARC genes (also referred to as MOSC1 and MOSC2), which share high sequence similarities. By RNAi experiments in two different human cell lines we demonstrate for the first time that both mARC proteins are capable of reducing N-hydroxylated substrates in cell metabolism. The extent of involvement is highly dependent on the expression level of the particular mARC protein. Furthermore, the mitochondrial isoform of CYB5 (CYB5B) is clearly identified as an essential component of the mARC-containing N-reductase system in human cells. The participation of the microsomal isoform (CYB5A) in N-reduction could be excluded by siRNA-mediated down-regulation in HEK-293 cells and knockout in mice. Using heme-free apo CYB5 the contribution of mitochondrial CYB5 to N-reductive catalysis was proven to strictly depend on heme. Finally, we created recombinant CYB5B variants corresponding to four nonsynonymous single nucleotide polymorphisms (SNPs). Investigated mutations of the heme protein seemed to have no significant impact on N-reductive activity of the reconstituted enzyme system.

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Major vault protein regulates class A scavenger receptor-mediated TNF-alpha synthesis and apoptosis in macrophages.

Publication Date: 2013 May 23 PMID: 23703615
Authors: Ben, J. - Zhang, Y. - Zhou, R. - Zhang, H. - Zhu, X. - Li, X. - Zhang, H. - Li, N. - Zhou, X. - Bai, H. - Yang, Q. - Li, D. - Xu, Y. - Chen, Q.
Journal: J Biol Chem

Atherosclerosis is considered a disease of chronic inflammation largely initiated and perpetuated by macrophage-dependent synthesis and release of pro-inflammatory mediators. Class A scavenger receptor (SR-A) expressed on macrophages plays a key role in this process. However, how SR-A mediated pro-inflammatory response is modulated in macrophages remains ill-defined. Here through immunoprecipitation coupled with mass spectrometry, we reported major vault protein (MVP) as a novel binding partner for SR-A. The interaction between SR-A and MVP was confirmed by immunofluorescence staining and chemical crosslinking assay. Treatment of macrophages with fucoidan, a SR-A ligand, led to a marked increase in TNF-alpha production, which was attenuated by MVP depletion. Further analysis revealed that SR-A stimulated TNF-alpha synthesis in macrophages via the caveolin- instead of clathrin-mediated endocytic pathway linked to p38- and JNK-, but not ERK-, signaling pathways. Importantly, fucoidan invoked an enrichment of MVP in lipid raft, a caveolin-reliant membrane structure, and enhanced the interaction between SR-A, caveolin, and MVP. Finally, we demonstrated that MVP elimination ameliorated SR-A-mediated apoptosis in macrophages. As such, MVP may fine-tune SR-A activity in macrophages and by which contributes to the development of atherosclerosis.

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N-glycosylation induces collagen triple helix repeat containing 1 (CTHRC1) and drives oral cancer cell migration.

Publication Date: 2013 May 23 PMID: 23703614
Authors: Liu, G. - Sengupta, P. K. - Jamal, B. - Yang, H. Y. - Bouchie, M. P. - Lindner, V. - Varelas, X. - Kukuruzinska, M. A.
Journal: J Biol Chem

Oral squamous cell carcinoma (OSCC) is one of the most pernicious malignancies, but the mechanisms underlying its development and progression are poorly understood. One of the key pathways implicated in OSCC is the canonical Wnt/beta-catenin signaling pathway. Previously, we reported that canonical Wnt signaling functioned in a positive feedback loop with the DPAGT1 gene, a principal regulator of the metabolic pathway of protein N-glycosylation, to hyper-glycosylate E-cadherin and reduce intercellular adhesion. Here, we show that in OSCC, DPAGT1 and canonical Wnt signaling converge to upregulate collagen triple helix repeat containing 1 (CTHRC1), an N-glycoprotein implicated in tumor invasion and metastasis. We find that in human OSCC specimens, amplification of CTHRC1 levels is associated with its hyper-glycosylation. Partial inhibition of DPAGT1 expression in OSCC CAL27 cells reduces CTHRC1 abundance by increasing protein turnover, indicating that N-glycosylation stabilizes CTHRC1. Additionally, canonical Wnt signaling promotes beta-catenin/TCF transcriptional activity at the CTHRC1 promoter to further elevate CTHRC1 levels. We demonstrate that DPAGT1 promotes cell migration and drives the localization of CTHRC1 to cells at the leading edge of a wound front coincident with drastic changes in cell morphology. We propose that in OSCC, dysregulation of canonical Wnt signaling and DPAGT1-dependent N-glycosylation induces CTHRC1, thereby driving OSCC cell migration and tumor spread.

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Structure and Function of Hainantoxin-III-a Selective Antagonist of Neuronal Tetrodotoxin-Sensitive Voltage-gated Sodium Channels Isolated from the Chinese Bird Spider Ornithoctonus hainana.

Publication Date: 2013 May 23 PMID: 23703613
Authors: Liu, Z. - Cai, T. - Zhu, Q. - Deng, M. - Li, J. - Zhou, X. - Zhang, F. - Li, D. - Li, J. - Liu, Y. - Hu, W. - Liang, S.
Journal: J Biol Chem

In the present study, we investigated the structure and function of Hainantoxin-III (HNTX-III), a 33-residue polypeptide from the venom of the spider Ornithoctonus hainana. It is a selective antagonist of neuronal tetrodotoxin-sensitive voltage-gated sodium channels. HNTX-III suppressed Nav1.7 current amplitude without significantly altering the activation, inactivation and repriming kinetics. Short extreme depolarizations partially activated the toxin-bound channel, indicating voltage-dependent inhibition of HNTX-III. HNTX-III increased the deactivation of the Nav1.7 current after extreme depolarizations. The HNTX-III-Nav1.7 complex was gradually dissociated upon prolonged strong depolarizations in a voltage-dependent manner, and the unbound toxin rebound to Nav1.7 after a long repolarization. Moreover, analysis of chimeric channels showed that the DIIS3-S4 linker was critical for HNTX-III binding to Nav1.7. These data are consistent with HNTX-III interacting with Nav1.7 site 4 and trapping the domain II voltage sensor in the closed state. The solution structure of HNTX-III was determined by 2D-NMR and shown to possess an inhibitor cystine knot motif. Structural analysis indicated that certain basic, hydrophobic and aromatic residues mainly localized in the C-terminus may constitute an amphiphilic surface potentially involved in HNTX-III binding to Nav1.7. Taken together, our results show that HNTX-III is distinct from beta-scorpion toxins and other beta-spider toxins in its mechanism of action and binding specificity and affinity. The present findings contribute to our understanding of the mechanism of toxin-sodium channel interaction and provide a useful tool for the investigation of the structure and function of sodium channel isoforms and for the development of analgesics.

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Interaction Between Her2 and Beclin-1 Underlies a New Mechanism of Reciprocal Regulation.

Publication Date: 2013 May 23 PMID: 23703612
Authors: Han, J. - Hou, W. - Lu, C. - Goldstein, L. A. - Stolz, D. B. - Watkins, S. C. - Rabinowich, H.
Journal: J Biol Chem

Beclin-1 is a key regulator of autophagy that functions in the context of two phase-specific complexes in the initiation and maturation of autophagosomes. Its known interacting proteins include autophagy effectors, Bcl-2 family members, and organelle membrane anchor proteins. Here we report a newly identified interaction between Beclin-1 and the protein tyrosine kinase receptor Her2. We demonstrate that in Her2-expressing breast carcinoma cells that do not succumb to lapatinib, this Her1/2 inhibitor disrupts the cell surface interaction between Her2 and Beclin-1. The data suggest that the ensuing autophagic response is correlatively associated with the release of Beclin-1 from its complex with Her2 and with the subsequent increase in cytosolic Beclin-1. Upon its interaction with Her2, Beclin-1 upregulates the phosphorylation levels of Her2 and Akt. The Beclin-1 evolutionarily conserved domain, is required for both Beclin-1 interaction with Her2 and for the increased Her2 and Akt phosphorylation. These findings shed new light on mechanisms involved in lapatinib-mediated autophagy in Her2-expressing breast carcinoma cell lines and in Beclin-1 signaling in these cells.

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Primuline Derivatives That Mimic RNA To Stimulate Hepatitis C Virus NS3 Helicase-Catalyzed ATP Hydrolysis.

Publication Date: 2013 May 23 PMID: 23703611
Authors: Sweeney, N. L. - Shadrick, W. R. - Mukherjee, S. - Li, K. - Frankowski, K. J. - Schoenen, F. J. - Frick, D. N.
Journal: J Biol Chem

ATP hydrolysis fuels the ability of helicases and related proteins to translocate on nucleic acids and separate base pairs. As a consequence, nucleic acid binding stimulates the rate at which a helicase catalyzes ATP hydrolysis. In this study, we searched a library of small molecule helicase inhibitors for compounds that stimulate ATP hydrolysis catalyzed by the hepatitis C virus (HCV) NS3 helicase, which is an important antiviral drug target. Two compounds were found that stimulate HCV helicase-catalyzed ATP hydrolysis, both of which are amide derivatives synthesized from the main component of the yellow dye primuline. Both compounds possess a terminal pyridine moiety, which was critical for stimulation. Analogs lacking a terminal pyridine inhibited HCV helicase catalyzed ATP hydrolysis. Unlike other HCV helicase inhibitors, the stimulatory compounds differentiate between helicases isolated from various HCV genotypes and related viruses. The compounds only stimulated ATP hydrolysis catalyzed by NS3 purified from HCV genotype 1b. They inhibited helicases from other HCV genotypes (e.g. 1a and 2a) or related flaviviruses (e.g. Dengue virus). The stimulatory compounds interacted with HCV helicase in the absence of ATP with dissocia-tion constants of about 2 muM. Molecular modeling and site-directed mutagenesis stud-ies suggest that the stimulatory compounds bind in the HCV helicase RNA-binding cleft near key residues Arg393, Glu493, and Ser231.

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Small Molecule Agonist of VLA-4 Integrin Induces Progenitor Cell Adhesion.

Publication Date: 2013 May 23 PMID: 23703610
Authors: Vanderslice, P. - Biediger, R. J. - Woodside, D. G. - Brown, W. S. - Khounlo, S. - Warier, N. D. - Gundlach, C. W. - Caivano, A. R. - Bornmann, W. G. - Maxwell, D. S. - McIntyre, B. W. - Willerson, J. T. - Dixon, R. A.
Journal: J Biol Chem

Activation of the integrin family of cell adhesion receptors on progenitor cells may be a viable approach to enhance the effects of stem cell-based therapies by improving cell retention and engraftment. Here, we describe the synthesis and characterization of the first small molecule agonist identified for the integrin alpha4beta1 (also known as very late antigen-4 or VLA-4). The agonist, THI0019, was generated via 2 structural modifications to a previously identified alpha4beta1 antagonist. THI0019 greatly enhanced the adhesion of cultured cell lines and primary progenitor cells to alpha4beta1 ligands VCAM-1 and CS1 under both static and flow conditions. Furthermore, THI0019 facilitated the rolling and spreading of cells on VCAM-1 and the migration of cells toward SDF-1alpha. Molecular modeling predicted that the compound binds at the alpha/beta subunit interface overlapping the ligand binding site thus indicating that the compound must be displaced upon ligand binding. In support of this model, an analog of THI0019 modified to contain a photoreactive group was used to demonstrate that when cross-linked to the integrin, the compound behaves as an antagonist instead of an agonist. In addition, THI0019 showed cross-reactivity with the related integrin alpha4beta7 as well as alpha5beta1 and alphaLbeta2. When cross-linked to alphaLbeta2, the photoreactive analog of THI0019 remained an agonist, consistent with it binding at the alpha/beta subunit interface and not at the ligand binding site in the inserted ("I") domain of the alphaL subunit. Co-administering progenitor cells with a compound such as THI0019 may provide a mechanism for enhancing stem cell therapy.

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TORC2 is required to maintain genome stability during the S phase in fission yeast.

Publication Date: 2013 May 23 PMID: 23703609
Authors: Schonbrun, M. - Kolesnikov, M. - Kupiec, M. - Weisman, R.
Journal: J Biol Chem

DNA damage can occur due to environmental insults or intrinsic metabolic processes and is a major threat to genome stability. The DNA damage response is composed of a series of well coordinated cellular processes that include activation of the DNA damage checkpoint, transient cell cycle arrest, DNA damage repair and re-entrance into the cell cycle. Here we demonstrate that mutant cells defective for TOR complex 2 (TORC2) or the downstream AGC-like kinase, Gad8, are highly sensitive to chronic replication stress, but are insensitive to ionizing radiation (IR). We show that in response to replication stress, TORC2 is dispensable for Chk1-mediated cell cycle arrest but is required for return to cell cycle progression. Rad52 is a DNA repair and recombination protein that forms foci at DNA damage sites and stalled replication forks. TORC2 mutant cells show increased spontaneous nuclear Rad52 foci, particularly during the S phase, suggesting that TORC2 protects cells from DNA damage that occurs during normal DNA replication. Consistently, the viability of TORC2-Gad8 mutant cells is dependent on the presence of the homologous recombination pathway and other proteins that are required for replication re-start following fork replication stalling. Our findings indicate that TORC2 is required for genome integrity. This may be relevant for the growing amount of evidence implicating TORC2 in cancer development.

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T-cell receptor specificity maintained by altered thermodynamics.

Publication Date: 2013 May 22 PMID: 23698002
Authors: Madura, F. - Rizkallah, P. J. - Miles, K. M. - Holland, C. J. - Bulek, A. M. - Fuller, A. - Schauenburg, A. J. - Miles, J. J. - Liddy, N. - Sami, M. - Li, Y. - Hossain, M. - Baker, B. M. - Jakobsen, B. K. - Sewell, A. K. - Cole, D. K.
Journal: J Biol Chem

The T-cell receptor (TCR) recognises peptides bound to major histocompatibility molecules (pMHC) and allows T-cells to interrogate the cellular proteaome for internal anomalies from the cell surface. The TCR contacts both MHC and peptide in an interaction characterised by weak affinity (KD >1 muM). We used phage-display to produce a melanoma-specific TCR (alpha24beta17) with a >30,000-fold enhanced binding affinity (KD = 600 pM) in order to aid our exploration of the molecular mechanisms utilised to maintain peptide specificity. Remarkably, although the enhanced affinity was mediated primarily through new TCR-MHC contacts, alpha24beta17 remained acutely sensitive to modifications at every position along the peptide backbone, mimicking the specificity of the wild type TCR. Thermodynamic analyses revealed an important role for solvation in directing peptide specificity. These findings advance our understanding of the molecular mechanisms that can govern the exquisite peptide specificity characteristic of TCR recognition.

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Structure and kinetic analysis of H2S production by human mercaptopyruvate sulfurtransferase.

Publication Date: 2013 May 22 PMID: 23698001
Authors: Yadav, P. K. - Yamada, K. - Chiku, T. - Koutmos, M. - Banerjee, R.
Journal: J Biol Chem

Mercaptopyruvate sulfurtransferase (MST) is a source of endogenous H2S, a gaseous signaling molecule implicated in a wide range of physiological processes. The contribution of MST versus the other two H2S generators, cystathionine beta-synthase and gamma-cystathionase has been difficult to evaluate since many studies on MST have been conducted at high pH and have used varied reaction conditions. In this study, we have expressed, purified and crystallized human MST in the presence of the substrate, 3-mercaptopyruvate (3-MP). The kinetics of H2S production by MST from 3-MP was studied at pH 7.4 in the presence of various physiological persulfide acceptors: cysteine, dihydrolipoic acid, glutathione, homocysteine and thioredoxin and in the presence of cyanide. The crystal structure of MST reveals a mixture of the product complex containing pyruvate and an active site cysteine persulfide (Cys248-SSH), and a nonproductive intermediate in which 3-MP is covalently linked via a disulfide bond to an active site cysteine. The crystal structure analysis allows us to propose a detailed mechanism for MST in which an Asp-His-Ser catalytic triad is positioned to activate the nucleophilic cysteine residue and participate in general acid-base chemistry while our kinetic analysis indicates that thioredoxin is likely to be the major physiological persulfide acceptor for MST.

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A substrate-induced biotin binding pocket in the carboxyl transferase domain of pyruvate carboxylase.

Publication Date: 2013 May 22 PMID: 23698000
Authors: Lietzan, A. D. - St Maurice, M.
Journal: J Biol Chem

Biotin-dependent enzymes catalyze carboxyl transfer reactions by efficiently coordinating multiple reactions between spatially distinct active sites. Pyruvate carboxylase (PC), a multifunctional biotin-dependent enzyme, catalyzes the bicarbonate- and MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To complete the overall reaction, the tethered biotin prosthetic group must first gain access to the biotin carboxylase domain and become carboxylated, and then translocate to the carboxyl transferase domain where the carboxyl group is transferred from biotin to pyruvate. Here, we report structural and kinetic evidence for the formation of a substrate-induced biotin binding pocket in the carboxyl transferase domain of PC from Rhizobium etli. Structures of the carboxyl transferase domain reveal that R. etli PC occupies a symmetrical conformation in the absence of the biotin carboxylase domain and that the carboxyl transferase domain active site is conformationally rearranged upon pyruvate binding. This conformational change is stabilized by the interaction of the conserved residues Asp590 and Tyr628 and results in the formation of the biotin binding pocket. Site-directed mutations at these residues reduce the rate of biotin-dependent reactions but have no effect on the rate of biotin-independent oxaloacetate decarboxylation. Given the conservation with carboxyl transferase domains in oxaloacetate decarboxylase and transcarboxylase, the structure-based mechanism described for PC may be applicable to the larger family of biotin-dependent enzymes.

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Functional modulation of the glutamate transporter variant GLT1b by the PDZ domain protein PICK1.

Publication Date: 2013 May 22 PMID: 23697999
Authors: Sogaard, R. - Borre, L. - Braunstein, T. H. - Madsen, K. L. - Macaulay, N.
Journal: J Biol Chem

The dominant glutamate transporter isoform in the mammalian brain, GLT1, exists as at least three splice variants, GLT1a, GLT1b and GLT1c. GLT1b interacts with the scaffold protein PICK1 (Protein interacting with kinase C 1), which is implicated in glutamatergic neurotransmission via its regulatory effect on trafficking of AMPA-type glutamate receptors. The 11 extreme C-terminal residues specific for the GLT1b variant are essential for its specific interaction with the PICK1 PDZ domain but a functional consequence of this interaction has remained unresolved. To identify a functional effect of PICK1 on GLT1a or GLT1b separately, we employed the Xenopus laevis expression system. GLT1a and GLT1b displayed similar electrophysiological properties and glutamate EC50. Co-expressed PICK1 localized efficiently to the plasma membrane and resulted in a 5-fold enhancement of the leak current in GLT1b-expressing oocytes with only a minor effect on [3H]-glutamate uptake. Three different GLT1 substrates all caused a slow TBOA-sensitive decay in the membrane current upon prolonged application, which provides support for the leak current being mediated by GLT1b itself. Leak and glutamate-evoked currents in GLT1a-expressing oocytes were unaffected by PICK1 co-expression. PKC activation down-regulated GLT1a and GLT1b activity to a similar extent, which was not affected by co-expression of PICK1. In conclusion, PICK1 may not only affect glutamatergic neurotransmission by its regulatory effect on glutamate receptors but also by affecting neuronal excitability via an increased GLT1b-mediated leak current. This may be particularly relevant in pathological conditions such as amyotropic lateral sclerosis and cerebral hypoxia which are associated with neuronal GLT1b up-regulation.

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Direct role for the replication protein Treslin (Ticrr) in the ATR-mediated checkpoint response.

Publication Date: 2013 May 21 PMID: 23696651
Authors: Hassan, B. H. - Lindsey-Boltz, L. A. - Kemp, M. G. - Sancar, A.
Journal: J Biol Chem

The topoisomerase IIbeta-binding protein 1 (TopBP1) is a dual replication/checkpoint protein. Treslin/Ticrr, an essential replication protein, was discovered as a binding partner for TopBP1 and also in a genetic screen for checkpoint regulators in zebrafish. Treslin is phosphorylated by CDK2/cyclinE in a cell cycle-dependent manner and its phosphorylation state dictates its interaction with TopBP1. The role of Treslin in the initiation of DNA replication has been partially elucidated; however its role in checkpoint response remained elusive. In this study we show that Treslin stimulates ATR phosphorylation of Chk1 both in vitro and in vivo in a TopBP1-dependent manner. Moreover, we show that the phosphorylation state of Treslin on Ser1000 is important for its checkpoint activity. Overall, our results indicate that Treslin, like TopBP1, is a dual replication/checkpoint protein that directly participates in ATR-mediated checkpoint signaling.

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Breaking the Carboxyl Rule: Lysine-96 Facilitates Reprotonation of the Schiff Base in the Photocycle of a Retinal Protein from Exiguobacterium sibiricum.

Publication Date: 2013 May 21 PMID: 23696649
Authors: Balashov, S. P. - Petrovskaya, L. E. - Imasheva, E. S. - Lukashev, E. P. - Dioumaev, A. K. - Wang, J. M. - Sychev, S. V. - Dolgikh, D. A. - Rubin, A. B. - Kirpichnikov, M. P. - Lanyi, J. K.
Journal: J Biol Chem

A lysine instead of the usual carboxyl group is in place of the internal proton donor to the retinal Schiff base in the light-driven proton pump of Exiguobacterium sibiricum, ESR. The involvement of this lysine in proton transfer is indicated by the finding that its substitution with alanine or other residues slows reprotonation of the Schiff base (decay of the M intermediate) by more than two orders of magnitude. In these mutants the rate constant of the M decay linearly decreases with decrease in proton concentration, as expected if reprotonation is limited by uptake of a proton from the bulk. In wild type ESR, M decay is biphasic, and the rate constants are nearly pH independent between pH 6 and 9. Proton uptake occurs after M formation but before M decay, especially evident in D2O and at high pH. Proton uptake is biphasic; the amplitude of the fast phase decreases with a pKa of 8.5+/-0.3, which reflects the pKa of the donor during proton uptake. Similarly, the fraction of the faster component of M decay decreases and the slower one increases, with pKa of 8.1+/-0.2. The data therefore suggest that the reprotonation of the Schiff base in ESR is preceded by transient protonation of an initially unprotonated donor, which is probably the epsilon-amino group of Lys96 or a water molecule in its vicinity, and it facilitates proton delivery from the bulk to the reaction center of the protein.

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An integrin-binding-defective mutant of insulin-like growth factor-1 (R36E/R37E IGF1) acts as a dominant-negative antagonist of IGF1R and suppresses tumorigenesis, while the mutant still binds to IGF1R.

Publication Date: 2013 May 21 PMID: 23696648
Authors: Fujita, M. - Ieguchi, K. - Cedano Prieto, D. - Fong, A. - Wilkerson, C. - Chen, J. Q. - Wu, M. - Lo, S. H. - Cheung, A. T. - Willson, M. D. - Cardiff, R. D. - Borowsky, A. D. - Takada, Y. K. - Takada, Y.
Journal: J Biol Chem

Insulin-like growth factor-1 (IGF1) is a major therapeutic target for cancer. We recently reported that IGF1 directly binds to integrins (alphavbeta3 and alpha6beta4) and induces ternary complex formation (integrin-IGF1-type I IGF receptor (IGF1R), and that the integrin-binding defective mutant of IGF1 (R36E/R37E) is defective in signaling and ternary complex formation. These findings predict that R36E/R37E competes with wt IGF1 for binding to IGF1R and inhibits IGF signaling. Here we describe that excess R36E/R37E suppressed cell viability increased by wt IGF1 in vitro in non-transformed cells. We studied the effect of R36E/R37E on viability and tumorigenesis in cancer cell lines. We did not detect the effect of wt IGF or R36E/R37E in cancer cells in anchorage-dependence. However, in anchorage-independence wt IGF1 enhanced cell viability and induced signals, while R36E/R37E did not. Notably, excess R36E/R37E suppressed cell viability and signaling induced by wt IGF1 in anchorage-independence. Using cancer cells that stably express wt IGF1 or R36E/R37E, we demonstrate that R36E/R37E suppressed tumorigenesis in vivo, while wt IGF1 markedly enhanced it. R36E/R37E suppressed the binding of wt IGF1 to the cell surface, and the subsequent ternary complex formation induced by wt IGF1. R36E/R37E suppressed activation of IGF1R by insulin. Wt IGF1, not R36E/R37E, induced ternary complex formation of IGF1R/ insulin receptor hybrid receptor. These findings suggest that 1) IGF1 induces signals in anchorage-independent conditions, and 2) R36E/R37E acts as a dominant-negative inhibitor of IGF1R (IGF1 decoy). Our results are consistent with a model in which ternary complex formation is critical for IGF signaling.

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Pyrococcus horikoshii TET2 peptidase assembling process and associated functional regulation.

Publication Date: 2013 May 21 PMID: 23696647
Authors: Appolaire, A. - Rosenbaum, E. - Dura, M. A. - Colombo, M. - Marty, V. - Noirclerc Savoye, M. - Godfroy, A. - Schoehn, G. - Girard, E. - Gabel, F. - Franzetti, B.
Journal: J Biol Chem

TET aminopeptidases are large polypeptide destruction machines present in prokaryotes and eukaryotes. Here, the rules governing their assembly into hollow 12-subunit tetrahedrons are addressed by using TET2 from Pyrococcus horikoshii (PhTET2) as a model. Point mutations allowed the capture of a stable, catalytically active precursor. Small angle X-ray scattering (SAXS) revealed that it is a dimer whose architecture in solution is identical to that determined by X-ray crystallography within the fully assembled TET particle. SAXS also showed that the reconstituted PhTET2 dodecameric particle displayed the same quaternary structure and thermal stability as the wild type complex. The PhTET2 assembly intermediates were characterized by analytical ultracentrifugation, native gel electrophoresis and electron microscopy. They revealed that PhTET2 assembling is a highly ordered process in which hexamers represent the main intermediate. Peptide degradation assays demonstrated that oligomerization triggers the activity of the TET enzyme toward large polypeptidic substrates. Fractionation experiments in Pyrococcus and Halobacterium cells revealed that, in vivo, the dimeric precursor co-exists together with assembled TET complexes. Taken together, our observations explain the biological significance of TET oligomerization and suggest the existence of a functional regulation of the dimer-dodecamer equilibrium in vivo.

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Ceramide-1-phosphate mediates endothelial cell invasion via the annexin a2/p11 heterotetrameric protein complex.

Publication Date: 2013 May 21 PMID: 23696646
Authors: Hankins, J. L. - Ward, K. E. - Linton, S. S. - Barth, B. M. - Stahelin, R. V. - Fox, T. E. - Kester, M.
Journal: J Biol Chem

The bioactive sphingolipid, ceramide-1-phosphate (C-1-P), has been implicated as an extracellular chemotactic agent directing cellular migration in hematopoietic stem/progenitor cells and macrophages. However, interacting proteins that could mediate these actions of C-1-P have, thus far, eluded identification. We have now identified and characterized interactions between ceramide-1-phosphate and the annexin a2/p11 heterotetramer constituents. This C-1-P-receptor complex is capable of facilitating cellular invasion. Herein, we demonstrate in both coronary artery macrovascular- and retinal microvascular-endothelial cells that C-1-P induces invasion through an extracellular matrix barrier. By employing surface plasmon resonance, lipid binding ELISA and mass spectrometry technologies, we have demonstrated that the heterotetramer constituents bind to C-1-P. While the annexin a2/p11 heterotetramer constituents do not bind the lipid C-1-P exclusively, other structurally similar lipids, such as phosphatidylserine, sphingosine-1-phosphate and phosphatidic acid, could not elicit the potent chemotactic stimulation observed with C-1-P. Further, we show that siRNA-mediated knockdown of either annexin a2 or p11 protein significantly inhibits C-1-P-directed invasion, indicating that the heterotetrameric complex is required for C-1-P-mediated chemotaxis. These results imply that extracellular C-1-P, acting through the extracellular annexin a2/p11 heterotetrameric protein, can mediate vascular endothelial cell invasion.

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Redox Proteomics uncovers peroxynitrite-sensitive proteins that help Escherichia coli to overcome nitrosative stress.

Publication Date: 2013 May 21 PMID: 23696645
Authors: Lindemann, C. - Lupilova, N. - Muller, A. - Warscheid, B. - Meyer, H. E. - Kuhlmann, K. - Eisenacher, M. - Leichert, L. I.
Journal: J Biol Chem

Peroxynitrite is a highly reactive chemical species with antibacterial properties that is synthesized in immune cells. In a proteomic approach, we identified specific target proteins of peroxynitrite-induced modifications in Escherichia coli. While peroxynitrite caused a fairly indiscriminate nitration of tyrosine residues, reversible modifications of protein thiols were highly specific. We used a quantitative redox-proteomic method based on isotope coded affinity tag (ICAT) chemistry and identified four proteins consistently thiol-modified in cells treated with peroxynitrite: AsnB, FrmA, MaeB, and RidA. All four were required for peroxynitrite stress tolerance in vivo. Three of the identified proteins were modified at highly conserved cysteines and MaeB and FrmA are known to be directly involved in the oxidative and nitrosative stress response in E. coli. In in vitro studies we could show that the activity of RidA, a recently discovered enamine/imine deaminase, is regulated in a specific manner by the modification of its single conserved cysteine. Mutation of this cysteine 107 to serine generated a constitutively active protein that was not susceptible to peroxynitrite.

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Drebrin-Induced Stabilization of Actin Filaments.

Publication Date: 2013 May 21 PMID: 23696644
Authors: Mikati, M. A. - Grintsevich, E. E. - Reisler, E.
Journal: J Biol Chem

Drebrin is a mammalian neuronal protein that binds to and organizes filamentous actin (F-actin) in dendritic spines, the receptive regions of most excitatory synapses which play a crucial role in higher brain functions. Here, the structural effects of drebrin on F-actin were examined in solution. Depolymerization and differential scanning calorimetry assays show that F-actin is stabilized by the binding of drebrin. Drebrin inhibits depolymerization mainly at the barbed end of F-actin. Full length drebrin and its C-terminal truncated constructs were used to clarify the domain requirements for these effects. Drebrin's actin binding domain decreases the intra-strand disulfide cross-linking of Cys41 (in the DNase I binding loop) to Cys374 (C-terminal), but increases the inter-strand disulfide cross-linking of Cys265 (Hydrophobic-loop) to Cys374 in yeast mutants Q41C and S265C, respectively. We also demonstrate, using solution biochemistry methods and EM, the rescue of filament formation by drebrin in different cases of longitudinal interprotomer contacts perturbation: T203C/C374S yeast actin mutant and grimelysin-cleaved skeletal actin (between Gly42 and Val43). Additionally, we show that drebrin rescues the polymerization of V266G/L267G, a hydrophobic-loop yeast actin mutant with an impaired lateral interface formation between the two filament strands. Overall, our data suggest that drebrin stabilizes actin filaments through its effect on their inter-strand and intra-strand contacts.

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Mechanism of Inducible Nitric-Oxide Synthase Dimerization Inhibition by Novel Pyrimidine Imidazoles.

Publication Date: 2013 May 21 PMID: 23696643
Authors: Nagpal, L. - Haque, M. M. - Saha, A. - Mukherjee, N. - Ghosh, A. - Ranu, B. C. - Stuehr, D. J. - Panda, K.
Journal: J Biol Chem

Over-production of Nitric-Oxide (NO) by inducible Nitric-Oxide Synthase (iNOS) has been etiologically linked to several inflammatory, immunological and neuro-degenerative diseases. As dimerization of NOS is required for its activity, several dimerization inhibitors including pyrimidine imidazoles are being evaluated for therapeutic inhibition of iNOS. However, the precise mechanism of their action is still unclear. Here, we examined the mechanism of iNOS inhibition by a pyrimidine imidazole core compound, PIC and its derivative, PID, having low cellular toxicity and high affinity for iNOS, using rapid stopped-flow kinetic, gel-filtration and spectro-photometric analysis. PID bound to iNOS heme to generate an irreversible PID-iNOS monomer complex that could not be converted to active dimers by tetrahydrobiopterin (H4B) and L-arginine (Arg). We utilized the iNOS oxygenase domain (iNOSoxy) and two monomeric mutants whose dimerization could be induced (K82AiNOSoxy) or not induced (D92AiNOSoxy) with H4B, to elucidate the kinetics of PID binding to the iNOS monomer and dimer. We observed that the apparent PID affinity for the monomer was eleven times higher than the dimer. PID binding rate was also sensitive to H4B and Arg site occupancy. PID could also interact with nascent iNOS monomers in iNOS-synthesizing RAW cells, to prevent their post-translational dimerization, and also caused irreversible monomerization of active iNOS dimers thereby accomplishing complete physiological inhibition of iNOS. Thus, our study establishes PID as a versatile iNOS inhibitor and therefore a potential in-vivo tool for examining the causal role of iNOS in diseases associated with its over-expression as well as therapeutic control of such diseases.

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Evidence for late resolution of the AUX codon box in evolution.

Publication Date: 2013 May 21 PMID: 23696642
Authors: Jones, T. E. - Ribas de Pouplana, L. - Alexander, R. W.
Journal: J Biol Chem

Recognition strategies for tRNA aminoacylation are ancient and highly conserved, having been selected very early in the evolution of the genetic code. In most cases, the trinucleotide anticodons of tRNA are important identity determinants for aminoacylation by cognate aminoacyl-tRNA synthetases (aaRSs). Yet a degree of ambiguity exists in the recognition of certain tRNAIle isoacceptors that are initially transcribed with the methionine-specifying CAU anticodon. In most organisms, the C34 wobble position in these tRNAIle precursors is rapidly modified to lysidine, to prevent recognition by methionyl-tRNA synthetase (MRS) and production of a chimeric Met-tRNAIle that would compromise translational fidelity. In certain bacteria, however, lysidine modification is not required for MRS rejection, indicating that this recognition strategy is not universally conserved and may be relatively recent. To explore the actual distribution of lysidine-dependent tRNAIle rejection by MRS, we have investigated the ability of bacterial MRSs from different clades to differentiate cognate tRNAMetCAU from near-cognate tRNAIleCAU. Discrimination abilities vary greatly and appear unrelated to phylogenetic or structural features of the enzymes or sequence determinants of the tRNA. Our data indicates that tRNAIle identity elements were established late and independently in different bacterial groups. We propose that the observed variation in MRS discrimination ability reflects differences in the evolution of the genetic code machineries of emerging bacterial clades.

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Translational redefinition of UGA codons is regulated by selenium availability.

Publication Date: 2013 May 21 PMID: 23696641
Authors: Howard, M. T. - Carlson, B. A. - Anderson, C. B. - Hatfield, D. L.
Journal: J Biol Chem

Incorporation of selenium into ~25 mammalian selenoproteins occurs by translational recoding whereby in-frame UGA codons are redefined to encode the selenium containing amino acid, selenocysteine (Sec). Here we applied ribosome profiling to examine the effect of dietary selenium levels on the translational mechanisms controlling selenoprotein synthesis in mouse liver. Dietary selenium levels were shown to control gene-specific selenoprotein expression primarily at the translation level by differential regulation of UGA redefinition and Sec incorporation efficiency, although effects on translation initiation and mRNA abundance were also observed. Direct evidence is presented that increasing dietary selenium causes a vast increase in ribosome density downstream of the UGA-Sec codons for a subset of selenoprotein mRNAs and that the selenium-dependent effects on Sec incorporation efficiency are mediated in part by the degree of Sec-tRNA[Ser]Sec Um34 methylation. Further, we find evidence for translation in the 5' UTRs for a subset of selenoproteins and for ribosome pausing near the UGA-Sec codon in those mRNAs encoding the selenoproteins most affected by selenium availability. These data illustrate how dietary levels of the trace element selenium can alter the readout of the genetic code to affect the expression of an entire class of proteins.

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alpha-Hemoglobin Stabilizing Protein (AHSP) Perturbs the Proximal Heme Pocket of Oxy-alpha-Hemoglobin and Weakens the Iron-Oxygen Bond.

Publication Date: 2013 May 21 PMID: 23696640
Authors: Dickson, C. F. - Rich, A. M. - D'Avigdor, W. M. - Collins, D. A. - Lowry, J. A. - Mollan, T. L. - Khandros, E. - Olson, J. S. - Weiss, M. J. - Mackay, J. P. - Lay, P. A. - Gell, D. A.
Journal: J Biol Chem

alpha-Hemoglobin (alphaHb) Stabilizing Protein (AHSP) is a molecular chaperone that assists hemoglobin assembly. AHSP induces changes in alphaHb heme coordination, but how these changes are facilitated by interactions at the alphaHb:AHSP interface is not well understood. To address this question we have used NMR, X-ray absorption spectroscopy and ligand binding measurements to probe alphaHb conformational changes induced by AHSP binding. NMR chemical shift analyses of free CO-alphaHb and CO-alphaHb:AHSP indicated that the seven helical elements of the native alphaHb structure are retained, and that the heme Fe(II) remains coordinated to the proximal His-87 side chain. However, chemical shift differences revealed alterations of the F, G and H helices and the heme pocket of CO-alphaHb bound to AHSP. Comparisons of iron-ligand geometry using extended X-ray absorption fine structure (EXAFS) spectroscopy showed that AHSP binding induces a small 0.03 A lengthening of the Fe-O2 bond, explaining previous reports that AHSP decreases alphaHb O2 affinity roughly four-fold and promotes autooxidation due primarily to a three to four-fold increase in the rate of O2 dissociation. Pro-30 mutations diminished NMR chemical shift changes in the proximal heme pocket, restored normal O2 dissociation rate and equilibrium constants, and reduced O2-alphaHb autooxidation rates. Thus, the contacts mediated by Pro-30 in wild-type AHSP promote alphaHb autooxidation by introducing strain into the proximal heme pocket. As a chaperone, AHSP facilitates rapid assembly of alphaHb into Hb when betaHb is abundant but diverts alphaHb to a redox resistant holding state when betaHb is limiting.

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Controlling cholesterol synthesis beyond 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR).

Publication Date: 2013 May 21 PMID: 23696639
Authors: Sharpe, L. J. - Brown, A. J.
Journal: J Biol Chem

HMGCR is the target of the statins, important drugs to lower blood cholesterol levels and treat cardiovascular disease. Consequently, the regulation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) has been investigated in detail. However, this enzyme acts very early in the cholesterol synthesis pathway, with approximately 20 subsequent enzymes needed to produce cholesterol. How they are regulated is largely unexplored territory, but there is growing evidence that enzymes beyond HMGCR serve as flux controlling points. Here, we introduce some of the known regulatory mechanisms affecting enzymes beyond HMGCR, and highlight the need to further investigate their control.

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The SEC14 and spectrin domains 1 (Sestd1) and Dapper antagonist of catenin 1 (Dact1) scaffold proteins cooperatively regulate the Van Gogh-like 2 (Vangl2) 4-pass transmembrane protein and planar cell polarity (PCP) pathway during embryonic development in mice.

Publication Date: 2013 May 21 PMID: 23696638
Authors: Yang, X. - Cheyette, B. N.
Journal: J Biol Chem

The planar cell polarity (PCP) pathway is a conserved non-canonical (beta-catenin-independent) branch of Wnt signaling crucial to embryogenesis, during which it regulates cell polarity and polarized cell movements. Disruption of PCP components in mice, including Vangl2 and Dact1, results in defective neural tube closure and other developmental defects. Here we show that Sestd1 is a novel binding partner of Vangl2 and Dact1. The Sestd1-Dact1 interface is formed by circumscribed regions of Sestd1 (the carboxyl-terminal region) and Dact1 (the amino-terminal region). Remarkably, we show that loss of Sestd1 precisely phenocopies loss of Dact1 during embryogenesis in mice, leading to a spectrum of birth malformations including neural tube defects, a shortened and/or curly tail, no genital tubercle, blind-ended colons, hydronephrotic kidneys, and no bladder. Moreover, as with the Dact1, a knockout mutation at the Sestd1 locus exhibits reciprocal genetic rescue interactions during development with a semidominant mutation at the Vangl2 locus. Consistent with this, examination of Wnt pathway activities in Sestd1 mutant mouse embryonic tissue reveals disrupted PCP pathway biochemistry similar to that characterized in Dact1 mutant embryos. The Sestd1 protein is a divergent member of the Trio family of GTPase regulatory proteins that lacks a guanine nucleotide exchange factor (GEF) domain. Nonetheless, in cell-based assays the Sestd1-Dact1 interaction can induce Rho GTPase activation. Together, our data indicate that Sestd1 cooperates with Dact1 in Vangl2 regulation and in the PCP pathway during mammalian embryonic development.

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Casitas B-lineage Lymphoma (CBL) linker region and Really Interesting New Gene (RING) finger mutations lead to enhanced Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) signaling via elevated levels of Janus Kinase 2 (JAK2) and Lck-Yes-related novel tyrosine kinase (LYN).

Publication Date: 2013 May 21 PMID: 23696637
Authors: Javadi, M. - Richmond, T. D. - Huang, K. - Barber, D. L.
Journal: J Biol Chem

Juvenile myelomonocytic leukemia (JMML) is characterized by hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). SHP2, NF-1, KRAS and NRAS are mutated in JMML patients, leading to aberrant regulation of RAS signaling. A subset of JMML patients harbor CBL mutations associated with 11q acquired uniparental disomy. Many of these mutations are in the linker region and the RING finger of CBL, leading to a loss of E3 ligase activity. We investigated the mechanism by which CBL-Y371H, a linker region mutant, and CBL-C384R, a RING finger mutant, lead to enhanced GM-CSF signaling. Expression of CBL mutants in the TF-1 cell line resulted in enhanced survival in the absence of GM-CSF. Cells expressing CBL mutations displayed increased phosphorylation of GM-CSF receptor betac subunit in response to stimulation, although expression of total GM-CSFR betac was lower. This suggested enhanced kinase activity downstream of GM-CSFR. JAK2 and LYN kinase expression is elevated in CBL-Y371H and CBL-C384R mutant cells, resulting in enhanced phosphorylation of CBL and S6 in response to GM-CSF stimulation. Incubation with the JAK2 inhibitor, TG101348, abolished the increased phosphorylation of GM-CSFR betac in cells expressing CBL mutants, whereas treatment with the SRC kinase inhibitor dasatinib resulted in equalization of GM-CSFR betac phosphorylation signal between wild type CBL and CBL mutant samples. Dasatinib treatment inhibited the elevated phosphorylation of CBL-Y371H and CBL-C384R mutants. Our study indicates that CBL linker and RING finger mutants lead to enhanced GM-CSF signaling due to elevated kinase expression, which can be blocked using small molecule inhibitors targeting specific downstream pathways.

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The ubiquitin conjugating enzyme (E2) Ube2w ubiquitinates the N-terminus of substrates.

Publication Date: 2013 May 21 PMID: 23696636
Authors: Scaglione, K. M. - Basrur, V. - Ashraf, N. S. - Konen, J. R. - Elenitoba-Johnson, K. S. - Todi, S. V. - Paulson, H. L.
Journal: J Biol Chem

Attachment of ubiquitin to substrate is typically thought to occur via formation of an isopeptide bond between the C-terminal glycine residue of ubiquitin and a lysine residue in the substrate. In vitro, Ube2w is nonreactive with free lysine yet readily ubiquitinates substrate. Ube2w also contains novel residues within its active site that are important for its ability to ubiquitinate substrate. To identify the site of modification we analyzed ubiquitinated substrates by mass spectrometry and found the N-terminal -NH2 group as the site of conjugation. To confirm N-terminal ubiquitination we generated lysine-less and N-terminally blocked versions of one substrate, the polyglutamine disease protein ataxin-3, and showed that Ube2w can ubiquitinate a lysine-less, but not N-terminally blocked ataxin-3. This was confirmed with a second substrate, the neurodegenerative disease protein tau. Finally we directly sequenced the N-terminus of unmodified and ubiquitinated ataxin-3 demonstrating that Ube2w attaches ubiquitin to the N-terminus of its substrates. Together these data demonstrate that Ube2w has novel enzymatic properties that direct ubiquitination of the N-terminus of substrates.

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Structural and Pharmacological Characterisation of Novel Potent and Selective Monoclonal Antibody Antagonists of Glucose-Dependent Insulinotropic Polypeptide Receptor.

Publication Date: 2013 May 20 PMID: 23689510
Authors: Ravn, P. - Madhurantakam, C. - Kunze, S. - Matthews, E. - Priest, C. - O'brien, S. - Collinson, A. - Papworth, M. - Fritsch-Fredin, M. - Jermutus, L. - Benthem, L. - Gruetter, M. - Jackson, R. H.
Journal: J Biol Chem

Glucose-dependent insulinotropic polypeptide (GIP) is an endogenous hormonal factor (incretin) that upon binding to its receptor (GIPr; a class B GPCR), stimulates insulin secretion by beta cells in the pancreas, There has been a lack of potent inhibitors of the GIPr with prolonged in vivo exposure, to investigate GIP biology. Here we describe here the generation of an antagonizing antibody to the GIPr, using phage and ribosome display libraries. Gipg013 is a specific competitive antagonist with equally high potencies to mouse, rat, dog and human GIP receptors with a Ki of 7 nM for the human GIPr. Gipg013 antagonises the GIP receptor and inhibits GIP induced insulin secretion in vivo. A crystal structure of Gipg013 Fab in complex with the human GIPr ECD indicates that the antibody binds through a series of hydrogen bonds from the CDRs of Gipg013 Fab to the N-terminal alpha-helix of GIPr extracellular domain (ECD) as well as to residues around its highly conserved glucagon receptor sub-family recognition fold. The antibody epitope overlaps with the GIP binding site on GIPr ECD ensuring competitive antagonism of the receptor. This well characterized antagonizing antibody to the GIPr will be useful as a tool to further understand the biological roles of GIP.

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COP9 signalosome subunit Csn8 is involved in maintaining proper duration of the G1 phase.

Publication Date: 2013 May 20 PMID: 23689509
Authors: Liu, C. - Guo, L. Q. - Menon, S. - Jin, D. - Pick, E. - Wang, X. J. - Deng, X. W. - Wei, N.
Journal: J Biol Chem

The COP9 signalosome (CSN) is a conserved protein complex known to be involved in developmental processes of eukaryotic organisms. Genetic disruption of a CSN gene causes arrest during early embryonic development in mice. The Csn8 subunit is the smallest and the least conserved subunit, being absent from the CSN complex of several fungal species. Nevertheless, Csn8 is an integral component of the CSN complex in higher eukaryotes, where it is essential for life. By characterizing the mouse embryonic fibroblasts (MEFs) that express Csn8 at a low level, we found that Csn8 plays an important role in maintaining the proper duration of the G1 phase of the cell cycle. A decreased level of Csn8, either in Csn8 hypomorphic MEFs or following siRNA mediated knockdown in HeLa cells, accelerated cell growth rate. Csn8 hypomorphic MEFs exhibited a shortened G1 duration and affected expression of G1 regulators. In contrast to Csn8, down-regulation of Csn5 impaired cell proliferation. Csn5 proteins were found both as a component of the CSN complex and outside of CSN (Csn5-f), and the amount of Csn5-f relative to CSN was increased in the Csn8 hypomorphic cells. We conclude that CSN harbors both positive and negative regulators of the cell cycle, and therefore is poised to influence the fate of a cell at the crossroad of cell division, differentiation, and senescence.

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Impact of peripheral ketolytic deficiency on hepatic ketogenesis and gluconeogenesis during the transition to birth.

Publication Date: 2013 May 20 PMID: 23689508
Authors: Cotter, D. G. - Ercal, B. - d'Avignon, D. A. - Dietzen, D. J. - Crawford, P. A.
Journal: J Biol Chem

Preservation of bioenergetic homeostasis during the transition from the carbohydrate-laden fetal diet to the high fat, low carbohydrate neonatal diet requires inductions of hepatic fatty acid oxidation, gluconeogenesis, and ketogenesis. Mice with loss-of-function mutation in the extrahepatic mitochondrial enzyme CoA transferase (succinyl-CoA:3-oxoacid CoA transferase, SCOT, encoded by nuclear Oxct1) cannot terminally oxidize ketone bodies and develop lethal hyperketonemic hypoglycemia within 48 hr of birth. Here we use this model to demonstrate that loss of ketone body oxidation, an exclusively extrahepatic process, disrupts hepatic intermediary metabolic homeostasis after high fat mother's milk is ingested. Livers of SCOT-knockout (SCOT-KO) neonates induce the expression of the genes encoding PGC-1alpha, PEPCK, pyruvate carboxylase, and glucose-6-phosphatase, and the neonate's pools of gluconeogenic alanine and lactate are each diminished by 50%. NMR-based quantitative fate mapping of 13C-labeled substrates revealed that livers SCOT-KO newborn mice synthesize glucose from exogenously administered pyruvate. However, the contribution of exogenous pyruvate to the TCA cycle as acetyl-CoA is increased in SCOT-KO livers, and is associated with diminished terminal oxidation of fatty acids. After mother's milk provokes hyperketonemia, livers of SCOT-KO mice diminish de novo hepatic beta-hydroxybutyrate synthesis by 90%. Disruption of beta-hydroxybutyrate production increases hepatic NAD+/NADH ratios three-fold, oxidizing redox potential in liver but not skeletal muscle. Together, these results indicate that peripheral ketone body oxidation prevents hypoglycemia and supports hepatic metabolic homeostasis, which is critical for the maintenance of glycemia during the adaptation to birth.

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Analysis of Polymorphic Residues Reveals Distinct Enzymatic and Cytotoxic Activities of the Streptococcus pyogenes NAD+ Glycohydrolase.

Publication Date: 2013 May 20 PMID: 23689507
Authors: Chandrasekaran, S. - Ghosh, J. - Port, G. C. - Koh, E. - Caparon, M. G.
Journal: J Biol Chem

The Streptococcus pyogenes NAD+ glycohydrolase (SPN) is secreted from the bacterial cell and translocated into the host cell cytosol where it contributes to cell death. Recent studies suggest that SPN is evolving and has diverged into NAD+ glycohydrolase inactive variants that correlate with tissue tropism. However, the role of SPN in both cytotoxicity and niche selection are unknown. To gain insight into the forces driving the adaptation of SPN, a detailed comparison of representative glycohydrolase activity-proficient and -deficient variants was conducted. Out of a total 454 amino acids the activity-deficient variants differed at only 9 highly conserved positions. Exchanging residues between variants revealed that no one single residue could account for the inability of the deficient variants to cleave the glycosidic bond of beta-NAD+ into nicotinamide and ADPribose. Rather, reciprocal changes at 3 specific residues were required to both abolish activity of the proficient version and restore full activity to the deficient variant. Changing any combination of one or two residues resulted in intermediate activity. However, a change to any one residue resulted in a significant decrease in enzyme efficiency. A similar pattern involving multiple residues was observed for comparison to a second highly conserved activity-deficient variant class. Remarkably, despite differences in glycohydrolase activity, all versions of SPN were equally cytotoxic to cultured epithelial cells. These data indicate that the glycohydrolase activity of SPN may not be the only contribution the toxin has to the pathogenesis of S. pyogenes and both versions of SPN play an important role during infection.

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Succinylated octopamine ascarosides and a new pathway of biogenic amine metabolism in C. elegans.

Publication Date: 2013 May 20 PMID: 23689506
Authors: Artyukhin, A. B. - Yim, J. J. - Srinivasan, J. - Izrayelit, Y. - Bose, N. - von Reuss, S. H. - Jo, Y. - Jordan, J. M. - Baugh, L. R. - Cheong, M. - Sternberg, P. W. - Avery, L. - Schroeder, F. C.
Journal: J Biol Chem

The ascarosides, small-molecule signals derived from combinatorial assembly of primary metabolism-derived building blocks, play a central role in C. elegans biology and regulate many aspects of development and behavior in this model organism as well as in other nematodes. Using HPLC-MS/MS-based targeted metabolomics, we identified novel ascarosides incorporating a side-chain derived from succinylation of the neurotransmitter octopamine. These compounds, named osas#2, osas#9, and osas#10, are produced predominantly by L1 larvae, where they serve as part of a dispersal signal, whereas these ascarosides are largely absent from the metabolomes of other life stages. Investigating the biogenesis of these octopamine-derived ascarosides, we found that succinylation represents a previously unrecognized pathway of biogenic amine metabolism. At physiological concentrations, the neurotransmitters serotonin, dopamine, and octopamine are converted to a large extent into the corresponding succinates, in addition to the previously described acetates. Chemically, bimodal deactivation of biogenic amines via acetylation and succinylation parallels posttranslational modification of proteins via acetylation and succinylation of L-lysine. Our results reveal a small-molecule connection between neurotransmitter signaling and interorganismal regulation of behavior and suggest that ascaroside biosynthesis is based in part on co-option of degradative biochemical pathways.

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Identification and functional characterization of a Ku-binding motif in Aprataxin Polynucleotide kinase/phosphatase-Like Factor (APLF).

Publication Date: 2013 May 20 PMID: 23689425
Authors: Shirodkar, P. - Fenton, A. L. - Meng, L. - Koch, C. A.
Journal: J Biol Chem

APLF facilitates nonhomologous end joining (NHEJ) and associates with the core NHEJ components XRCC4/DNA ligase IV and Ku. The APLF forkhead-associated (FHA) domain directs interactions with XRCC4, but the APLF-Ku interaction has not been well-characterized. Here we describe an evolutionary conserved amino acid motif within APLF that is required for mediating the physical interaction between APLF and Ku. This APLF Ku-binding motif possesses similarity to regions identified in other NHEJ factors, WRN and XLF, which also direct interactions with Ku. Indeed, peptides derived from the Ku-binding regions of APLF, WRN or XLF were sufficient to reconstitute the interaction with Ku in vitro. Although APLF is predominantly localized to the nucleus, it does not possess a nuclear localization signal (NLS). Interestingly, the disruption of the APLF-Ku interaction by substituting key residues in the APLF Ku-binding motif was associated with increased relocalization of APLF to the cytoplasm and reduced association with XRCC4, that were rescued by the introduction of an NLS onto APLF. When human cells stably depleted of APLF were reconstituted with APLF Ku-binding mutants, or with an APLF FHA mutant that is known to disrupt interactions with XRCC4, APLF-dependent NHEJ and the retention of APLF at sites of laser generated DNA damage were impaired. These data suggest functional requirements for Ku and XRCC4 in APLF-dependent NHEJ, and a unique role for Ku as a factor required to facilitate the nuclear retention of APLF.

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Simultaneous Surface Display and Secretion of Proteins from Mammalian Cells Facilitates Efficient in vitro Selection and Maturation of Antibodies.

Publication Date: 2013 May 20 PMID: 23689374
Authors: Horlick, R. A. - Macomber, J. L. - Bowers, P. M. - Neben, T. Y. - Tomlinson, G. L. - Krapf, I. P. - Dalton, J. L. - Verdino, P. - King, D. J.
Journal: J Biol Chem

A mammalian expression system has been developed that permits simultaneous cell surface display and secretion of the same protein through alternate splicing of pre-mRNA. This enables a flexible system for in vitro protein evolution in mammalian cells where the displayed protein phenotype remains linked to genotype, but with the advantage of soluble protein also being produced without the requirement for any further recloning to allow a wide range of assays, including biophysical and cell-based functional assays, to be used during the selection process. This system has been used for the simultaneous surface presentation and secretion of IgG during antibody discovery and maturation. Presentation and secretion of monomeric Fab can also be achieved to minimize avidity effects. Manipulation of the splice donor site sequence enables control of the relative amounts of cell surface and secreted antibody. Multi-domain proteins may be presented and secreted in different formats to enable flexibility in experimental design, and secreted proteins may be produced with epitope tags to facilitate high-throughput testing. This system is particularly useful in the context of in situ mutagenesis, as in the case of in vitro somatic hypermutation.

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Comparison of predicted epimerases and reductases of the Campylobacter jejuni D-altro- and L-gluco- heptose synthesis pathways.

Publication Date: 2013 May 20 PMID: 23689373
Authors: McCallum, M. - Shaw, G. S. - Creuzenet, C.
Journal: J Biol Chem

Uniquely modified heptoses found in surface carbohydrates of bacterial pathogens are potential therapeutic targets against such pathogens. Our recent biochemical characterization of the GDP-6-deoxy-D-manno- and GDP-6-deoxy-D-altro-heptose biosynthesis pathways has provided the foundation for elucidation of the more complex L-gluco-heptose synthesis pathway of Campylobacter jejuni strain NCTC 11168. In this work, we use GDP-4-keto, 6-deoxy-D-lyxo-heptose as a surrogate substrate to characterize three enzymes predicted to be involved in this pathway: WcaGNCTC (also known as Cj1427), MlghB (Cj1430) and MlghC (Cj1428). We compare them with homologues involved in D-altro-heptose production: WcaG81176 (formerly WcaG), DdahB (Cjj1430) and DdahC (Cjj1427). We show that despite high levels of similarity, the enzymes have pathway-specific catalytic activities and substrate specificities. MlghB forms three products via C3 and C5 epimerisation activities while its DdahB homologue only had C3 epimerase activity along its cognate pathway. MlghC is specific for the double C3/C5 epimer generated by MlghB and produces L-gluco-heptose via stereospecific C4 reductase activity. In contrast, its homologue DdahC only uses the C3 epimer to yield D-altro-heptose via C4 reduction. Finally, we show that WcaGNCTC is not necessary for L-gluco-heptose synthesis and does not affect its production by MlghB and MlghC in contrast to its homologue WcaG81176 that has regulatory activity on D-altro-heptose synthesis. These studies expand our fundamental understanding of heptose modification, provide new glycobiology tools to synthesize novel heptose derivatives with biomedical applications, and provide a foundation for the structure function analysis of these enzymes.

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Dismantling promoter-driven RNA polymerase II transcription complexes in vitro by the termination factor Rat1.

Publication Date: 2013 May 20 PMID: 23689372
Authors: Pearson, E. L. - Moore, C. L.
Journal: J Biol Chem

Proper RNA Polymerase II (Pol II) transcription termination is essential to generate stable transcripts, to prevent interference at downstream loci, and to recycle Pol II back to the promoter (1-3). As such, termination is an intricately controlled process that is tightly regulated by a variety of different cis- and trans-acting factors (4, 5). Although many eukaryotic termination factors have been identified to date, the details of the precise molecular mechanisms governing termination remain to be elucidated. To tease apart these details, we have devised an in vitro transcription system to assay for specific Pol II termination. We show for the first time that the exonucleolytic Rat1/Rai1 complex can elicit release of stalled Pol II in vitro and can do so in the absence of other factors. We also find that Rtt103, which interacts with the Pol II C-terminal domain (CTD) and with Rat1, can rescue termination activity of an exonucleolytically deficient Rat1 mutant. In light of our findings, we posit a model whereby functional nucleolytic activity is not the feature of Rat1 that ultimately promotes termination. Degradation of the nascent transcript allows Rat1 to pursue Pol II in a guided fashion and to arrive at the site of RNA exit from Pol II. Upon this arrival, however, it is perhaps the specific and direct contact between Rat1 and Pol II that transmits the signal to terminate transcription.

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Structural Transformation of the Amyloidogenic Core Region of TAR DNA Binding Protein of 43 kDa (TDP-43) Initiates Its Aggregation and Cytoplasmic Inclusion.

Publication Date: 2013 May 20 PMID: 23689371
Authors: Jiang, L. L. - Che, M. X. - Zhao, J. - Zhou, C. J. - Xie, M. Y. - Li, H. Y. - He, J. H. - Hu, H. Y.
Journal: J Biol Chem

TAR DNA binding protein of 43 kDa (TDP-43) is a major deposited protein in amyotrophic lateral sclerosis and frontotemporal dementia with ubiquitin. A great number of genetic mutations identified in the flexible C-terminal part are associated with the disease pathologies. We investigated the molecular determinants of TDP-43 aggregation and its underlying mechanisms. A hydrophobic patch, ranging from residue 318 to 343, has been identified to be the amyloidogenic core essential for TDP-43 aggregation. Biophysical studies demonstrate that the homologous peptide forms a helix-turn-helix structure in solution, while it undergoes structural transformation from alpha-helix to beta-sheet during aggregation. Mutation or deletion of this core region significantly reduces the aggregation and cytoplasmic inclusions of full-length TDP-43 (or TDP-35 fragment) in cells. Thus, structural transformation of the amyloidogenic core initiates the aggregation and cytoplasmic inclusion formation of TDP-43. This particular core region provides a potential therapeutic target to design small-molecule compounds for mitigating TDP-43 proteinopathies.

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Single-cell analysis of RNA-mediated histone H3.3 recruitment to a cytomegalovirus promoter-regulated transcription site.

Publication Date: 2013 May 20 PMID: 23689370
Authors: Newhart, A. - Rafalska-Metcalf, I. U. - Yang, T. - Joo, L. M. - Powers, S. L. - Kossenkov, A. V. - Lopez-Jones, M. - Singer, R. H. - Showe, L. C. - Skordalakes, E. - Janicki, S. M.
Journal: J Biol Chem

Unlike the core histones, which are incorporated into nucleosomes concomitant with DNA replication, histone H3.3 is synthesized throughout the cell cycle and utilized for replication-independent (RI) chromatin assembly. The RI incorporation of H3.3 into nucleosomes is highly conserved and occurs at both euchromatin and heterochromatin. However, neither the mechanism of H3.3 recruitment nor its essential function is well understood. Several different chaperones regulate H3.3 assembly at distinct sites. The H3.3 chaperone, Daxx, and the chromatin-remodeling factor, ATRX, are required for H3.3 incorporation and heterochromatic silencing at telomeres, pericentromeres, and the cytomegalovirus (CMV) promoter. By evaluating H3.3 dynamics at a CMV-promoter-regulated transcription site in a genetic background in which RI chromatin assembly is blocked, we have been able to decipher the regulatory events upstream of RI nucleosomal deposition. We find that at the activated transcription site, H3.3 accumulates with sense and antisense RNA suggesting that it is recruited through an RNA-mediated mechanism. Sense and antisense transcription also increases after H3.3 knockdown suggesting that the RNA signal is amplified when chromatin assembly is blocked and attenuated by nucleosomal deposition. Additionally, we find that H3.3 is still recruited after Daxx knockdown supporting a chaperone-independent recruitment mechanism. Sequences in the H3.3 N-terminal tail and alphaN helix mediate both its recruitment to RNA at the activated transcription site and its interaction with double-stranded RNA in vitro. Interestingly, the H3.3 gain-of-function pediatric glioblastoma mutations, G34R and K27M, differentially affect H3.3 affinity in these assays suggesting that disruption of an RNA-mediated regulatory event could drive malignant transformation.

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The lectin domain of the polypeptide GalNAc transferase family of glycosyltransferases (ppGalNAc T's) acts as a switch directing glycopeptide substrate glycosylation in an N- or C- Direction, further controlling mucin-type O-glycosylation.

Publication Date: 2013 May 20 PMID: 23689369
Authors: Gerken, T. A. - Revoredo, L. - Thome, J. J. - Tabak, L. A. - Vester-Christensen, M. B. - Clausen, H. - Gahlay, G. K. - Jarvis, D. L. - Johnson, R. W. - Moniz, H. A. - Moremen, K.
Journal: J Biol Chem

Mucin type O-glycosylation is initiated by a large family of polypeptide GalNAc transferases (ppGalNAc T's) that add alpha-GalNAc to the Ser and Thr residues of peptides. Of the 20 human isoforms all but 1 are comprised of 2 globular domains linked by a short flexible linker: a catalytic domain and a ricin-like lectin carbohydrate binding domain. Presently the roles of the catalytic and lectin domains in peptide and glycopeptide recognition and specificity remain unclear. To systematically study the role of the lectin domain on ppGalNAc T glycopeptide substrate utilization we have developed a series of novel random glycopeptide substrates containing a single GalNAc-O-Thr residue placed near either the N- or C- terminus of the glycopeptide substrate. Our results reveal that the presence and amino or carboxyl terminal placement of the GalNAc-O-Thr can be important determinants of overall catalytic activity and specificity which differ between transferase isoforms. For example ppGalNAc T1, T2 and T14 perfer C-terminal placed GalNAc-O-Thr while ppGalNAc T3 and T6 perfer N-terminal placed GalNAc-O-Thr. Several transferase isoforms, ppGalNAc T5, T13 and T16, display equally enhanced N- or C-terminal activities relative to the nonglycosylated control peptides. This N- and/or C- terminal selectivity is presumably due to weak glycopeptide binding to the lectin domain whose orientation relative to the catalytic domain is dynamic and isoform dependent. Such N- or C-terminal glycopeptide selectivity provides an additional level of control or fidelity for the O-glycosylation of biologically significant sites and suggests that O-glycosylation may in some instances be exquisitely controlled.

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CCN3 participates in bone regeneration as an inhibitory factor.

Publication Date: 2013 May 21 PMID: 23653360
Authors: Matsushita, Y. - Sakamoto, K. - Tamamura, Y. - Shibata, Y. - Minamizato, T. - Kihara, T. - Ito, M. - Katsube, K. I. - Hiraoka, S. - Koseki, H. - Harada, K. - Yamaguchi, A.
Journal: J Biol Chem

CCN3, a member of the CCN protein family, inhibits osteoblast differentiation in vitro. However, the role of CCN3 in bone regeneration has not been well elucidated. In this study, we investigated the role of CCN3 in bone regeneration. We identified Ccn3 gene by microarray analysis as a highly expressed gene at the early phase of bone regeneration in a mouse bone regeneration model. We confirmed the upregulation of Ccn3 at the early phase of bone regeneration by RT-PCR, western blot, and immunofluorescence analyses. Ccn3 transgenic mice, in which Ccn3 expression was driven by 2.3-kb Col1a1 promoter, showed osteopenia compared with wild-type mice, but Ccn3 knockout mice showed no skeletal changes compared with wild-type mice. We analyzed bone regeneration process in Ccn3 transgenic mice and Ccn3 knockout mice by microcomputed tomography and histological analyses. Bone regeneration in Ccn3 knockout mice was accelerated compared with that in wild-type mice. The mRNA expression levels of osteoblast-related genes (Runx2, Sp7, Col1a1, Alpl, Bglap) in Ccn3 knockout mice were upregulated earlier than those in wild-type mice, as demonstrated by RT-PCR. Bone regeneration in Ccn3 transgenic mice showed no significant changes compared with that in wild-type mice. Phosphorylation of Smad1/5 was highly upregulated at bone regeneration sites in Ccn3 KO mice compared with wild-type mice. These results indicate that CCN3 is upregulated in the early phase of bone regeneration, and acts as a negative regulator for bone regeneration. This study may contribute to the development of new strategies for bone regeneration therapy.

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A mechanism for protein monoubiquitination dependent on a trans-acting ubiquitin binding domain.

Publication Date: 2013 May 3 PMID: 23645667
Authors: Herrador, A. - Leon, S. - Haguenauer-Tsapis, R. - Vincent, O.
Journal: J Biol Chem

The length of the ubiquitin chain on a substrate dictates various functional outcomes, yet little is known about its regulation in vivo. The yeast arrestin-related protein Rim8/Art9 is monoubiquitinated in vivo by the Rsp5 ubiquitin ligase. This also requires Vps23, a protein that displays an ubiquitin-E2 variant (UEV) domain. Here, we report that binding of the UEV domain to Rim8 interferes with ubiquitin chain elongation and directs Rim8 monoubiquitination. We propose that Vps23 UEV competes with Rsp5 HECT N-lobe for binding to the first conjugated ubiquitin, thereby preventing polyubiquitination. These findings reveal a novel mechanism to control ubiquitin chain length on substrates in vivo.

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The Interaction of Caveolin 3 with the Inward Rectifier Channel Kir2.1; Physiology and Pathology related to LQT9.

Publication Date: 2013 May 29 PMID: 23640888
Authors: Vaidyanathan, R. - Vega, A. L. - Song, C. - Zhou, Q. - Tan, B. H. - Berger, S. - Makielski, J. C. - Eckhardt, L. L.
Journal: J Biol Chem

Background: Mutations in CAV3 cause LQT Syndrome-9 (LQT9). A previously reported LQT9 patient had prominent U-waves on ECG, a feature correlated with Kir2.1 loss of function. Our objective was to determine if Cav3 associates with Kir2.1 and if CAV3 mutations LQT9 associated affect Kir2.1 biophysical properties. Methods and Results: Kir2.1 current (IK1) density was measured using the whole-cell voltage clamp technique. WT-Cav3 did not affect IK1, however F97C-Cav3 and T78M-Cav3 significantly decreased IK1 density by ~60%, and P104L-Cav3 significantly decreased IK1 density by ~30%. Rat heart cryosections and HEK293 cells co-transfected with Kir2.1 and WT-Cav3 were immunostained and both demonstrated co-localization of Kir2.1 and WT-Cav3 by confocal imaging. Cav3 co-immunoprecipitated with Kir2.1 both in human ventricular myocytes and in heterologous expression systems. Additionally, FRET efficacy was highly specific, with a molecular distance of 5.6 +/- 0.4 nm, indicating close protein location. Co-localization experiments found that Cav3 and Kir2.1 accumulated in the Golgi compartment. On-cell Western showed a ~60% and 20% decrease in Kir2.1 cell-surface expression when expressed with F97C-Cav3 and P104L-Cav3 vs. WT-Cav3 respectively. Conclusions: This is the first report of an association between Cav3 and Kir2.1. Cav3 mutations F97C-Cav3, P104L-Cav3 and T78M-Cav3 induced a decrease in IK1 density. This effect was related to a decrease in cell-surface expression of Kir2.1. Our finding of Kir2.1 loss-of-function in association with these Cav3 mutations may combine with previously reported increase in late INa, to result in prolonged repolarization and the arrhythmia generation in Cav3-mediated LQTS9.

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Matrix metalloproteinase-8 (collagenase-2) induces the expression of interleukins-6 and -8 in breast cancer cells.

Publication Date: 2013 Apr 30 PMID: 23632023
Authors: Thirkettle, S. - Decock, J. - Arnold, H. - Pennington, C. J. - Jaworski, D. M. - Edwards, D. R.
Journal: J Biol Chem

Matrix metalloproteinase-8 (MMP-8) is a tumor suppressive protease that cleaves numerous substrates including matrix proteins and chemokines. In particular MMP-8 proteolytically activates interleukin-8 (IL-8) and thereby regulates neutrophil chemotaxis in vivo. We have explored the effects of expression of either a wild-type (wt) or catalytically inactive (E198A) mutant version of MMP-8 in human breast cancer cell lines. Analysis of serum-free conditioned media from three breast cancer cell lines (MCF-7, SK-BR-3 and MDA-MB-231) expressing wt MMP-8 revealed elevated levels of interleukin-6 (IL-6) and IL-8. This increase was mirrored at the mRNA level, and was dependent on MMP-8 catalytic activity. However, sustained expression of wt MMP-8 by breast cancer cells was non-permissive for long-term growth, as shown by reduced colony formation compared to cells expressing either control vector or E198A mutant MMP-8. In long term culture of transfected MDA-MB-231 cells expression of wt but not E198A mutant MMP8 was lost, with IL-6 and IL-8 levels returning to baseline. Rare clonal isolates of MDA-MB-231 cells expressing wt MMP-8 were generated and these showed constitutively high levels of IL-6 and IL-8, though production of the interleukins was no longer dependent upon MMP-8 activity. These studies support a causal connection between MMP-8 activity and the IL-6/IL-8 network, with an acute response to MMP-8 involving induction of the pro-inflammatory mediators, which may in part serve to compensate for the deleterious effects of MMP-8 on breast cancer cell growth. This axis may be relevant to the recognised ability of MMP-8 to orchestrate the innate immune system in inflammation in vivo.

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Investigations of the contribution of a putative glycine hinge to ryanodine receptor channel gating.

Publication Date: 2013 Apr 30 PMID: 23632022
Authors: Euden, J. - Mason, S. A. - Viero, C. - Thomas, N. L. - Williams, A. J.
Journal: J Biol Chem

Ryanodine receptor channels (RyR) are key components of striated muscle excitation-contraction coupling and alterations in their function underlie both inherited and acquired disease. A full understanding of the disease process will require a detailed knowledge of the mechanisms and structures involved in RyR function. Unfortunately high-resolution structural data, such as exists for K+-selective channels, is not available for RyR. In its absence we have used modelling to identify similarities in the structural elements of K+ channel pore-forming regions (PFR) and postulated equivalent regions of RyR. This has identified a sequence of residues in the cytosolic cavity-lining trans-membrane helix of RyR (G4864LIIDA4869 in RyR2) analogous to the glycine hinge motif present in many K+ channels. Gating in these K+ channels can be disrupted by substitution of residues for the hinge glycine. We investigated the involvement of glycine4864 in RyR2 gating by monitoring properties of recombinant human RyR2 channels in which this glycine is replaced by residues that alter gating in K+ channels. Our data demonstrate that introducing alanine at position 4864 produces no significant change in RyR2 function. In contrast function is altered when glycine4864 is replaced by either valine or proline. The former preventing channel opening, the latter modifying both ion translocation and gating. Our studies reveal novel information on the structural basis of RyR gating, identifying both similarities with, and differences from, K+ channels. Glycine4864 is not absolutely required for channel gating but some flexibility at this point in the cavity-lining trans-membrane helix is necessary for normal RyR function.

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Rapid Accumulation of Endogenous Tau Oligomers in a Rat Model of Traumatic Brain Injury: Possible Link Between TBI and Sporadic Tauopathies.

Publication Date: 2013 Apr 30 PMID: 23632019
Authors: Hawkins, B. E. - Krishnamurthy, S. - Castillo-Carranza, D. L. - Sengupta, U. - Prough, D. S. - Jackson, G. R. - Dewitt, D. S. - Kayed, R.
Journal: J Biol Chem

Traumatic brain injury (TBI) is a serious problem affecting millions of people in the United States alone. Multiple concussions or even a single moderate-severe TBI can also predispose individuals to develop a pathologically distinct form of tauopathy-related dementia at an early age. No effective treatments are currently available for TBI or TBI-related dementia; moreover, only recently has insight been gained regarding the mechanisms behind their connection. Here, we used antibodies to detect oligomeric and phosphorylated tau proteins in a non-transgenic rodent model of parasagittal fluid percussion injury. Oligomeric and phosphorylated tau proteins were detected 4 and 24 hours and 2 weeks post-TBI in injured, but not sham control rats. These findings suggest that diagnostic tools and therapeutics that target only toxic forms of tau may provide earlier detection and safe, more effective treatments for tauopathies associated with repetitive neurotrauma.

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Large terminase conformational change induced by connector binding in bacteriophage T7.

Publication Date: 2013 Apr 30 PMID: 23632014
Authors: Dauden, M. I. - Martin-Benito, J. - Sanchez-Ferrero, J. C. - Pulido-Cid, M. - Valpuesta, J. M. - Carrascosa, J. L.
Journal: J Biol Chem

During bacteriophage morphogenesis DNA is translocated into a preformed prohead by the complex formed by the portal protein, or connector, plus the terminase, which are located at an especial prohead vertex. The terminase is a powerful motor that converts ATP hydrolysis into mechanical movement of the DNA. Here, we have determined the structure of the T7 large terminase by electron microscopy. The five terminase subunits assemble in a toroid which encloses a channel wide enough to accommodate dsDNA. The structure of the complete connector/terminase complex is also reported, revealing the coupling between the terminase and the connector forming a continuous channel. The structure of the terminase assembled into the complex showed a different conformation when compared to the isolated terminase pentamer. To understand in molecular terms the terminase morphological change we generated the terminase atomic model based on the crystallographic structure of its phage T4 counterpart. The docking of the threaded model in both terminase conformations showed that the transition between the two states can be achieved by rigid body subunit rotation in the pentameric assembly. The existence of two terminase conformations and its possible relation to the sequential DNA translocation may shed light into the molecular bases of the packaging mechanism of bacteriophage T7.

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Myocilin stimulates osteogenic differentiation of mesenchymal stem cells through MAPK signaling.

Publication Date: 2013 Apr 29 PMID: 23629661
Authors: Kwon, H. S. - Johnson, T. V. - Tomarev, S. I.
Journal: J Biol Chem

Myocilin is a secreted glycoprotein that is expressed in ocular and non-ocular tissues. Mutations in the MYOCILIN gene may lead to juvenile and adult onset primary open-angle glaucoma. Here we report that myocilin is expressed in bone marrow-derived mesenchymal stem cells (MSCs) and plays a role in their differentiation into osteoblasts in vitro and in osteogenesis in vivo. Expression of myocilin was detected in MSCs derived from mouse, rat and human bone marrow with human MSCs exhibiting the highest level of myocilin expression. Expression of myocilin rose during the course of human MSC differentiation into osteoblasts but not into adipocytes, and treatment with exogenous myocilin further enhanced osteogenesis. MSCs derived from Myoc null mice had a reduced ability to differentiate into the osteoblastic lineage, which was partially rescued by exogenous extracellular myocilin treatment. Myocilin also stimulated osteogenic differentiation of wild type MSCs, which was associated with activation of the p38, Erk1/2, and JNK MAP kinase signaling pathways as well as upregulated expression of the osteogenic transcription factors Runx2 and Dlx5. Finally, cortical bone thickness and trabecular volume, as well as the expression level of osteopontin, a known factor of bone remodeling and osteoblast differentiation, were dramatically reduced in the femurs of Myoc null mice compared with wild-type mice. These data suggest that myocilin should be considered as a target for improving the bone regenerative potential of MSCs, and may identify a new role for myocilin in bone formation and/or maintenance in vivo.

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Nuclear Corepressors Mediate the Repression of Phospholipase A2 Group IIa Gene Transcription by Thyroid Hormone.

Publication Date: 2013 Apr 29 PMID: 23629656
Authors: Sharma, P. - Thakran, S. - Deng, X. - Elam, M. B. - Park, E. A.
Journal: J Biol Chem

Secretory phospholipase A2 group IIa (PLA2g2a) is associated with inflammation, hyperlipidemia and atherogenesis. Transcription of the PLA2g2a gene is induced by multiple cytokines. Here, we report the surprising observation that thyroid hormone (T3) inhibited PLA2g2a gene expression in human and rat hepatocytes as well as in rat liver. Moreover, T3 reduced the cytokine mediated induction of PLA2g2a suggesting that the thyroid status may modulate aspects of the inflammatory response. In an effort to dissect the mechanism of repression by T3, we cloned the PLA2g2a gene and identified a negative T3 response element in the promoter. This T3 receptor (TRbeta) binding site differed considerably from consensus T3 stimulatory elements. Using in vitro and in vivo binding assays, we found that TRbeta bound directly to the PLA2g2a promoter as a heterodimer with the retinoid X receptor. Knockdown of nuclear corepressor (NCoR1) or silencing mediator for retinoid and thyroid receptors (SMRT) by siRNA blocked the T3 inhibition of PLA2g2a. Using chromatin immunoprecipitation assays, we showed that NCoR1 and SMRT were associated with the PLA2g2a gene in the presence of T3. In contrast with the established role of T3 to promote coactivator association with TRbeta, our experiments demonstrate a novel inverse recruitment mechanism in which liganded TRbeta recruits corepressors to inhibit PLA2g2a expression.

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Cystatin C properties crucial for uptake and inhibition of intracellular target enzymes.

Publication Date: 2013 Apr 29 PMID: 23629651
Authors: Wallin, H. - Abrahamson, M. - Ekstrom, U.
Journal: J Biol Chem

To elucidate the molecular requirements for cancer cell internalization of the extracellular cysteine protease inhibitor cystatin C, 12 variants of the protein were produced and used for uptake experiments in MCF-7 cells. Variants with alterations in the cysteine cathepsin binding region ((Delta1-10)-, K5A-, R8G-, (R8G,L9G,V10G)-, (R8G,L9G,V10G, W106G)-, and W106G-cystatin C) were internalized to a very low extent compared to the wild-type inhibitor. Substitutions of N39 in the legumain binding region (N39K- and N39A-cystatin C) decreased the internalization and (R24A,R25A)-cystatin C, with substitutions of charged residues not involved in enzyme inhibition, was not taken up at all. Two variants, W106F- and K75A-cystatin C, showed that the internalization can be positively affected by engineering of the cystatin molecule. Microscopy revealed vesicular co-localization of internalized cystatin C with the lysosomal marker proteins cathepsin D and legumain. Activities of both cysteine cathepsins and legumain, possible target enzymes associated with cancer cell invasion and metastasis, were down-regulated in cell homogenates following cystatin C uptake. A positive effect on regulation of intracellular enzyme activity by a cystatin variant selected from uptake properties was illustrated by incubating cells with W106F-cystatin C. This resulted in more efficient down-regulation of intracellular legumain activity than when cells were incubated with wild-type cystatin C. Uptake experiments in prostate cancer cells corroborated that the cystatin C internalization is generally relevant and confirmed an increased uptake of W106F-cystatin C, in PC3 cells. Thus, intracellular cysteine proteases involved in cancer-promoting processes might be controled by cystatin uptake.

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Stabilization, Characterization and Selective Removal of Cystatin C Amyloid Oligomers.

Publication Date: 2013 Apr 29 PMID: 23629649
Authors: Ranheimer Ostner, G. - Lindstrom, V. - Hjort Christensen, P. - Kozak, M. - Abrahamson, M. - Grubb, A.
Journal: J Biol Chem

The pathophysiological process in amyloid disorders usually involves the transformation of a functional monomeric protein via potentially toxic oligomers into amyloid fibrils. The structure and properties of the intermediary oligomers have been difficult to study due to their instability and dynamic equilibrium with smaller and larger species. In hereditary cystatin C amyloid angiopathy, a cystatin C variant is deposited in arterial walls and cause brain hemorrhage in young adults. In the present investigation, we use redox experiments of monomeric cystatin C, stabilized against domain swapping by an intramolecular disulfide bond, to generate stable oligomers (dimers, trimers, tetramers, decamers and high molecular weight oligomers). These oligomers were characterized concerning size by gel filtration, polyacrylamide gel electrophoresis and mass spectrometry, concerning shape by electron and atomic force microscopy, and, concerning function, by assays of their capacity to inhibit proteases. The results showed the oligomers to be highly ordered, domainswapped assemblies of cystatin C and that the oligomers could not build larger oligomers, or fibrils, without domain swapping. The stabilized oligomers were used to induce antibody formation in rabbits. After immunosorption, using immobilized monomeric cystatin C, and elution from columns with immobilized cystatin C oligomers, oligomer-specific antibodies were obtained. These could be used to selectively remove cystatin C dimers from biological fluids containing both dimers and monomers.

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WDR26 functions as a scaffolding protein to promote Gbetagamma-mediated PLCbeta2 activation in leukocytes.

Publication Date: 2013 Apr 26 PMID: 23625927
Authors: Sun, Z. - Smrcka, A. V. - Chen, S.
Journal: J Biol Chem

We have recently identified WDR26 as a novel WD40 repeat protein that binds Gbetagamma and promotes Gbetagamma signaling during leukocyte migration. Here, we have determined the mechanism by which WDR26 enhances Gbetagamma-mediated phospholipase C beta2 (PLCbeta2) activation. We show that WDR26 not only directly bound Gbetagamma but also PLCbetagamma2. The binding sites of WDR26 and PLCbeta2 on Gbeta1gamma2 were overlapping, but not identical. WDR26 used the same domains for binding Gbetagamma and PLCbeta2, but still formed a signaling complex with Gbetagamma and PLCbeta2, probably due to the fact that WDR26 formed a high-ordered oligomer through its LisH-CTLH and WD40 domains. Additional studies indicated that the formation of high-ordered oligomers was required for WDR26 to promote PLCbeta2 interaction with and activation by Gbetagamma. Moreover, WDR26 was required for PLCbeta2 translocation from the cytosol to the membrane in polarized leukocytes, and the translocation of PLCbeta2 was sufficient to cause partial activation of PLCbeta2. Collectively, our data indicate that WDR26 functions as a scaffolding protein to promote PLCbeta2 membrane translocation and interaction with Gbetagamma, thereby enhancing PLCbeta2 activation in leukocytes. These findings have identified a novel mechanism of regulating Gbetagamma signaling through a scaffolding protein.

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Reduced immune-suppressive properties of axitinib in comparison to other tyrosine kinase inhibitors.

Publication Date: 2013 Apr 26 PMID: 23625925
Authors: Stehle, F. - Schulz, K. - Fahldieck, C. - Kalich, J. - Lichtenfels, R. - Riemann, D. - Seliger, B.
Journal: J Biol Chem

The multikinase inhibitors sunitinib, sorafenib and axitinib do not only have an impact on tumor growth and angiogenesis, but also on the activity and function of immune effector cells. In this study a comparative analysis of the growth inhibitory properties and apoptosis induction potentials of tyrosine kinase inhibitors on T cells was performed. TKI treatment resulted in a dramatic decrease in T cell proliferation along with distinct impacts on the cell cycle progression. This was at least partially associated with an enhanced induction of apoptosis, although triggered by distinct apoptotic mechanisms. In contrast to sunitinib and sorafenib, axitinib did not affect the mitochondrial membrane potential (Deltapsim), but resulted in an induction or stabilization of the induced myeloid leukemia cell differentiation protein (Mcl 1) leading to an irreversible arrest in the G2/M cell cycle phase and delayed apoptosis. Furthermore, the sorafenib-mediated suppression of immune effector cells, in particular the reduction of the CD8+ T cell subset along with the down-regulation of key immune cell markers such as CCR7, CD26, CD69, CD25 and CXCR3 was not observed in axitinib-treated immune effector cells. Therefore, rather axitinib than sorafenib seems to be suitable for implementation in complex treatment regimens of cancer patients including immunotherapy.

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IgG1 Thioether Bond Formation in Vivo.

Publication Date: 2013 Apr 25 PMID: 23625924
Authors: Zhang, Q. - Schenauer, M. R. - McCarter, J. D. - Flynn, G. C.
Journal: J Biol Chem

During either production or storage, the LC214-HC220 disulfide in therapeutic antibodies can convert to a thioether bond. Here we report that a thioether forms at the same position on antibodies in vivo. An IgG1kappa therapeutic antibody dosed in humans formed a thioether at this position at a rate of about 0.1%/day while circulating in blood. Thioether modifications were also found at this position in endogenous antibodies isolated from healthy human subjects, at levels consistent with this conversion rate. For both endogenous antibodies and recombinant antibodies studied in vivo, thioether conversion rates were faster for IgG1 antibodies containing lambda light chains than those containing kappa light chains. These light chain reaction rate differences were replicated in vitro. Additional mechanistic studies showed that base-catalyzed thioether formation through the light chain dehydrogenation was more preferred on antibodies with lambda light chains, which may help explain the observed reaction rate differences.

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Tiered Assembly of the Yeast Far3-7-8-9-10-11 Complex at the Endoplasmic Reticulum.

Publication Date: 2013 Apr 26 PMID: 23625923
Authors: Pracheil, T. - Liu, Z.
Journal: J Biol Chem

TOR (target of rapamycin) signaling is a conserved, essential pathway integrating nutritional cues with cell growth and proliferation. The TOR kinase exists in two distinct complexes, TORC1 and TORC2. It has been reported that protein phosphatase 2A (PP2A) and the Far3-7-8-9-10-11 complex (Far complex) negatively regulate TORC2 signaling in yeast. The Far complex, originally identified as factors required for pheromone-induced cell cycle arrest, and PP2A form the yeast counterpart of the STRIPAK complex, which was first isolated in mammals. The cellular localization of the Far complex has yet to be fully characterized. Here we show that the Far complex localizes to the endoplasmic reticulum (ER) by analyzing functional, GFP-tagged Far proteins in vivo. We found that Far9 and Far10, two homologous proteins each with a tail-anchor domain, localize to the ER in mutant cells lacking the other Far complex components. Far3, Far7, and Far8 form a subcomplex, which is recruited to the ER by Far9/10. The Far3-7-8 complex in turn recruits Far11 to the ER. Finally, we show that the tail-anchor domain of Far9 is required for its optimal function in TORC2 signaling. Our study reveals tiered assembly of the yeast Far complex at the ER and a function for Far complex's ER localization in TORC2 signaling.

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MicroRNA-33a Mediates the Regulation of High Mobility Group AT-Hook 2 Gene (HMGA2) by Thyroid Transcription Factor 1 (TTF-1/NKX2-1).

Publication Date: 2013 Apr 26 PMID: 23625920
Authors: Rice, S. J. - Lai, S. C. - Wood, L. W. - Helsley, K. R. - Runkle, E. A. - Winslow, M. M. - Mu, D.
Journal: J Biol Chem

In lung cancers, TTF-1 displays seemingly paradoxical activities. While TTF-1 is amplified in primary human lung cancers, it inhibits primary lung tumors from metastasizing in a mouse model system. It was reported that the oncogenic pro-epithelial mesenchymal transition (EMT) high-mobility group AT-hook 2 gene (HMGA2) mediates the anti-metastatic function of TTF-1. To gain mechanistic insight into the metastasis critical signaling axis of TTF-1 to HMGA2, we used both reverse and forward strategies and discovered that microRNA-33a (miR-33a) is under direct positive regulation of TTF-1. By chromatin immunoprecipitation, we determined that TTF-1 binds to the promoter of SREBF2, the host gene of miR-33a. The 3'untranslated region (UTR) of HMGA2 contains three predicted binding sites of miR-33a. We showed that the first two highly conserved sites are conducive to HMGA2 repression by miR-33a, establishing HMGA2 as a genuine target of miR-33a. Functional studies revealed that enforced expression of miR-33a inhibits the motility of lung cancer cells and this inhibition can be rescued by overexpression of the form of HMGA2 without 3'UTR, suggesting that TTF-1 keeps the pro-metastasis gene HMGA2 in check via upregulating miR-33a. This study reports the first miRNAs directly regulated by TTF-1 and clarifies how TTF-1 controls HMGA2 expression. Moreover, the documented importance of SREBF2 and miR-33a in regulating cholesterol metabolism suggests that TTF-1 may be a modulator of cholesterol homeostasis in the lung. Future studies will be dedicated to understanding how miRNAs influence the oncogenic activity of TTF-1 and the role of TTF-1 in cholesterol metabolism.

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Role of Phosphatidylethanolamine in the Biogenesis of Mitochondrial Outer Membrane Proteins.

Publication Date: 2013 Apr 26 PMID: 23625917
Authors: Becker, T. - Horvath, S. E. - Bottinger, L. - Gebert, N. - Daum, G. - Pfanner, N.
Journal: J Biol Chem

The mitochondrial outer membrane contains proteinaceous machineries for the import and assembly of proteins, including the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It has been shown that the dimeric phospholipid cardiolipin (CL) is required for the stability of TOM and SAM complexes and thus for the efficient import and assembly of beta-barrel proteins and some alpha-helical proteins of the outer membrane. Here we report that mitochondria deficient in phosphatidylethanolamine (PE), the second non-bilayer forming phospholipid, are impaired in the biogenesis of beta-barrel proteins, but not of alpha-helical outer membrane proteins. The stability of TOM and SAM complexes is not disturbed by the lack of PE. By dissecting the import steps of beta-barrel proteins, we show that an early import stage involving translocation through the TOM complex is affected. In PE-depleted mitochondria, the TOM complex binds precursor proteins with reduced efficiency. We conclude that PE is required for the proper function of the TOM complex.

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Store-operated Ca2+ Entry (SOCE) Induced by Protease-activated Receptor-1 mediates STIM1 Phosphorylation to inhibit SOCE in Endothelial Cells through AMP-activated Protein Kinase and p38beta Mitogen-activated Protein Kinase.

Publication Date: 2013 Apr 26 PMID: 23625915
Authors: Sundivakkam, P. C. - Natarajan, V. - Malik, A. B. - Tiruppathi, C.
Journal: J Biol Chem

The Ca2+ sensor STIM1 is crucial for activation of store-operated Ca2+ entry (SOCE) through TRPC and Orai channels. STIM1 phosphorylation serves as an off-switch for SOCE. However, the signaling pathway for STIM1 phosphorylation is unknown. Here we show that SOCE activates AMP-activated protein kinase (AMPK); its effector p38beta mitogen-activated protein kinase (p38beta MAPK) phosphorylates STIM1, thus inhibiting SOCE in human lung microvascular endothelial cells (ECs). Activation of AMPK using AICAR resulted in STIM1 phosphorylation on serine residues and prevented PAR-1-induced Ca2+ entry. Further, AICAR pretreatment blocked PAR-1-induced increase in permeability of mouse-lung microvessels. Activation of SOCE with thrombin caused phosphorylation of isoform alpha1, but not alpha2 of the AMPK catalytic subunit. Moreover, knockdown of AMPKalpha1 augmented SOCE induced by thrombin. Interestingly, SB203580, a selective inhibitor of p38 MAPK, blocked STIM1 phosphorylation and led to sustained STIM1-puncta formation and Ca2+ entry. Of the three p38 MAPK isoforms expressed in ECs, p38beta knockdown prevented PAR-1-mediated STIM1 phosphorylation and potentiated SOCE. In addition, inhibition of the SOCE downstream target CaMKKbeta or knockdown of AMPKalpha1 suppressed PAR-1-mediated phosphorylation of p38beta and hence STIM1. Thus, our findings demonstrate that SOCE activates CaMKKbeta-AMPKalpha1-p38beta MAPK signaling to phosphorylate STIM1, thereby suppressing endothelial SOCE and permeability responses.

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Substrate-Modulated Cytochrome P450 17A1 and Cytochrome b5 Interactions Revealed by NMR.

Publication Date: 2013 Apr 25 PMID: 23620596
Authors: Estrada, D. F. - Laurence, J. S. - Scott, E. E.
Journal: J Biol Chem

The membrane heme protein cytochrome b5 (b5) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Herein the interactions between the soluble domain of microsomal b5 and the catalytic domain of the bi-functional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b5, and an androgen-forming lyase reaction which is facilitated 10-fold by b5. NMR chemical shift mapping of b5 titrations with CYP17A1 indicate that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b5 residues (E48 or E49) or the corresponding cationic CYP17A1 residues (R347, R358, or R449). Cytochrome b5 binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase (CPR). To probe the differential effects of b5 on the two CYP17A1-mediated reactions and thus communication between the superficial b5 binding site and the buried CYP17A1 active site, CYP17A1/b5 complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17alpha-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b5 binding site.

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HMGN Proteins Modulate The Fidelity Of The Cellular Transcriptional Profile In A Tissue And Variant Specific Manner.

Publication Date: 2013 Apr 24 PMID: 23620591
Authors: Kugler, J. E. - Horsch, M. - Huang, D. - Furusawa, T. - Rochman, M. - Garrett, L. - Becker, L. - Bohla, A. - Holter, S. M. - Prehn, C. - Rathkolb, B. - Racz, I. - Aguilar-Pimentel, J. A. - Adler, T. - Adamski, J. - Beckers, J. - Busch, D. H. - Eickelberg, O. - Klopstock, T. - Ollert, M. - Stoeger, T. - Wolf, E. - Wurst, W. - Yildirim, A. O. - Zimmer, A. - Gailus-Durner, V. - Fuchs, H. - Hrabe de Angelis, M. - Garfinkel, B. - Orly, J. - Ovcherenko, I. - Bustin, M.
Journal: J Biol Chem

The nuclei of most vertebrate cells contain members of the high mobility group N (HMGN) protein family which bind specifically to nucleosome core particles and affect the structure and function of chromatin, including transcription. Here we study the biological role of this protein family by systematic analysis of phenotypes and tissue transcription profiles in mice lacking functional HMGN variants. Phenotypic analysis of Hmgn1-/-, Hmgn3-/-, and Hmgn5-/- mice and their wild type littermates with a battery of standardized tests uncovered variant-specific abnormalities. Gene expression analysis of four different tissues in each of the Hmgn-/- lines reveals very little overlap between the genes affected by the specific variants in the different tissues. Pathway analysis revels that loss of an HMGN variant affects subtly the expression of numerous genes in specific biological processes. We conclude that within the biological framework of an entire organism, HMGNs modulate the fidelity of the cellular transcriptional profile in a tissue- and HMGN variant-specific manner.

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Structural insights into histone demethylase NO66 in interaction with osteoblast specific transcription factor Osterix and gene repression.

Publication Date: 2013 Apr 24 PMID: 23620590
Authors: Tao, Y. - Wu, M. - Zhou, X. - Yin, W. - Hu, B. - de Crombrugghe, B. - Sinha, K. M. - Zang, J.
Journal: J Biol Chem

Osterix (Osx) is an osteoblast-specific transcriptional factor and is required for osteoblast differentiation and bone formation. A JmjC domain-containing protein NO66 was previously found to participate in regulation of Osx transcriptional activity and plays an important role in osteoblast differentiation through interaction with Osx. Here we report the crystal structure of NO66 forming in a functional tetramer. A hinge domain links the N-terminal JmjC domain and C-terminal wHTH domain of NO66, and both domains are essential for tetrameric assembly. The oligomerization interface of NO66 interacts with a conserved fragment of Osx. We show that the hinge domain-dependent oligomerization of NO66 is essential for inhibition of Osx-dependent gene activation. Our findings suggest that homo-oligomerization of JmjC domain containing proteins might play a physiological role through interactions with other regulatory factors during gene expression.

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Two distinct modes of exocytotic fusion pore expansion in large astrocytic vesicles.

Publication Date: 2013 Apr 25 PMID: 23620588
Authors: Peng, H. - Kang, N. - Xu, J. - Stanton, P. K. - Kang, J.
Journal: J Biol Chem

Formation of the fusion pore is a central question for regulated exocytosis by which secretory cells release neurotransmitters or hormones. Here, by dynamically monitoring exocytosis of large vesicles (~3 mum) in astrocytes with two-photon microscopy imaging, we found that the exocytotic fusion pore was generated from the SNARE-dependent fusion at a ring shape of the docked plasma-vesicular membrane and movement of a fusion-produced membrane fragment. We observed two modes of fragment movements, 1) a shift fragment that shifted to expand the fusion pore and 2) a fall-in fragment that fell into the collapsed vesicle to expand the fusion pore. The shift and fall-in modes are associated with full and partial collapses of large vesicles, respectively. The astrocytic marker, sulforhodamine 101 (SR101), stained the fusion-produced membrane fragment more brightly than FM 1-43. SR101 imaging showed that double fusion pores could simultaneously occur in a single vesicle (16% of large vesicles) to accelerate discharge of vesicular contents. Electron microscopy of large astrocytic vesicles showed shift and fall-in membrane fragments. Two modes of fusion pore formation demonstrate a novel mechanism for fusion pore expansion, as well as full and partial collapses of large secretory vesicles.

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Coordinated interactions of multiple POT1-TPP1 proteins with telomere DNA.

Publication Date: 2013 Apr 24 PMID: 23616058
Authors: Corriveau, M. - Mullins, M. R. - Baus, D. - Harris, M. E. - Taylor, D. J.
Journal: J Biol Chem

Telomeres are macromolecular nucleoprotein complexes that protect the ends of eukaryotic chromosomes from degradation, end-to-end fusion events, and from engaging DNA damage response. However, the assembly of this essential DNA-protein complex is poorly understood. Telomere DNA consists of the repeated sequence 5'-TTAGGG-3' in vertebrates followed by a single-stranded DNA overhang with the same sequence. Both double- and single-stranded regions are coated with high specificity by telomere end-binding proteins, including POT1 and TPP1, that bind as a heterodimer to single-stranded telomeric DNA. Multiple POT1-TPP1 proteins must fully coat the single-stranded telomere DNA to form a functional telomere. To better understand the mechanism of multiple binding we mutated or deleted the two guanosine nucleotides residing between adjacent POT1-TPP1 recognition-sites in single-stranded telomere DNA that are not required for multiple POT1-TPP1 binding events. Circular dichroism demonstrated that spectra from the native telomere sequence are characteristic of G-quadruplex secondary structure, whereas the altered telomere sequences were absent of these signatures. The altered telomere strands, however, facilitated more cooperative loading of multiple POT1-TPP1 proteins compared to wildtype telomere sequence. Finally, we show that a 48-nt DNA with telomere sequence is more susceptible to nuclease digestion when coated with POT1-TPP1 proteins than when it is left uncoated. Together, these data suggest that POT1-TPP1 binds telomeric DNA in a coordinated manner to facilitate assembly of the nucleoprotein complexes into a state that is more accessible to enzymatic activity.

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The Deubiquitylating Enzyme USP44 Counteracts the DNA Double-strand Break Response Mediated by the RNF8 and RNF168 Ubiquitin Ligases.

Publication Date: 2013 Jun 7 PMID: 23615962
Authors: Mosbech, A. - Lukas, C. - Bekker-Jensen, S. - Mailand, N.
Journal: J Biol Chem

Protein recruitment to DNA double-strand breaks (DSBs) relies on ubiquitylation of the surrounding chromatin by the RING finger ubiquitin ligases RNF8 and RNF168. Flux through this pathway is opposed by several deubiquitylating enzymes (DUBs), including OTUB1 and USP3. By analyzing the effect of individually overexpressing the majority of human DUBs on RNF8/RNF168-mediated 53BP1 retention at DSB sites, we found that USP44 and USP29 powerfully inhibited this response at the level of RNF168 accrual. Both USP44 and USP29 promoted efficient deubiquitylation of histone H2A, but unlike USP44, USP29 displayed nonspecific reactivity toward ubiquitylated substrates. Moreover, USP44 but not other H2A DUBs was recruited to RNF168-generated ubiquitylation products at DSB sites. Individual depletion of these DUBs only mildly enhanced accumulation of ubiquitin conjugates and 53BP1 at DSBs, suggesting considerable functional redundancy among cellular DUBs that restrict ubiquitin-dependent protein assembly at DSBs. Our findings implicate USP44 in negative regulation of the RNF8/RNF168 pathway and illustrate the usefulness of DUB overexpression screens for identification of antagonizers of ubiquitin-dependent cellular responses.

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Overexpression of USP14 Protease Reduces I-kappaB Protein Levels and Increases Cytokine Release in Lung Epithelial Cells.

Publication Date: 2013 May 31 PMID: 23615914
Authors: Mialki, R. K. - Zhao, J. - Wei, J. - Mallampalli, D. F. - Zhao, Y.
Journal: J Biol Chem

The ubiquitin-proteasome system is the major pathway of non-lysosomal intracellular protein degradation, playing an important role in a variety of cellular responses including cell division, proliferation, and apoptosis. Ubiquitin-specific protease 14 (USP14) is a component of proteasome regulatory subunit 19 S that regulates deubiquitinated proteins entering inside the proteasome core 20 S. The role of USP14 in protein degradation is still controversial. Several studies suggest that USP14 plays an inhibitory role in protein degradation. Here, in contrast, overexpression of USP14 induced I-kappaB degradation, which increased cytokine release in lung epithelial cells. Overexpression of HA-tagged USP14 (HA-USP14) reduced I-kappaB protein levels by increasing the I-kappaB degradation rate in mouse lung epithelial cells (MLE12). I-kappaB polyubiquitination was reduced in HA-USP14-overexpressed MLE12 cells, suggesting that USP14 regulates I-kappaB degradation by removing its ubiquitin chain, thus promoting the deubiquitinated I-kappaB degradation within the proteasome. Interestingly, we found that USP14 was associated with RelA, a binding partner of I-kappaB, suggesting that RelA is the linker between USP14 and I-kappaB. Lipopolysaccharide (LPS) treatment induced serine phosphorylation of USP14 as well as further reducing I-kappaB levels in HA-USP14-overexpressed MLE12 cells as compared with empty vector transfected cells. Further, overexpression of HA-USP14 increased the LPS-, TNFalpha-, or Escherichia coli-induced IL-8 release in human lung epithelial cells. This study suggests that USP14 removes the ubiquitin chain of I-kappaB, therefore inducing I-kappaB degradation and increasing cytokine release in lung epithelial cells.

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Phosphorylation of Serine 1137/1138 of Mouse Insulin Receptor Substrate (IRS) 2 Regulates cAMP-dependent Binding to 14-3-3 Proteins and IRS2 Protein Degradation.

Publication Date: 2013 Jun 7 PMID: 23615913
Authors: Neukamm, S. S. - Ott, J. - Dammeier, S. - Lehmann, R. - Haring, H. U. - Schleicher, E. - Weigert, C.
Journal: J Biol Chem

Insulin receptor substrate (IRS) 2 as intermediate docking platform transduces the insulin/IGF-1 (insulin like growth factor 1) signal to intracellular effector molecules that regulate glucose homeostasis, beta-cell growth, and survival. Previously, IRS2 has been identified as a 14-3-3 interaction protein. 14-3-3 proteins can bind their target proteins via phosphorylated serine/threonine residues located within distinct motifs. In this study the binding of 14-3-3 to IRS2 upon stimulation with forskolin or the cAMP analog 8-(4-chlorophenylthio)-cAMP was demonstrated in HEK293 cells. Binding was reduced with PKA inhibitors H89 or Rp-8-Br-cAMPS. Phosphorylation of IRS2 on PKA consensus motifs was induced by forskolin and the PKA activator N(6)-Phe-cAMP and prevented by both PKA inhibitors. The amino acid region after position 952 on IRS2 was identified as the 14-3-3 binding region by GST-14-3-3 pulldown assays. Mass spectrometric analysis revealed serine 1137 and serine 1138 as cAMP-dependent, potential PKA phosphorylation sites. Mutation of serine 1137/1138 to alanine strongly reduced the cAMP-dependent 14-3-3 binding. Application of cycloheximide revealed that forskolin enhanced IRS2 protein stability in HEK293 cells stably expressing IRS2 as well as in primary hepatocytes. Stimulation with forskolin did not increase protein stability either in the presence of a 14-3-3 antagonist or in the double 1137/1138 alanine mutant. Thus the reduced IRS2 protein degradation was dependent on the interaction with 14-3-3 proteins and the presence of serine 1137/1138. We present serine 1137/1138 as novel cAMP-dependent phosphorylation sites on IRS2 and show their importance in 14-3-3 binding and IRS2 protein stability.

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Engineering a Monomeric Fc Domain Modality by N-Glycosylation for the Half-life Extension of Biotherapeutics.

Publication Date: 2013 Jun 7 PMID: 23615911
Authors: Ishino, T. - Wang, M. - Mosyak, L. - Tam, A. - Duan, W. - Svenson, K. - Joyce, A. - O'Hara, D. M. - Lin, L. - Somers, W. S. - Kriz, R.
Journal: J Biol Chem

Human IgG is a bivalent molecule that has two identical Fab domains connected by a dimeric Fc domain. For therapeutic purposes, however, the bivalency of IgG and Fc fusion proteins could cause undesired properties. We therefore engineered the conversion of the natural dimeric Fc domain to a highly soluble monomer by introducing two Asn-linked glycans onto the hydrophobic CH3-CH3 dimer interface. The monomeric Fc (monoFc) maintained the binding affinity for neonatal Fc receptor (FcRn) in a pH-dependent manner. We solved the crystal structure of monoFc, which explains how the carbohydrates can stabilize the protein surface and provides the rationale for molecular recognition between monoFc and FcRn. The monoFc prolonged the in vivo half-life of an antibody Fab domain, and a tandem repeat of the monoFc further prolonged the half-life. This monoFc modality can be used to improve the pharmacokinetics of monomeric therapeutic proteins with an option to modulate the degree of half-life extension.

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Crystal Structure of Mycobacterium tuberculosis Polyketide Synthase 11 (PKS11) Reveals Intermediates in the Synthesis of Methyl-branched Alkylpyrones.

Publication Date: 2013 Jun 7 PMID: 23615910
Authors: Gokulan, K. - O'Leary, S. E. - Russell, W. K. - Russell, D. H. - Lalgondar, M. - Begley, T. P. - Ioerger, T. R. - Sacchettini, J. C.
Journal: J Biol Chem

PKS11 is one of three type III polyketide synthases (PKSs) identified in Mycobacterium tuberculosis. Although many PKSs in M. tuberculosis have been implicated in producing complex cell wall glycolipids, the biological function of PKS11 is unknown. PKS11 has previously been proposed to synthesize alkylpyrones from fatty acid substrates. We solved the crystal structure of M. tuberculosis PKS11 and found the overall fold to be similar to other type III PKSs. PKS11 has a deep hydrophobic tunnel proximal to the active site Cys-138 to accommodate substrates. We observed electron density in this tunnel from a co-purified molecule that was identified by mass spectrometry to be palmitate. Co-crystallization with malonyl-CoA (MCoA) or methylmalonyl-CoA (MMCoA) led to partial turnover of the substrate, resulting in trapped intermediates. Reconstitution of the reaction in solution confirmed that both co-factors are required for optimal activity, and kinetic analysis shows that MMCoA is incorporated first, then MCoA, followed by lactonization to produce methyl-branched alkylpyrones.

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A LewisX Glycoprotein Screen Identifies the Low Density Lipoprotein Receptor-related Protein 1 (LRP1) as a Modulator of Oligodendrogenesis in Mice.

Publication Date: 2013 Jun 7 PMID: 23615909
Authors: Hennen, E. - Safina, D. - Haussmann, U. - Worsdorfer, P. - Edenhofer, F. - Poetsch, A. - Faissner, A.
Journal: J Biol Chem

In the developing and adult CNS multipotent neural stem cells reside in distinct niches. Specific carbohydrates and glycoproteins are expressed in these niche microenvironments which are important regulators of stem cell maintenance and differentiation fate. LewisX (LeX), also known as stage-specific embryonic antigen-1 or CD15, is a defined carbohydrate moiety expressed in niche microenvironments of the developing and adult CNS. LeX-glycans are involved in stem cell proliferation, migration, and stemness. A few LeX carrier proteins are known, but a systematic analysis of the targets of LeX glycosylation in vivo has not been performed so far. Using LeX glycosylation as a biomarker we aimed to discover new glycoproteins with a potential functional relevance for CNS development. By immunoaffinity chromatography we enriched LeX glycoproteins from embryonic and postnatal mouse brains and used one-dimensional nLC-ESI-MS/MS for their identification. We could validate phosphacan, tenascin-C, and L1-CAM as major LeX carrier proteins present in vivo. Furthermore, we identified LRP1, a member of the LDL receptor family, as a new LeX carrier protein expressed by mouse neural stem cells. Surprisingly, little is known about LRP1 function for neural stem cells. Thus, we generated Lrp1 knock-out neural stem cells by Cre-mediated recombination and investigated their properties. Here, we provide first evidence that LRP1 is necessary for the differentiation of neural stem cells toward oligodendrocytes. However, this function is independent of LeX glycosylation.

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Biosynthesis of GDP-fucose and Other Sugar Nucleotides in the Blood Stages of Plasmodium falciparum.

Publication Date: 2013 Jun 7 PMID: 23615908
Authors: Sanz, S. - Bandini, G. - Ospina, D. - Bernabeu, M. - Marino, K. - Fernandez-Becerra, C. - Izquierdo, L.
Journal: J Biol Chem

Carbohydrate structures play important roles in many biological processes, including cell adhesion, cell-cell communication, and host-pathogen interactions. Sugar nucleotides are activated forms of sugars used by the cell as donors for most glycosylation reactions. Using a liquid chromatography-tandem mass spectrometry-based method, we identified and quantified the pools of UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, GDP-mannose, and GDP-fucose in Plasmodium falciparum intraerythrocytic life stages. We assembled these data with the in silico functional reconstruction of the parasite metabolic pathways obtained from the P. falciparum annotated genome, exposing new active biosynthetic routes crucial for further glycosylation reactions. Fucose is a sugar present in glycoconjugates often associated with recognition and adhesion events. Thus, the GDP-fucose precursor is essential in a wide variety of organisms. P. falciparum presents homologues of GDP-mannose 4,6-dehydratase and GDP-l-fucose synthase enzymes that are active in vitro, indicating that most GDP-fucose is formed by a de novo pathway that involves the bioconversion of GDP-mannose. Homologues for enzymes involved in a fucose salvage pathway are apparently absent in the P. falciparum genome. This is in agreement with in vivo metabolic labeling experiments showing that fucose is not significantly incorporated by the parasite. Fluorescence microscopy of epitope-tagged versions of P. falciparum GDP-mannose 4,6-dehydratase and GDP-l-fucose synthase expressed in transgenic 3D7 parasites shows that these enzymes localize in the cytoplasm of P. falciparum during the intraerythrocytic developmental cycle. Although the function of fucose in the parasite is not known, the presence of GDP-fucose suggests that the metabolite may be used for further fucosylation reactions.

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Functional Analysis of Paralogous Thiol-disulfide Oxidoreductases in Streptococcus gordonii.

Publication Date: 2013 Jun 7 PMID: 23615907
Authors: Davey, L. - Ng, C. K. - Halperin, S. A. - Lee, S. F.
Journal: J Biol Chem

Disulfide bonds are important for the stability of many extracellular proteins, including bacterial virulence factors. Formation of these bonds is catalyzed by thiol-disulfide oxidoreductases (TDORs). Little is known about their formation in Gram-positive bacteria, particularly among facultative anaerobic Firmicutes, such as streptococci. To investigate disulfide bond formation in Streptococcus gordonii, we identified five putative TDORs from the sequenced genome. Each of the putative TDOR genes was insertionally inactivated with an erythromycin resistance cassette, and the mutants were analyzed for autolysis, extracellular DNA release, biofilm formation, bacteriocin production, and genetic competence. This analysis revealed a single TDOR, SdbA, which exhibited a pleiotropic mutant phenotype. Using an in silico analysis approach, we identified the major autolysin AtlS as a natural substrate of SdbA and showed that SdbA is critical to the formation of a disulfide bond that is required for autolytic activity. Analysis by BLAST search revealed homologs to SdbA in other Gram-positive species. This study provides the first in vivo evidence of an oxidoreductase, SdbA, that affects multiple phenotypes in a Gram-positive bacterium. SdbA shows low sequence homology to previously identified oxidoreductases, suggesting that it may belong to a different class of enzymes. Our results demonstrate that SdbA is required for disulfide bond formation in S. gordonii and indicate that this enzyme may represent a novel type of oxidoreductase in Gram-positive bacteria.

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Endoplasmic Reticulum Glycoprotein Quality Control Regulates CD1d Assembly and CD1d-mediated Antigen Presentation.

Publication Date: 2013 Jun 7 PMID: 23615906
Authors: Kunte, A. - Zhang, W. - Paduraru, C. - Veerapen, N. - Cox, L. R. - Besra, G. S. - Cresswell, P.
Journal: J Biol Chem

The non-classical major histocompatibility complex (MHC) homologue CD1d presents lipid antigens to innate-like lymphocytes called natural-killer T (NKT) cells. These cells, by virtue of their broad cytokine repertoire, shape innate and adaptive immune responses. Here, we have assessed the role of endoplasmic reticulum glycoprotein quality control in CD1d assembly and function, specifically the role of a key component of the quality control machinery, the enzyme UDP glucose glycoprotein glucosyltransferase (UGT1). We observe that in UGT1-deficient cells, CD1d associates prematurely with beta2-microglobulin (beta2m) and is able to rapidly exit the endoplasmic reticulum. At least some of these CD1d-beta2m heterodimers are shorter-lived and can be rescued by provision of a defined exogenous antigen, alpha-galactosylceramide. Importantly, we show that in UGT1-deficient cells the CD1d-beta2m heterodimers have altered antigenicity despite the fact that their cell surface levels are unchanged. We propose that UGT1 serves as a quality control checkpoint during CD1d assembly and further suggest that UGT1-mediated quality control can shape the lipid repertoire of newly synthesized CD1d. The quality control process may play a role in ensuring stability of exported CD1d-beta2m complexes, in facilitating presentation of low abundance high affinity antigens, or in preventing deleterious responses to self lipids.

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The A128T Resistance Mutation Reveals Aberrant Protein Multimerization as the Primary Mechanism of Action of Allosteric HIV-1 Integrase Inhibitors.

Publication Date: 2013 May 31 PMID: 23615903
Authors: Feng, L. - Sharma, A. - Slaughter, A. - Jena, N. - Koh, Y. - Shkriabai, N. - Larue, R. C. - Patel, P. A. - Mitsuya, H. - Kessl, J. J. - Engelman, A. - Fuchs, J. R. - Kvaratskhelia, M.
Journal: J Biol Chem

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are a very promising new class of anti-HIV-1 agents that exhibit a multimodal mechanism of action by allosterically modulating IN multimerization and interfering with IN-lens epithelium-derived growth factor (LEDGF)/p75 binding. Selection of viral strains under ALLINI pressure has revealed an A128T substitution in HIV-1 IN as a primary mechanism of resistance. Here, we elucidated the structural and mechanistic basis for this resistance. The A128T substitution did not affect the hydrogen bonding between ALLINI and IN that mimics the IN-LEDGF/p75 interaction but instead altered the positioning of the inhibitor at the IN dimer interface. Consequently, the A128T substitution had only a minor effect on the ALLINI IC50 values for IN-LEDGF/p75 binding. Instead, ALLINIs markedly altered the multimerization of IN by promoting aberrant higher order WT (but not A128T) IN oligomers. Accordingly, WT IN catalytic activities and HIV-1 replication were potently inhibited by ALLINIs, whereas the A128T substitution in IN resulted in significant resistance to the inhibitors both in vitro and in cell culture assays. The differential multimerization of WT and A128T INs induced by ALLINIs correlated with the differences in infectivity of HIV-1 progeny virions. We conclude that ALLINIs primarily target IN multimerization rather than IN-LEDGF/p75 binding. Our findings provide the structural foundations for developing improved ALLINIs with increased potency and decreased potential to select for drug resistance.

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Structures of Arenaviral Nucleoproteins with Triphosphate dsRNA Reveal a Unique Mechanism of Immune Suppression.

Publication Date: 2013 Jun 7 PMID: 23615902
Authors: Jiang, X. - Huang, Q. - Wang, W. - Dong, H. - Ly, H. - Liang, Y. - Dong, C.
Journal: J Biol Chem

A hallmark of severe Lassa fever is the generalized immune suppression, the mechanism of which is poorly understood. Lassa virus (LASV) nucleoprotein (NP) is the only known 3'-5' exoribonuclease that can suppress type I interferon (IFN) production possibly by degrading immune-stimulatory RNAs. How this unique enzymatic activity of LASV NP recognizes and processes RNA substrates is unknown. We provide an atomic view of a catalytically active exoribonuclease domain of LASV NP (LASV NP-C) in the process of degrading a 5' triphosphate double-stranded (ds) RNA substrate, a typical pathogen-associated molecular pattern molecule, to induce type I IFN production. Additionally, we provide for the first time a high-resolution crystal structure of an active exoribonuclease domain of Tacaribe arenavirus (TCRV) NP. Coupled with the in vitro enzymatic and cell-based interferon suppression assays, these structural analyses strongly support a unified model of an exoribonuclease-dependent IFN suppression mechanism shared by all known arenaviruses. New knowledge learned from these studies should aid the development of therapeutics against pathogenic arenaviruses that can infect hundreds of thousands of individuals and kill thousands annually.

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Structure of Sorting Nexin 11 (SNX11) Reveals a Novel Extended Phox Homology (PX) Domain Critical for Inhibition of SNX10-induced Vacuolation.

Publication Date: 2013 Jun 7 PMID: 23615901
Authors: Xu, J. - Xu, T. - Wu, B. - Ye, Y. - You, X. - Shu, X. - Pei, D. - Liu, J.
Journal: J Biol Chem

Sorting nexins are phox homology (PX) domain-containing proteins involved in diverse intracellular endosomal trafficking pathways. The PX domain binds to certain phosphatidylinositols and is recruited to vesicles rich in these lipids. The structure of the PX domain is highly conserved, containing a three-stranded beta-sheet, followed by three alpha-helices. Here, we report the crystal structures of truncated human SNX11 (sorting nexin 11). The structures reveal that SNX11 contains a novel PX domain, hereby named the extended PX (PXe) domain, with two additional alpha-helices at the C terminus. We demonstrate that these alpha-helices are indispensible for the in vitro functions of SNX11. We propose that this PXe domain is present in SNX10 and is responsible for the vacuolation activity of SNX10. Thus, this novel PXe domain constitutes a structurally and functionally important PX domain subfamily.

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Protein Kinase CK2 Regulates the Dimerization of Histone Deacetylase 1 (HDAC1) and HDAC2 during Mitosis.

Publication Date: 2013 Jun 7 PMID: 23612983
Authors: Khan, D. H. - He, S. - Yu, J. - Winter, S. - Cao, W. - Seiser, C. - Davie, J. R.
Journal: J Biol Chem

Histone deacetylase 1 (HDAC1) and HDAC2 are components of corepressor complexes that are involved in chromatin remodeling and regulation of gene expression by regulating dynamic protein acetylation. HDAC1 and -2 form homo- and heterodimers, and their activity is dependent upon dimer formation. Phosphorylation of HDAC1 and/or HDAC2 in interphase cells is required for the formation of HDAC corepressor complexes. In this study, we show that during mitosis, HDAC2 and, to a lesser extent, HDAC1 phosphorylation levels dramatically increase. When HDAC1 and -2 are displaced from the chromosome during metaphase, they dissociate from each other, but each enzyme remains in association with components of the HDAC corepressor complexes Sin3, NuRD, and CoREST as homodimers. Enzyme inhibition studies and mutational analyses demonstrated that protein kinase CK2-catalyzed phosphorylation of HDAC1 and -2 is crucial for the dissociation of these two enzymes. These results suggest that corepressor complexes, including HDAC1 or HDAC2 homodimers, might target different cellular proteins during mitosis.

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The in Vivo TRPV6 Protein Starts at a Non-AUG Triplet, Decoded as Methionine, Upstream of Canonical Initiation at AUG.

Publication Date: 2013 Jun 7 PMID: 23612980
Authors: Fecher-Trost, C. - Wissenbach, U. - Beck, A. - Schalkowsky, P. - Stoerger, C. - Doerr, J. - Dembek, A. - Simon-Thomas, M. - Weber, A. - Wollenberg, P. - Ruppert, T. - Middendorff, R. - Maurer, H. H. - Flockerzi, V.
Journal: J Biol Chem

TRPV6 channels function as epithelial Ca(2+) entry pathways in the epididymis, prostate, and placenta. However, the identity of the endogenous TRPV6 protein relies on predicted gene coding regions and is only known to a certain level of approximation. We show that in vivo the TRPV6 protein has an extended N terminus. Translation initiates at a non-AUG codon, at ACG, which is decoded by methionine and which is upstream of the annotated AUG, which is not used for initiation. The in vitro properties of channels formed by the extended full-length TRPV6 proteins and the so-far annotated and smaller TRPV6 are similar, but the extended N terminus increases trafficking to the plasma membrane and represents an additional scaffold for channel assembly. The increased translation of the smaller TRPV6 cDNA version may overestimate the in vivo situation where translation efficiency may represent an additional mechanism to tightly control the TRPV6-mediated Ca(2+) entry to prevent deleterious Ca(2+) overload.

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Mammalian Target of Rapamycin Complex 1 (mTORC1) Enhances Bortezomib-induced Death in Tuberous Sclerosis Complex (TSC)-null Cells by a c-MYC-dependent Induction of the Unfolded Protein Response.

Publication Date: 2013 May 31 PMID: 23612979
Authors: Babcock, J. T. - Nguyen, H. B. - He, Y. - Hendricks, J. W. - Wek, R. C. - Quilliam, L. A.
Journal: J Biol Chem

Many factors, including duration and intensity of the unfolded protein response (UPR), dictate whether cells will adapt to endoplasmic reticulum stress or undergo apoptosis. In tuberous sclerosis (TSC), elevation of mammalian target of rapamycin complex 1 (mTORC1) activity has been proposed to compound the induction of UPR transcription factors ATF4 and CHOP, suggesting that the UPR could be targeted to eradicate TSC1/2-null cells during patient therapy. Here we report that control of c-MYC translation by mTORC1 plays a key role in determining whether TSC2-null Elt3 rat leiomyoma cells apoptose in response to UPR induction by the proteasome inhibitor bortezomib. Although bortezomib induces eukaryotic initiating factor 2alpha phosphorylation, mTORC1 activity was also required for downstream induction of the UPR transcription factors ATF4 and CHOP by a mechanism involving increased expression of c-MYC. Although bortezomib-induced c-MYC transcription was resistant to rapamycin treatment, mTORC1 activity was required for efficient c-MYC translation. c-MYC subsequently bound to the ATF4 promoter, suggesting direct involvement of an mTORC1/c-MYC-driven signaling pathway in the activation of the UPR. Consistent with this notion, exogenously expressed c-MYC reversed the ability of rapamycin to prevent bortezomib-induced CHOP and ATF4 expression as well as apoptosis. These findings indicate that the induction of ATF4/CHOP expression occurs via mTORC1 regulation of c-MYC and that this signaling pathway is a major determinant in the ability of bortezomib to induce apoptosis.

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HIV-1 Vpr Protein Inhibits Telomerase Activity via the EDD-DDB1-VPRBP E3 Ligase Complex.

Publication Date: 2013 May 31 PMID: 23612978
Authors: Wang, X. - Singh, S. - Jung, H. Y. - Yang, G. - Jun, S. - Sastry, K. J. - Park, J. I.
Journal: J Biol Chem

Viral pathogens utilize host cell machinery for their benefits. Herein, we identify that HIV-1 Vpr (viral protein R) negatively modulates telomerase activity. Telomerase enables stem and cancer cells to evade cell senescence by adding telomeric sequences to the ends of chromosomes. We found that Vpr inhibited telomerase activity by down-regulating TERT protein, a catalytic subunit of telomerase. As a molecular adaptor, Vpr enhanced the interaction between TERT and the VPRBP substrate receptor of the DYRK2-associated EDD-DDB1-VPRBP E3 ligase complex, resulting in increased ubiquitination of TERT. In contrast, the Vpr mutant identified in HIV-1-infected long-term nonprogressors failed to promote TERT destabilization. Our results suggest that Vpr inhibits telomerase activity by hijacking the host E3 ligase complex, and we propose the novel molecular mechanism of telomerase deregulation in possibly HIV-1 pathogenesis.

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NHERF2 Protein Mobility Rate Is Determined by a Unique C-terminal Domain That Is Also Necessary for Its Regulation of NHE3 Protein in OK Cells.

Publication Date: 2013 Jun 7 PMID: 23612977
Authors: Yang, J. - Singh, V. - Cha, B. - Chen, T. E. - Sarker, R. - Murtazina, R. - Jin, S. - Zachos, N. C. - Patterson, G. H. - Tse, C. M. - Kovbasnjuk, O. - Li, X. - Donowitz, M.
Journal: J Biol Chem

Na(+)/H(+) exchanger regulatory factor (NHERF) proteins are a family of PSD-95/Discs-large/ZO-1 (PDZ)-scaffolding proteins, three of which (NHERFs 1-3) are localized to the brush border in kidney and intestinal epithelial cells. All NHERF proteins are involved in anchoring membrane proteins that contain PDZ recognition motifs to form multiprotein signaling complexes. In contrast to their predicted immobility, NHERF1, NHERF2, and NHERF3 were all shown by fluorescence recovery after photobleaching/confocal microscopy to be surprisingly mobile in the microvilli of the renal proximal tubule OK cell line. Their diffusion coefficients, although different among the three, were all of the same magnitude as that of the transmembrane proteins, suggesting they are all anchored in the microvilli but to different extents. NHERF3 moves faster than NHERF1, and NHERF2 moves the slowest. Several chimeras and mutants of NHERF1 and NHERF2 were made to determine which part of NHERF2 confers the slower mobility rate. Surprisingly, the slower mobility rate of NHERF2 was determined by a unique C-terminal domain, which includes a nonconserved region along with the ezrin, radixin, moesin (ERM) binding domain. Also, this C-terminal domain of NHERF2 determined its greater detergent insolubility and was necessary for the formation of larger multiprotein NHERF2 complexes. In addition, this NHERF2 domain was functionally significant in NHE3 regulation, being necessary for stimulation by lysophosphatidic acid of activity and increased mobility of NHE3, as well as necessary for inhibition of NHE3 activity by calcium ionophore 4-Br-A23187. Thus, multiple functions of NHERF2 require involvement of an additional domain in this protein.

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Down-regulation of Wild-type p53-induced Phosphatase 1 (Wip1) Plays a Critical Role in Regulating Several p53-dependent Functions in Premature Senescent Tumor Cells.

Publication Date: 2013 Jun 7 PMID: 23612976
Authors: Crescenzi, E. - Raia, Z. - Pacifico, F. - Mellone, S. - Moscato, F. - Palumbo, G. - Leonardi, A.
Journal: J Biol Chem

Premature or drug-induced senescence is a major cellular response to chemotherapy in solid tumors. The senescent phenotype develops slowly and is associated with chronic DNA damage response. We found that expression of wild-type p53-induced phosphatase 1 (Wip1) is markedly down-regulated during persistent DNA damage and after drug release during the acquisition of the senescent phenotype in carcinoma cells. We demonstrate that down-regulation of Wip1 is required for maintenance of permanent G2 arrest. In fact, we show that forced expression of Wip1 in premature senescent tumor cells induces inappropriate re-initiation of mitosis, uncontrolled polyploid progression, and cell death by mitotic failure. Most of the effects of Wip1 may be attributed to its ability to dephosphorylate p53 at Ser(15) and to inhibit DNA damage response. However, we also uncover a regulatory pathway whereby suppression of p53 Ser(15) phosphorylation is associated with enhanced phosphorylation at Ser(46), increased p53 protein levels, and induction of Noxa expression. On the whole, our data indicate that down-regulation of Wip1 expression during premature senescence plays a pivotal role in regulating several p53-dependent aspects of the senescent phenotype.

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Phosphorylation of Serine 399 in LKB1 Protein Short Form by Protein Kinase Czeta Is Required for Its Nucleocytoplasmic Transport and Consequent AMP-activated Protein Kinase (AMPK) Activation.

Publication Date: 2013 Jun 7 PMID: 23612973
Authors: Zhu, H. - Moriasi, C. M. - Zhang, M. - Zhao, Y. - Zou, M. H.
Journal: J Biol Chem

Two splice variants of LKB1 exist: LKB1 long form (LKB1L) and LKB1 short form (LKB1S). In a previous study, we demonstrated that phosphorylation of Ser-428/431 (in LKB1L) by protein kinase Czeta (PKCzeta) was essential for LKB1-mediated activation of AMP-activated protein kinase (AMPK) in response to oxidants or metformin. Paradoxically, LKB1S also activates AMPK although it lacks Ser-428/431. Thus, we hypothesized that LKB1S contained additional phosphorylation sites important in AMPK activation. Truncation analysis and site-directed mutagenesis were used to identify putative PKCzeta phosphorylation sites in LKB1S. Substitution of Ser-399 to alanine did not alter the activity of LKB1S, but abolished peroxynitrite- and metformin-induced activation of AMPK. Furthermore, the phosphomimetic mutation (S399D) increased the phosphorylation of AMPK and its downstream target phospho-acetyl-coenzyme A carboxylase (ACC). PKCzeta-dependent phosphorylation of Ser-399 triggered nucleocytoplasmic translocation of LKB1S in response to metformin or peroxynitrite treatment. This effect was ablated by pharmacological and genetic inhibition of PKCzeta, by inhibition of CRM1 activity and by substituting Ser-399 with alanine (S399A). Overexpression of PKCzeta up-regulated metformin-mediated phosphorylation of both AMPK (Thr-172) and ACC (Ser-79), but the effect was ablated in the S399A mutant. We conclude that, similar to Ser-428/431 (in LKB1L), Ser-399 (in LKB1S) is a PKCzeta-dependent phosphorylation site essential for nucleocytoplasmic export of LKB1S and consequent AMPK activation.

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Site-specific Acetylation of the Proteasome Activator REGgamma Directs Its Heptameric Structure and Functions.

Publication Date: 2013 Jun 7 PMID: 23612972
Authors: Liu, J. - Wang, Y. - Li, L. - Zhou, L. - Wei, H. - Zhou, Q. - Liu, J. - Wang, W. - Ji, L. - Shan, P. - Wang, Y. - Yang, Y. - Jung, S. Y. - Zhang, P. - Wang, C. - Long, W. - Zhang, B. - Li, X.
Journal: J Biol Chem

The proteasome activator REGgamma has been reported to promote degradation of steroid receptor coactivator-3 and cyclin-dependent kinase inhibitors p21, p16, and p19 in a ubiquitin- and ATP-independent manner. A recent comparative analysis of REGgamma expression in mouse and human tissues reveals a unique pattern of REGgamma in specific cell types, suggesting undisclosed functions and biological importance of this molecule. Despite the emerging progress made in REGgamma-related studies, how REGgamma function is regulated remains to be explored. In this study, we report for the first time that REGgamma can be acetylated mostly on its lysine 195 (Lys-195) residue by CREB binding protein (CBP), which can be reversed by sirtuin 1 (SIRT1) in mammalian cells. Site-directed mutagenesis abrogated acetylation at Lys-195 and significantly attenuated the capability of REGgamma to degrade its target substrates, p21 and hepatitis C virus core protein. Mechanistically, acetylation at Lys-195 is important for the interactions between REGgamma monomers and ultimately influences REGgamma heptamerization. Biological analysis of cells containing REGgamma-WT or REGgamma-K195R mutant indicates an impact of acetylation on REGgamma-mediated regulation of cell proliferation and cell cycle progression. These findings reveal a previously unknown mechanism in the regulation of REGgamma assembly and activity, suggesting a potential venue for the intervention of the ubiquitin-independent REGgamma proteasome activity.

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The von Hippel-Lindau Protein pVHL Inhibits Ribosome Biogenesis and Protein Synthesis.

Publication Date: 2013 Jun 7 PMID: 23612971
Authors: Zhao, W. T. - Zhou, C. F. - Li, X. B. - Zhang, Y. F. - Fan, L. - Pelletier, J. - Fang, J.
Journal: J Biol Chem

pVHL, the product of von Hippel-Lindau (VHL) tumor suppressor gene, functions as the substrate recognition component of an E3-ubiquitin ligase complex that targets hypoxia inducible factor alpha (HIF-alpha) for ubiquitination and degradation. Besides HIF-alpha, pVHL also interacts with other proteins and has multiple functions. Here, we report that pVHL inhibits ribosome biogenesis and protein synthesis. We find that pVHL associates with the 40S ribosomal protein S3 (RPS3) but does not target it for destruction. Rather, the pVHL-RPS3 association interferes with the interaction between RPS3 and RPS2. Expression of pVHL also leads to nuclear retention of pre-40S ribosomal subunits, diminishing polysomes and 18S rRNA levels. We also demonstrate that pVHL suppresses both cap-dependent and cap-independent protein synthesis. Our findings unravel a novel function of pVHL and provide insight into the regulation of ribosome biogenesis by the tumor suppressor pVHL.

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Batroxobin binds fibrin with higher affinity and promotes clot expansion to a greater extent than thrombin.

Publication Date: 2013 Jun 7 PMID: 23612970
Authors: Vu, T. T. - Stafford, A. R. - Leslie, B. A. - Kim, P. Y. - Fredenburgh, J. C. - Weitz, J. I.
Journal: J Biol Chem

Batroxobin is a thrombin-like serine protease from the venom of Bothrops atrox moojeni that clots fibrinogen. In contrast to thrombin, which releases fibrinopeptide A and B from the NH2-terminal domains of the Aalpha- and Bbeta-chains of fibrinogen, respectively, batroxobin only releases fibrinopeptide A. Because the mechanism responsible for these differences is unknown, we compared the interactions of batroxobin and thrombin with the predominant gammaA/gammaA isoform of fibrin(ogen) and the gammaA/gamma' variant with an extended gamma-chain. Thrombin binds to the gamma'-chain and forms a higher affinity interaction with gammaA/gamma'-fibrin(ogen) than gammaA/gammaA-fibrin(ogen). In contrast, batroxobin binds both fibrin(ogen) isoforms with similar high affinity (Kd values of about 0.5 mum) even though it does not interact with the gamma'-chain. The batroxobin-binding sites on fibrin(ogen) only partially overlap with those of thrombin because thrombin attenuates, but does not abrogate, the interaction of gammaA/gammaA-fibrinogen with batroxobin. Furthermore, although both thrombin and batroxobin bind to the central E-region of fibrinogen with a Kd value of 2-5 mum, the alpha(17-51) and Bbeta(1-42) regions bind thrombin but not batroxobin. Once bound to fibrin, the capacity of batroxobin to promote fibrin accretion is 18-fold greater than that of thrombin, a finding that may explain the microvascular thrombosis that complicates envenomation by B. atrox moojeni. Therefore, batroxobin binds fibrin(ogen) in a manner distinct from thrombin, which may contribute to its higher affinity interaction, selective fibrinopeptide A release, and prothrombotic properties.

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A Novel p53 Mutant Found in Iatrogenic Urothelial Cancers Is Dysfunctional and Can Be Rescued by a Second-site Global Suppressor Mutation.

Publication Date: 2013 Jun 7 PMID: 23612969
Authors: Odell, A. F. - Odell, L. R. - Askham, J. M. - Alogheli, H. - Ponnambalam, S. - Hollstein, M.
Journal: J Biol Chem

Exposure to herbal remedies containing the carcinogen aristolochic acid (AA) has been widespread in some regions of the world. Rare A-->T TP53 mutations were recently discovered in AA-associated urothelial cancers. The near absence of these mutations among all other sequenced human tumors suggests that they could be biologically silent. There are no cell banks with established lines derived from human tumors with which to explore the influence of the novel mutants on p53 function and cellular behavior. To investigate their impact, we generated isogenic mutant clones by integrase-mediated cassette exchange at the p53 locus of platform (null) murine embryonic fibroblasts and kidney epithelial cells. Common tumor mutants (R248W, R273C) were compared with the AA-associated mutants N131Y, R249W, and Q104L. Assays of cell proliferation, migration, growth in soft agar, apoptosis, senescence, and gene expression revealed contrasting outcomes on cellular behavior following introduction of N131Y or Q104L. The N131Y mutant demonstrated a phenotype akin to common tumor mutants, whereas Q104L clone behavior resembled that of cells with wild-type p53. Wild-type p53 responses were restored in double-mutant cells harboring N131Y and N239Y, a second-site rescue mutation, suggesting that pharmaceutical reactivation of p53 function in tumors expressing N131Y could have therapeutic benefit. N131Y is likely to contribute directly to tumor phenotype and is a promising candidate biomarker of AA exposure and disease. Rare mutations thus do not necessarily point to sites where amino acid exchanges are phenotypically neutral. Encounter with mutagenic insults targeting cryptic sites can reveal specific signature hotspots.

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Recruitment of Nox4 to a Plasma Membrane Scaffold Is Required for Localized Reactive Oxygen Species Generation and Sustained Src Activation in Response to Insulin-like Growth Factor-I.

Publication Date: 2013 May 31 PMID: 23612968
Authors: Xi, G. - Shen, X. C. - Wai, C. - Clemmons, D. R.
Journal: J Biol Chem

Nox4-derived ROS is increased in response to hyperglycemia and is required for IGF-I-stimulated Src activation. This study was undertaken to determine the mechanism by which Nox4 mediates sustained Src activation. IGF-I stimulated sustained Src activation, which occurred primarily on the SHPS-1 scaffold protein. In vitro oxidation experiments indicated that Nox4-derived ROS was able to oxidize Src when they are in close proximity, and Src oxidation leads to its activation. Therefore we hypothesized that Nox4 recruitment to the plasma membrane scaffold SHPS-1 allowed localized ROS generation to mediate sustained Src oxidation and activation. To determine the mechanism of Nox4 recruitment, we analyzed the role of Grb2, a component of the SHPS-1 signaling complex. We determined that Nox4 Tyr-491 was phosphorylated after IGF-I stimulation and was responsible for Nox4 binding to the SH2 domain of Grb2. Overexpression of a Nox4 mutant, Y491F, prevented Nox4/Grb2 association. Importantly, it also prevented Nox4 recruitment to SHPS-1. The role of Grb2 was confirmed using a Pyk2 Y881F mutant, which blocked Grb2 recruitment to SHPS-1. Cells expressing this mutant had impaired Nox4 recruitment to SHPS-1. IGF-I-stimulated downstream signaling and biological actions were also significantly impaired in Nox4 Y491F-overexpressing cells. Disruption of Nox4 recruitment to SHPS-1 in aorta from diabetic mice inhibited IGF-I-stimulated Src oxidation and activation as well as cell proliferation. These findings provide insight into the mechanism by which localized Nox4-derived ROS regulates the sustained activity of a tyrosine kinase that is critical for mediating signal transduction and biological actions.

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A Molecular Mechanism for Therapeutic Effects of cGMP-elevating Agents in Pulmonary Arterial Hypertension.

Publication Date: 2013 Jun 7 PMID: 23612967
Authors: Schwappacher, R. - Kilic, A. - Kojonazarov, B. - Lang, M. - Diep, T. - Zhuang, S. - Gawlowski, T. - Schermuly, R. T. - Pfeifer, A. - Boss, G. R. - Pilz, R. B.
Journal: J Biol Chem

Pulmonary arterial hypertension (PAH) is a progressive, usually fatal disease with abnormal vascular remodeling. Pulmonary artery smooth muscle cells (PASMCs) from PAH patients are hyperproliferative and apoptosis-resistant and demonstrate decreased signaling in response to bone morphogenetic proteins (BMPs). Cyclic GMP-elevating agents are beneficial in PAH, but their mechanism(s) of action are incompletely understood. Here we show that BMP signaling via Smad1/5/8 requires cGMP-dependent protein kinase isotype I (PKGI) to maintain PASMCs in a differentiated, low proliferative state. BMP cooperation with cGMP/PKGI was crucial for transcription of contractile genes and suppression of pro-proliferative and anti-apoptotic genes. Lungs from mice with low or absent PKGI (Prkg1(+/-) and Prkg1(-/-) mice) exhibited impaired BMP signaling, decreased contractile gene expression, and abnormal vascular remodeling. Conversely, cGMP stimulation of PKGI restored defective BMP signaling in rats with hypoxia-induced PAH, consistent with cGMP-elevating agents reversing vascular remodeling in this PAH model. Our results provide a mechanism for the therapeutic effects of cGMP-elevating agents in PAH and suggest that combining them with BMP mimetics may provide a novel, disease-modifying approach to PAH therapy.

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Feedback Inhibition of Deoxy-D-xylulose-5-phosphate Synthase Regulates the Methylerythritol 4-Phosphate Pathway.

Publication Date: 2013 Jun 7 PMID: 23612965
Authors: Banerjee, A. - Wu, Y. - Banerjee, R. - Li, Y. - Yan, H. - Sharkey, T. D.
Journal: J Biol Chem

The 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway leads to the biosynthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the precursors for isoprene and higher isoprenoids. Isoprene has significant effects on atmospheric chemistry, whereas other isoprenoids have diverse roles ranging from various biological processes to applications in commercial uses. Understanding the metabolic regulation of the MEP pathway is important considering the numerous applications of this pathway. The 1-deoxy-d-xylulose-5-phosphate synthase (DXS) enzyme was cloned from Populus trichocarpa, and the recombinant protein (PtDXS) was purified from Escherichia coli. The steady-state kinetic parameters were measured by a coupled enzyme assay. An LC-MS/MS-based assay involving the direct quantification of the end product of the enzymatic reaction, 1-deoxy-d-xylulose 5-phosphate (DXP), was developed. The effect of different metabolites of the MEP pathway on PtDXS activity was tested. PtDXS was inhibited by IDP and DMADP. Both of these metabolites compete with thiamine pyrophosphate for binding with the enzyme. An atomic structural model of PtDXS in complex with thiamine pyrophosphate and Mg(2+) was built by homology modeling and refined by molecular dynamics simulations. The refined structure was used to model the binding of IDP and DMADP and indicated that IDP and DMADP might bind with the enzyme in a manner very similar to the binding of thiamine pyrophosphate. The feedback inhibition of PtDXS by IDP and DMADP constitutes an important mechanism of metabolic regulation of the MEP pathway and indicates that thiamine pyrophosphate-dependent enzymes may often be affected by IDP and DMADP.

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Acquired Substrate Preference for GAB1 Protein Bestows Transforming Activity to ERBB2 Kinase Lung Cancer Mutants.

Publication Date: 2013 Jun 7 PMID: 23612964
Authors: Fan, Y. X. - Wong, L. - Marino, M. P. - Ou, W. - Shen, Y. - Wu, W. J. - Wong, K. K. - Reiser, J. - Johnson, G. R.
Journal: J Biol Chem

Activating mutations in the alphaC-beta4 loop of the ERBB2 kinase domain, such as ERBB2(YVMA) and ERBB2(G776VC), have been identified in human lung cancers and found to drive tumor formation. Here we observe that the docking protein GAB1 is hyper-phosphorylated in carcinomas from transgenic mice and in cell lines expressing these ERBB2 cancer mutants. Using dominant negative GAB1 mutants lacking canonical tyrosine residues for SHP2 and PI3K interactions or lentiviral shRNA that targets GAB1, we demonstrate that GAB1 phosphorylation is required for ERBB2 mutant-induced cell signaling, cell transformation, and tumorigenesis. An enzyme kinetic analysis comparing ERBB2(YVMA) to wild type using physiologically relevant peptide substrates reveals that ERBB2(YVMA) kinase adopts a striking preference for GAB1 phosphorylation sites as evidenced by approximately 150-fold increases in the specificity constants (kcat/Km) for several GAB1 peptides, and this change in substrate selectivity was predominantly attributed to the peptide binding affinities as reflected by the apparent Km values. Furthermore, we demonstrate that ERBB2(YVMA) phosphorylates GAB1 protein approximately 70-fold faster than wild type ERBB2 in vitro. Notably, the mutation does not significantly alter the Km for ATP or sensitivity to lapatinib, suggesting that, unlike EGFR lung cancer mutants, the ATP binding cleft of the kinase is not significantly changed. Taken together, our results indicate that the acquired substrate preference for GAB1 is critical for the ERBB2 mutant-induced oncogenesis.

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Diverse Sequence Determinants Control Human and Mouse Receptor Interacting Protein 3 (RIP3) and Mixed Lineage Kinase domain-Like (MLKL) Interaction in Necroptotic Signaling.

Publication Date: 2013 Jun 7 PMID: 23612963
Authors: Chen, W. - Zhou, Z. - Li, L. - Zhong, C. Q. - Zheng, X. - Wu, X. - Zhang, Y. - Ma, H. - Huang, D. - Li, W. - Xia, Z. - Han, J.
Journal: J Biol Chem

Receptor interacting protein 3 (RIP3) is a protein kinase essential for TNF-induced necroptosis. Phosphorylation on Ser-227 in human RIP3 (hRIP3) is required for its interaction with human mixed lineage kinase domain-like (MLKL) in the necrosome, a signaling complex induced by TNF stimulation. RIP1 and RIP3 mediate necrosome aggregation leading to the formation of amyloid-like signaling complexes. We found that TNF induces Thr-231 and Ser-232 phosphorylation in mouse RIP3 (mRIP3) and this phosphorylation is required for mRIP3 to interact with mMLKL. Ser-232 in mRIP3 corresponds to Ser-227 in hRIP3, whereas Thr-231 is not conserved in hRIP3. Although the RIP3-MLKL interaction is required for necroptosis in both human and mouse cells, hRIP3 does not interact with mMLKL and mRIP3 cannot bind to hMLKL. The species specificity of the RIP3-MLKL interaction is primarily determined by the sequence differences in the phosphorylation sites and the flanking sequence around the phosphorylation sites in hRIP3 and mRIP3. It appears that the RIP3-MLKL interaction has been selected as an evolutionarily conserved mechanism in mediating necroptosis signaling despite that differing structural and mechanistic bases for this interaction emerged simultaneously in different organisms. In addition, we further revealed that the interaction of RIP3 with MLKL prevented massive abnormal RIP3 aggregation, and therefore should be crucial for formation of the amyloid signaling complex of necrosomes. We also found that the interaction between RIP3 and MLKL is required for the translocation of necrosomes to mitochondria-associated membranes. Our data demonstrate the importance of the RIP3-MLKL interaction in the formation of functional necrosomes and suggest that translocation of necrosomes to mitochondria-associated membranes is essential for necroptosis signaling.

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60-kDa Tat-interactive Protein (TIP60) Positively Regulates Th-inducing POK (ThPOK)-mediated Repression of Eomesodermin in Human CD4+ T Cells.

Publication Date: 2013 May 31 PMID: 23609452
Authors: Li, Y. - Tsun, A. - Gao, Z. - Han, Z. - Gao, Y. - Li, Z. - Lin, F. - Wang, Y. - Wei, G. - Yao, Z. - Li, B.
Journal: J Biol Chem

The abundant expression of IFNgamma in Th-inducing POK (ThPOK)-deficient CD4(+) T cells requires the activation of Eomesodermin (Eomes); however, the underlying mechanism of this phenomenon remains unclear. Here we report that ThPOK binds directly to the promoter region of the Eomes gene to repress its expression in CD4(+) T cells. We identified the histone acetyltransferase TIP60 as a co-repressor of ThPOK-target genes, where ectopically expressed TIP60 increased ThPOK protein stability by promoting its acetylation at its Lys(360) residue to then augment the transcriptional repression of Eomes. Moreover, knockdown of endogenous TIP60 abolished the stabilization of ThPOK in CD4(+) T cells, which led to the transcriptional activation of Eomes and increased production of IFNgamma. Our results reveal a novel pathway by which TIP60 and ThPOK synergistically suppresses Eomes function and IFNgamma production, which could contribute to the regulation of inflammation.

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Increased Chemosensitivity via Targeting Testicular Nuclear Receptor 4 (TR4)-Oct4-Interleukin 1 Receptor Antagonist (IL1Ra) Axis in Prostate Cancer CD133+ Stem/Progenitor Cells to Battle Prostate Cancer.

Publication Date: 2013 Jun 7 PMID: 23609451
Authors: Yang, D. R. - Ding, X. F. - Luo, J. - Shan, Y. X. - Wang, R. - Lin, S. J. - Li, G. - Huang, C. K. - Zhu, J. - Chen, Y. - Lee, S. O. - Chang, C.
Journal: J Biol Chem

Prostate cancer (PCa) stem/progenitor cells are known to have higher chemoresistance than non-stem/progenitor cells, but the underlying molecular mechanism remains unclear. We found the expression of testicular nuclear receptor 4 (TR4) is significantly higher in PCa CD133(+) stem/progenitor cells compared with CD133(-) non-stem/progenitor cells. Knockdown of TR4 levels in the established PCa stem/progenitor cells and the CD133(+) population of the C4-2 PCa cell line with lentiviral TR4 siRNA led to increased drug sensitivity to the two commonly used chemotherapeutic drugs, docetaxel and etoposide, judging from significantly reduced IC50 values and increased apoptosis in the TR4 knockdown cells. Mechanism dissection studies found that suppression of TR4 in these stem/progenitor cells led to down-regulation of Oct4 expression, which, in turn, down-regulated the IL-1 receptor antagonist (IL1Ra) expression. Neutralization experiments via adding these molecules into the TR4 knockdown PCa stem/progenitor cells reversed the chemoresistance, suggesting that the TR4-Oct4-IL1Ra axis may play a critical role in the development of chemoresistance in the PCa stem/progenitor cells. Together, these studies suggest that targeting TR4 may alter chemoresistance of PCa stem/progenitor cells, and this finding provides the possibility of targeting TR4 as a new and better approach to overcome the chemoresistance problem in PCa therapeutics.

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Immune Responsive Gene 1 (IRG1) Promotes Endotoxin Tolerance by Increasing A20 Expression in Macrophages through Reactive Oxygen Species.

Publication Date: 2013 Jun 7 PMID: 23609450
Authors: Li, Y. - Zhang, P. - Wang, C. - Han, C. - Meng, J. - Liu, X. - Xu, S. - Li, N. - Wang, Q. - Shi, X. - Cao, X.
Journal: J Biol Chem

Sepsis-associated immunosuppression (SAIS) is regarded as one of main causes for the death of septic patients at the late stage because of the decreased innate immunity with a more opportunistic infection. LPS-tolerized macrophages, which are re-challenged by LPS after prior exposure to LPS, are regarded as the common model of hypo-responsiveness for SAIS. However, the molecular mechanisms of endotoxin tolerance and SAIS remain to be fully elucidated. In addition, negative regulation of the Toll-like receptor (TLR)-triggered innate inflammatory response needs further investigation. Here we show that expression of immune responsive gene 1 (IRG1) was highly up-regulated in the peripheral blood mononuclear cells of septic patients and in LPS-tolerized mouse macrophages. IRG1 significantly suppressed TLR-triggered production of proinflammatory cytokines TNF-alpha, IL-6, and IFN-beta in LPS-tolerized macrophages, with the elevated expression of reactive oxygen species (ROS) and A20. Moreover, ROS enhanced A20 expression by increasing the H3K4me3 modification of histone on the A20 promoter domain, and supplement of the ROS abrogated the IRG1 knockdown function in breaking endotoxin tolerance by increasing A20 expression. Our results demonstrate that inducible IRG1 promotes endotoxin tolerance by increasing A20 expression through ROS, indicating a new molecular mechanism regulating hypoinflammation of sepsis and endotoxin tolerance.

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Structure of the Lectin Mannose 6-Phosphate Receptor Homology (MRH) Domain of Glucosidase II, an Enzyme That Regulates Glycoprotein Folding Quality Control in the Endoplasmic Reticulum.

Publication Date: 2013 Jun 7 PMID: 23609449
Authors: Olson, L. J. - Orsi, R. - Alculumbre, S. G. - Peterson, F. C. - Stigliano, I. D. - Parodi, A. J. - D'Alessio, C. - Dahms, N. M.
Journal: J Biol Chem

Here we report for the first time the three-dimensional structure of a mannose 6-phosphate receptor homology (MRH) domain present in a protein with enzymatic activity, glucosidase II (GII). GII is involved in glycoprotein folding in the endoplasmic reticulum. GII removes the two innermost glucose residues from the Glc3Man9GlcNAc2 transferred to nascent proteins and the glucose added by UDP-Glc:glycoprotein glucosyltransferase. GII is composed of a catalytic GIIalpha subunit and a regulatory GIIbeta subunit. GIIbeta participates in the endoplasmic reticulum localization of GIIalpha and mediates in vivo enhancement of N-glycan trimming by GII through its C-terminal MRH domain. We determined the structure of a functional GIIbeta MRH domain by NMR spectroscopy. It adopts a beta-barrel fold similar to that of other MRH domains, but its binding pocket is the most shallow known to date as it accommodates a single mannose residue. In addition, we identified a conserved residue outside the binding pocket (Trp-409) present in GIIbeta but not in other MRHs that influences GII glucose trimming activity.

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Mycobacterium tuberculosis Maltosyltransferase GlgE, a Genetically Validated Antituberculosis Target, Is Negatively Regulated by Ser/Thr Phosphorylation.

Publication Date: 2013 Jun 7 PMID: 23609448
Authors: Leiba, J. - Syson, K. - Baronian, G. - Zanella-Cleon, I. - Kalscheuer, R. - Kremer, L. - Bornemann, S. - Molle, V.
Journal: J Biol Chem

GlgE is a maltosyltransferase involved in the biosynthesis of alpha-glucans that has been genetically validated as a potential therapeutic target against Mycobacterium tuberculosis. Despite also making alpha-glucan, the GlgC/GlgA glycogen pathway is distinct and allosterically regulated. We have used a combination of genetics and biochemistry to establish how the GlgE pathway is regulated. M. tuberculosis GlgE was phosphorylated specifically by the Ser/Thr protein kinase PknB in vitro on one serine and six threonine residues. Furthermore, GlgE was phosphorylated in vivo when expressed in Mycobacterium bovis bacillus Calmette-Guerin (BCG) but not when all seven phosphorylation sites were replaced by Ala residues. The GlgE orthologues from Mycobacterium smegmatis and Streptomyces coelicolor were phosphorylated by the corresponding PknB orthologues in vitro, implying that the phosphorylation of GlgE is widespread among actinomycetes. PknB-dependent phosphorylation of GlgE led to a 2 orders of magnitude reduction in catalytic efficiency in vitro. The activities of phosphoablative and phosphomimetic GlgE derivatives, where each phosphorylation site was substituted with either Ala or Asp residues, respectively, correlated with negative phosphoregulation. Complementation studies of a M. smegmatis glgE mutant strain with these GlgE derivatives, together with both classical and chemical forward genetics, were consistent with flux through the GlgE pathway being correlated with GlgE activity. We conclude that the GlgE pathway appears to be negatively regulated in actinomycetes through the phosphorylation of GlgE by PknB, a mechanism distinct from that known in the classical glycogen pathway. Thus, these findings open new opportunities to target the GlgE pathway therapeutically.

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Dectin-1 Activation Controls Maturation of beta-1,3-Glucan-containing Phagosomes.

Publication Date: 2013 May 31 PMID: 23609446
Authors: Mansour, M. K. - Tam, J. M. - Khan, N. S. - Seward, M. - Davids, P. J. - Puranam, S. - Sokolovska, A. - Sykes, D. B. - Dagher, Z. - Becker, C. - Tanne, A. - Reedy, J. L. - Stuart, L. M. - Vyas, J. M.
Journal: J Biol Chem

Elimination of fungal pathogens by phagocytes requires phagosome maturation, a process that involves the recruitment and fusion of intracellular proteins. The role of Dectin-1, a beta-1,3-glucan receptor, critical for fungal recognition and triggering of Th17 responses, to phagosomal maturation has not been defined. We show that GFP-Dectin-1 translocates to the fungal phagosome, but its signal decays after 2 h. Inhibition of acidification results in retention of GFP-Dectin-1 to phagosome membranes highlighting the requirement for an acidic pH. Following beta-1,3-glucan recognition, GFP-Dectin-1 undergoes tyrosine phosphorylation by Src kinases with subsequent Syk activation. Our results demonstrate that Syk is activated independently of intraphagosomal pH. Inhibition of Src or Syk results in prolonged retention of GFP-Dectin-1 to the phagosome signifying a link between Syk and intraphagosomal pH. beta-1,3-glucan phagosomes expressing a signaling incompetent Dectin-1 failed to mature as demonstrated by prolonged Dectin-1 retention, presence of Rab5B, failure to acquire LAMP-1 and inability to acidify. Phagosomes containing Candida albicans also require Dectin-1-dependent Syk activation for phagosomal maturation. Taken together, these results support a model where Dectin-1 not only controls internalization of beta-1,3-glucan containing cargo and triggers proinflammatory cytokines, but also acts as a master regulator for subsequent phagolysosomal maturation through Syk activation.

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YidC Occupies the Lateral Gate of the SecYEG Translocon and Is Sequentially Displaced by a Nascent Membrane Protein.

Publication Date: 2013 Jun 7 PMID: 23609445
Authors: Sachelaru, I. - Petriman, N. A. - Kudva, R. - Kuhn, P. - Welte, T. - Knapp, B. - Drepper, F. - Warscheid, B. - Koch, H. G.
Journal: J Biol Chem

Most membrane proteins are co-translationally inserted into the lipid bilayer via the universally conserved SecY complex and they access the lipid phase presumably via a lateral gate in SecY. In bacteria, the lipid transfer of membrane proteins from the SecY channel is assisted by the SecY-associated protein YidC, but details on the SecY-YidC interaction are unknown. By employing an in vivo and in vitro site-directed cross-linking approach, we have mapped the SecY-YidC interface and found YidC in contact with all four transmembrane domains of the lateral gate. This interaction did not require the SecDFYajC complex and was not influenced by SecA binding to SecY. In contrast, ribosomes dissociated the YidC contacts to lateral gate helices 2b and 8. The major contact between YidC and the lateral gate was lost in the presence of ribosome nascent chains and new SecY-YidC contacts appeared. These data demonstrate that the SecY-YidC interaction is influenced by nascent-membrane-induced lateral gate movements.

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Large lateral movement of transmembrane helix s5 is not required for substrate access to the active site of rhomboid intramembrane protease.

Publication Date: 2013 Jun 7 PMID: 23609444
Authors: Xue, Y. - Ha, Y.
Journal: J Biol Chem

Rhomboids represent an evolutionarily ancient protease family. Unlike most other proteases, they are polytopic membrane proteins and specialize in cleaving transmembrane protein substrates. The polar active site of rhomboid protease is embedded in the membrane and normally closed. For the bacterial rhomboid GlpG, it has been proposed that one of the transmembrane helices (S5) of the protease can rotate to open a lateral gate, enabling substrate to enter the protease from inside the membrane. Here, we studied the conformational change in GlpG by solving the cocrystal structure of the protease with a mechanism-based inhibitor. We also examined the lateral gating model by cross-linking S5 to a neighboring helix (S2). The crystal structure shows that inhibitor binding displaces a capping loop (L5) from the active site but causes only minor shifts in the transmembrane helices. Cross-linking S5 and S2, which not only restricts the lateral movement of S5 but also prevents substrate from passing between the two helices, does not hinder the ability of the protease to cleave a membrane protein substrate in detergent solution and in reconstituted membrane vesicles. Taken together, these data suggest that a large lateral movement of the S5 helix is not required for substrate access to the active site of rhomboid protease.

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Stabilization of the Conductive Conformation of a Voltage-gated K+ (Kv) Channel: THE LID MECHANISM.

Publication Date: 2013 Jun 7 PMID: 23609443
Authors: Santos, J. S. - Syeda, R. - Montal, M.
Journal: J Biol Chem

Voltage-gated K(+) (Kv) channels are molecular switches that sense membrane potential and in response open to allow K(+) ions to diffuse out of the cell. In these proteins, sensor and pore belong to two distinct structural modules. We previously showed that the pore module alone is a robust yet dynamic structural unit in lipid membranes and that it senses potential and gates open to conduct K(+) with unchanged fidelity. The implication is that the voltage sensitivity of K(+) channels is not solely encoded in the sensor. Given that the coupling between sensor and pore remains elusive, we asked whether it is then possible to convert a pore module characterized by brief openings into a conductor with a prolonged lifetime in the open state. The strategy involves selected probes targeted to the filter gate of the channel aiming to modulate the probability of the channel being open assayed by single channel recordings from the sensorless pore module reconstituted in lipid bilayers. Here we show that the premature closing of the pore is bypassed by association of the filter gate with two novel open conformation stabilizers: an antidepressant and a peptide toxin known to act selectively on Kv channels. Such stabilization of the conductive conformation of the channel is faithfully mimicked by the covalent attachment of fluorescein at a cysteine residue selectively introduced near the filter gate. This modulation prolongs the occupancy of permeant ions at the gate. It is this longer embrace between ion and gate that we conjecture underlies the observed stabilization of the conductive conformation. This study provides a new way of thinking about gating.

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Dynamic Structure of the Translocon SecYEG in Membrane: DIRECT SINGLE MOLECULE OBSERVATIONS.

Publication Date: 2013 Jun 7 PMID: 23609442
Authors: Sanganna Gari, R. R. - Frey, N. C. - Mao, C. - Randall, L. L. - King, G. M.
Journal: J Biol Chem

Purified SecYEG was reconstituted into liposomes and studied in near-native conditions using atomic force microscopy. These SecYEG proteoliposomes were active in translocation assays. Changes in the structure of SecYEG as a function of time were directly visualized. The dynamics observed were significant in magnitude ( approximately 1-10 A) and were attributed to the two large loops of SecY linking transmembrane helices 6-7 and 8-9. In addition, we identified a distribution between monomers and dimers of SecYEG as well as a smaller population of higher order oligomers. This work provides a new vista of the flexible and dynamic structure of SecYEG, an intricate and vital membrane protein.

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alpha-1,6-Fucosyltransferase (FUT8) Inhibits Hemoglobin Production during Differentiation of Murine and K562 Human Erythroleukemia Cells.

Publication Date: 2013 Jun 7 PMID: 23609441
Authors: Sasaki, H. - Toda, T. - Furukawa, T. - Mawatari, Y. - Takaesu, R. - Shimizu, M. - Wada, R. - Kato, D. - Utsugi, T. - Ohtsu, M. - Murakami, Y.
Journal: J Biol Chem

Erythropoiesis results from a complex combination of the expression of several transcription factor genes and cytokine signaling. However, the overall view of erythroid differentiation remains unclear. First, we screened for erythroid differentiation-related genes by comparing the expression profiles of high differentiation-inducible and low differentiation-inducible murine erythroleukemia cells. We identified that overexpression of alpha-1,6-fucosyltransferase (Fut8) inhibits hemoglobin production. FUT8 catalyzes the transfer of a fucose residue to N-linked oligosaccharides on glycoproteins via an alpha-1,6 linkage, leading to core fucosylation in mammals. Expression of Fut8 was down-regulated during chemically induced differentiation of murine erythroleukemia cells. Additionally, expression of Fut8 was positively regulated by c-Myc and c-Myb, which are known as suppressors of erythroid differentiation. Second, we found that FUT8 is the only fucosyltransferase family member that inhibits hemoglobin production. Functional analysis of FUT8 revealed that the donor substrate-binding domain and a flexible loop play essential roles in inhibition of hemoglobin production. This result clearly demonstrates that core fucosylation inhibits hemoglobin production. Third, FUT8 also inhibited hemoglobin production of human erythroleukemia K562 cells. Finally, a short hairpin RNA study showed that FUT8 down-regulation induced hemoglobin production and increase of transferrin receptor/glycophorin A-positive cells in human erythroleukemia K562 cells. Our findings define FUT8 as a novel factor for hemoglobin production and demonstrate that core fucosylation plays an important role in erythroid differentiation.

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Sphingolipid domains in the plasma membranes of fibroblasts are not enriched with cholesterol.

Publication Date: 2013 Jun 7 PMID: 23609440
Authors: Frisz, J. F. - Klitzing, H. A. - Lou, K. - Hutcheon, I. D. - Weber, P. K. - Zimmerberg, J. - Kraft, M. L.
Journal: J Biol Chem

The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. Thus, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize the cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton.

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Conserved Asp-137 Is Important for both Structure and Regulatory Functions of Cardiac alpha-Tropomyosin (alpha-TM) in a Novel Transgenic Mouse Model Expressing alpha-TM-D137L.

Publication Date: 2013 Jun 7 PMID: 23609439
Authors: Yar, S. - Chowdhury, S. A. - Davis, R. T. 3rd - Kobayashi, M. - Monasky, M. M. - Rajan, S. - Wolska, B. M. - Gaponenko, V. - Kobayashi, T. - Wieczorek, D. F. - Solaro, R. J.
Journal: J Biol Chem

alpha-Tropomyosin (alpha-TM) has a conserved, charged Asp-137 residue located in the hydrophobic core of its coiled-coil structure, which is unusual in that the residue is found at a position typically occupied by a hydrophobic residue. Asp-137 is thought to destabilize the coiled-coil and so impart structural flexibility to the molecule, which is believed to be crucial for its function in the heart. A previous in vitro study indicated that the conversion of Asp-137 to a more typical canonical Leu alters flexibility of TM and affects its in vitro regulatory functions. However, the physiological importance of the residue Asp-137 and altered TM flexibility is unknown. In this study, we further analyzed structural properties of the alpha-TM-D137L variant and addressed the physiological importance of TM flexibility in cardiac function in studies with a novel transgenic mouse model expressing alpha-TM-D137L in the heart. Our NMR spectroscopy data indicated that the presence of D137L introduced long range rearrangements in TM structure. Differential scanning calorimetry measurements demonstrated that alpha-TM-D137L has higher thermal stability compared with alpha-TM, which correlated with decreased flexibility. Hearts of transgenic mice expressing alpha-TM-D137L showed systolic and diastolic dysfunction with decreased myofilament Ca(2+) sensitivity and cardiomyocyte contractility without changes in intracellular Ca(2+) transients or post-translational modifications of major myofilament proteins. We conclude that conversion of the highly conserved Asp-137 to Leu results in loss of flexibility of TM that is important for its regulatory functions in mouse hearts. Thus, our results provide insight into the link between flexibility of TM and its function in ejecting hearts.

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The Intermediate Conductance Calcium-activated Potassium Channel KCa3.1 Regulates Vascular Smooth Muscle Cell Proliferation via Controlling Calcium-dependent Signaling.

Publication Date: 2013 May 31 PMID: 23609438
Authors: Bi, D. - Toyama, K. - Lemaitre, V. - Takai, J. - Fan, F. - Jenkins, D. P. - Wulff, H. - Gutterman, D. D. - Park, F. - Miura, H.
Journal: J Biol Chem

The intermediate conductance calcium-activated potassium channel KCa3.1 contributes to a variety of cell activation processes in pathologies such as inflammation, carcinogenesis, and vascular remodeling. We examined the electrophysiological and transcriptional mechanisms by which KCa3.1 regulates vascular smooth muscle cell (VSMC) proliferation. Platelet-derived growth factor-BB (PDGF)-induced proliferation of human coronary artery VSMCs was attenuated by lowering intracellular Ca(2+) concentration ([Ca(2+)]i) and was enhanced by elevating [Ca(2+)]i. KCa3.1 blockade or knockdown inhibited proliferation by suppressing the rise in [Ca(2+)]i and attenuating the expression of phosphorylated cAMP-response element-binding protein (CREB), c-Fos, and neuron-derived orphan receptor-1 (NOR-1). This antiproliferative effect was abolished by elevating [Ca(2+)]i. KCa3.1 overexpression induced VSMC proliferation, and potentiated PDGF-induced proliferation, by inducing CREB phosphorylation, c-Fos, and NOR-1. Pharmacological stimulation of KCa3.1 unexpectedly suppressed proliferation by abolishing the expression and activity of KCa3.1 and PDGF beta-receptors and inhibiting the rise in [Ca(2+)]i. The stimulation also attenuated the levels of phosphorylated CREB, c-Fos, and cyclin expression. After KCa3.1 blockade, the characteristic round shape of VSMCs expressing high l-caldesmon and low calponin-1 (dedifferentiation state) was maintained, whereas KCa3.1 stimulation induced a spindle-shaped cellular appearance, with low l-caldesmon and high calponin-1. In conclusion, KCa3.1 plays an important role in VSMC proliferation via controlling Ca(2+)-dependent signaling pathways, and its modulation may therefore constitute a new therapeutic target for cell proliferative diseases such as atherosclerosis.

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Structural Reevaluation of Streptococcus pneumoniae Lipoteichoic Acid and New Insights into Its Immunostimulatory Potency.

Publication Date: 2013 May 31 PMID: 23603911
Authors: Gisch, N. - Kohler, T. - Ulmer, A. J. - Muthing, J. - Pribyl, T. - Fischer, K. - Lindner, B. - Hammerschmidt, S. - Zahringer, U.
Journal: J Biol Chem

Streptococcus pneumoniae is a Gram-positive human pathogen with a complex lipoteichoic acid (pnLTA) structure. Because the current structural model for pnLTA shows substantial inconsistencies, we reinvestigated purified and, more importantly, O-deacylated pnLTA, which is most suitable for NMR spectroscopy and electrospray ionization-MS spectrometry. We analyzed pnLTA of nonencapsulated pneumococcal strains D39Deltacps and TIGR4Deltacps, respectively. The data obtained allowed us to (re)define (i) the position and linkage of the repeating unit, (ii) the putative alpha-GalpNAc substitution at the ribitiol 5-phosphate (Rib-ol-5-P), and (iii) the length of (i.e. the number of repeating units in) the pnLTA chain. We here also describe for the first time that the terminal sugar residues in the pnLTA (Forssman disaccharide; alpha-d-GalpNAc-(1-->3)-beta-d-GalpNAc-(1-->)), responsible for the cross-reactivity with anti-Forssman antigen antibodies, can be heterogeneous with respect to its degree of phosphorylcholine substitution in both O-6-positions. To assess the proinflammatory potency of pnLTA, we generated a (lipopeptide-free) Deltalgt mutant of strain D39Deltacps, isolated its pnLTA, and showed that it is capable of inducing IL-6 release in human mononuclear cells, independent of TLR2 activation. This finding was quite in contrast to LTA of the Staphylococcus aureus SA113Deltalgt mutant, which did not activate human mononuclear cells in our experiments. Remarkably, this is also contrary to various other reports showing a proinflammatory potency of S. aureus LTA. Taken together, our study refines the structure of pnLTA and indicates that pneumococcal and S. aureus LTAs differ not only in their structure but also in their bioactivity.

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Signal Relay by CC Chemokine Receptor 2 (CCR2) and Formylpeptide Receptor 2 (Fpr2) in the Recruitment of Monocyte-derived Dendritic Cells in Allergic Airway Inflammation.

Publication Date: 2013 Jun 7 PMID: 23603910
Authors: Chen, K. - Liu, M. - Liu, Y. - Wang, C. - Yoshimura, T. - Gong, W. - Le, Y. - Tessarollo, L. - Wang, J. M.
Journal: J Biol Chem

Chemoattractant receptors regulate leukocyte accumulation at sites of inflammation. In allergic airway inflammation, although a chemokine receptor CCR2 was implicated in mediating monocyte-derived dendritic cell (DC) recruitment into the lung, we previously also discovered reduced accumulation of DCs in the inflamed lung in mice deficient in formylpeptide receptor Fpr2 (Fpr2(-/-)). We therefore investigated the role of Fpr2 in the trafficking of monocyte-derived DCs in allergic airway inflammation in cooperation with CCR2. We report that in allergic airway inflammation, CCR2 mediated the recruitment of monocyte-derived DCs to the perivascular region, and Fpr2 was required for further migration of the cells into the bronchiolar area. We additionally found that the bronchoalveolar lavage liquid from mice with airway inflammation contained both the CCR2 ligand CCL2 and an Fpr2 agonist CRAMP. Furthermore, similar to Fpr2(-/-) mice, in the inflamed airway of CRAMP(-/-) mice, DC trafficking into the peribronchiolar areas was diminished. Our study demonstrates that the interaction of CCR2 and Fpr2 with their endogenous ligands sequentially mediates the trafficking of DCs within the inflamed lung.

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Ubiquitin-specific Protease 7 Is a Regulator of Ubiquitin-conjugating Enzyme UbE2E1.

Publication Date: 2013 Jun 7 PMID: 23603909
Authors: Sarkari, F. - Wheaton, K. - La Delfa, A. - Mohamed, M. - Shaikh, F. - Khatun, R. - Arrowsmith, C. H. - Frappier, L. - Saridakis, V. - Sheng, Y.
Journal: J Biol Chem

Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme found in all eukaryotes that catalyzes the removal of ubiquitin from specific target proteins. Here, we report that UbE2E1, an E2 ubiquitin conjugation enzyme with a unique N-terminal extension, is a novel USP7-interacting protein. USP7 forms a complex with UbE2E1 in vitro and in vivo through the ASTS USP7 binding motif within its N-terminal extension in an identical manner with other known USP7 binding proteins. We show that USP7 attenuates UbE2E1-mediated ubiquitination, an effect that requires the N-terminal ASTS sequence of UbE2E1 as well as the catalytic activity of USP7. Additionally, USP7 is critical in maintaining the steady state levels of UbE2E1 in cells. This study reveals a new cellular mechanism that couples the opposing activities of the ubiquitination machinery and a deubiquitinating enzyme to maintain and modulate the dynamic balance of the ubiquitin-proteasome system.

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Sulfo-N-succinimidyl Oleate (SSO) Inhibits Fatty Acid Uptake and Signaling for Intracellular Calcium via Binding CD36 Lysine 164: SSO ALSO INHIBITS OXIDIZED LOW DENSITY LIPOPROTEIN UPTAKE BY MACROPHAGES.

Publication Date: 2013 May 31 PMID: 23603908
Authors: Kuda, O. - Pietka, T. A. - Demianova, Z. - Kudova, E. - Cvacka, J. - Kopecky, J. - Abumrad, N. A.
Journal: J Biol Chem

FAT/CD36 is a multifunctional glycoprotein that facilitates long-chain fatty acid (FA) uptake by cardiomyocytes and adipocytes and uptake of oxidized low density lipoproteins (oxLDL) by macrophages. CD36 also mediates FA-induced signaling to increase intracellular calcium in various cell types. The membrane-impermeable sulfo-N-hydroxysuccinimidyl (NHS) ester of oleate (SSO) irreversibly binds CD36 and has been widely used to inhibit CD36-dependent FA uptake and signaling to calcium. The inhibition mechanism and whether SSO modification of CD36 involves the FA-binding site remain unexplored. CHO cells expressing human CD36 were SSO-treated, and the protein was pulled down, deglycosylated, and resolved by electrophoresis. The CD36 band was extracted from the gel and digested for analysis by mass spectrometry. NHS derivatives react with primary or secondary amines on proteins to yield stable amide or imide bonds. Two oleoylated peptides, found only in SSO-treated samples, were identified with high contribution and confidence scores as carrying oleate modification of Lys-164. Lysine 164 lies within a predicted CD36 binding domain for FA and oxLDL. CHO cells expressing CD36 with mutated Lys-164 had impaired CD36 function in FA uptake and FA-induced calcium release from the endoplasmic reticulum, supporting the importance of Lys-164 for both FA effects. Furthermore, consistent with the importance of Lys-164 for oxLDL binding, SSO inhibited oxLDL uptake by macrophages. In conclusion, SSO accesses Lys-164 in the FA-binding site on CD36, and initial modeling of this site is presented. The data suggest competition between FA and oxLDL for access to the CD36 binding pocket.

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FOXO3 Growth Inhibition of Colonic Cells Is Dependent on Intraepithelial Lipid Droplet Density.

Publication Date: 2013 Jun 7 PMID: 23603907
Authors: Qi, W. - Fitchev, P. S. - Cornwell, M. L. - Greenberg, J. - Cabe, M. - Weber, C. R. - Roy, H. K. - Crawford, S. E. - Savkovic, S. D.
Journal: J Biol Chem

Forkhead transcription factor FOXO3 plays a critical role in suppressing tumor growth, in part, by increasing the cell cycle inhibitor p27kip1, and Foxo3 deficiency in mice results in marked colonic epithelial proliferation. Here, we show in Foxo3-deficient colonic epithelial cells a striking increase in intracytoplasmic lipid droplets (LDs), a dynamic organelle recently observed in human tumor tissue. Although the regulation and function of LDs in non-adipocytes is unclear, we hypothesize that the anti-proliferative effect of FOXO3 was dependent on lowering LD density, thus decreasing fuel energy in both normal and colon cancer cells. In mouse colonic tumors, we found an increased expression of LD coat protein PLIN2 compared with normal colonic epithelial cells. Stimulation of LD density in human colon cancer cells led to a PI3K-dependent loss of FOXO3 and a decrease in the negative regulator of lipid metabolism in Sirtuin6 (SIRT6). Foxo3 deficiency also led to a decrease in SIRT6, revealing the existence of LD and FOXO3 feedback regulation in colonic cells. In parallel, LD-dependent loss of FOXO3 led to its dissociation from the promoter and decreased expression of the cell cycle inhibitor p27kip1. Stimulation of LD density promoted proliferation in colon cancer cells, whereas silencing PLIN2 or overexpression of FOXO3 inhibited proliferation. Taken together, FOXO3 and LDs might serve as new targets for therapeutic intervention of colon cancer.

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The Choline-binding Protein PspC of Streptococcus pneumoniae Interacts with the C-terminal Heparin-binding Domain of Vitronectin.

Publication Date: 2013 May 31 PMID: 23603906
Authors: Voss, S. - Hallstrom, T. - Saleh, M. - Burchhardt, G. - Pribyl, T. - Singh, B. - Riesbeck, K. - Zipfel, P. F. - Hammerschmidt, S.
Journal: J Biol Chem

Adherence of Streptococcus pneumoniae is directly mediated by interactions of adhesins with eukaryotic cellular receptors or indirectly by exploiting matrix and serum proteins as molecular bridges. Pneumococci engage vitronectin, the human adhesive glycoprotein and complement inhibitor, to facilitate attachment to epithelial cells of the mucosal cavity, thereby modulating host cell signaling. In this study, we identified PspC as a vitronectin-binding protein interacting with the C-terminal heparin-binding domain of vitronectin. PspC is a multifunctional surface-exposed choline-binding protein displaying various adhesive properties. Vitronectin binding required the R domains in the mature PspC protein, which are also essential for the interaction with the ectodomain of the polymeric immunoglobulin receptor and secretory IgA. Consequently, secretory IgA competitively inhibited binding of vitronectin to purified PspC and to PspC-expressing pneumococci. In contrast, Factor H, which binds to the N-terminal part of mature PspC molecules, did not interfere with the PspC-vitronectin interaction. Using a series of vitronectin peptides, the C-terminal heparin-binding domain was shown to be essential for the interaction of soluble vitronectin with PspC. Binding experiments with immobilized vitronectin suggested a region N-terminal to the identified heparin-binding domain as an additional binding region for PspC, suggesting that soluble, immobilized, as well as cellularly bound vitronectin possesses different conformations. Finally, vitronectin bound to PspC was functionally active and inhibited the deposition of the terminal complement complex. In conclusion, this study identifies and characterizes (on the molecular level) the interaction between the pneumococcal adhesin PspC and the human glycoprotein vitronectin.

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A Nucleus-targeted Alternately Spliced Nix/Bnip3L Protein Isoform Modifies Nuclear Factor kappaB (NFkappaB)-mediated Cardiac Transcription.

Publication Date: 2013 May 31 PMID: 23603904
Authors: Chen, Y. - Decker, K. F. - Zheng, D. - Matkovich, S. J. - Jia, L. - Dorn, G. W. 2nd
Journal: J Biol Chem

Several Bcl2 family proteins are expressed both as mitochondrial-targeted full-length and as cytosolic truncated alternately spliced isoforms. Recombinantly expressed shorter Bcl2 family isoforms can heterotypically bind to and prevent mitochondrial localization of their full-length analogs, thus suppressing their activity by sequestration. This "sponge" role requires 1:1 expression stoichiometry; absent this an alternate role is suggested. Here, RNA sequencing revealed coordinate regulation of BH3-only protein Nix/Bnip3L (Nix) and its alternately spliced soluble form (sNix) in hearts, but relative sNix/Nix expression of approximately 1:10. Accordingly, we examined other putative functions of sNix. Although Nix expressed in H9c2 rat myoblasts localized to mitochondria, sNix showed variable cytoplasmic and nuclear distribution. Tumor necrosis factor alpha (TNFalpha) induced rapid and complete sNix nucleoplasmic translocation concomitant with nuclear translocation of the p65/RelA subunit of NFkappaB. sNix co-localized and co-precipitated with p65/RelA after TNFalpha stimulation; TNFalpha-induced sNix nuclear translocation did not occur in p65/RelA null murine embryonic fibroblasts. ChIP sequencing of TNFalpha-stimulated H9c2 cells revealed sNix suppression of p65/RelA binding to a subset of weaker DNA binding sites, accounting for its ability to alter gene expression in cultured cells and in vivo mouse hearts. These findings reveal TNFalpha-stimulated cytoplasmic-nuclear shuttling of the alternately spliced non-mitochondrial Nix isoform and uncover a role for sNix as a modulator of TNFalpha/NFkappaB-stimulated cardiac gene expression. Transcriptional co-regulation of sNix and Nix, combined with sNix posttranslational regulation by TNFalpha, comprises a previously unknown mechanism for molecular cross-talk between extrinsic death receptor and intrinsic mitochondrial apoptosis pathways.

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Structure of the biliverdin cofactor in the pfr state of bathy and prototypical phytochromes.

Publication Date: 2013 Jun 7 PMID: 23603902
Authors: Salewski, J. - Escobar, F. V. - Kaminski, S. - von Stetten, D. - Keidel, A. - Rippers, Y. - Michael, N. - Scheerer, P. - Piwowarski, P. - Bartl, F. - Frankenberg-Dinkel, N. - Ringsdorf, S. - Gartner, W. - Lamparter, T. - Mroginski, M. A. - Hildebrandt, P.
Journal: J Biol Chem

Phytochromes act as photoswitches between the red- and far-red absorbing parent states of phytochromes (Pr and Pfr). Plant phytochromes display an additional thermal conversion route from the physiologically active Pfr to Pr. The same reaction pattern is found in prototypical biliverdin-binding bacteriophytochromes in contrast to the reverse thermal transformation in bathy bacteriophytochromes. However, the molecular origin of the different thermal stabilities of the Pfr states in prototypical and bathy bacteriophytochromes is not known. We analyzed the structures of the chromophore binding pockets in the Pfr states of various bathy and prototypical biliverdin-binding phytochromes using a combined spectroscopic-theoretical approach. For the Pfr state of the bathy phytochrome from Pseudomonas aeruginosa, the very good agreement between calculated and experimental Raman spectra of the biliverdin cofactor is in line with important conclusions of previous crystallographic analyses, particularly the ZZEssa configuration of the chromophore and its mode of covalent attachment to the protein. The highly homogeneous chromophore conformation seems to be a unique property of the Pfr states of bathy phytochromes. This is in sharp contrast to the Pfr states of prototypical phytochromes that display conformational equilibria between two sub-states exhibiting small structural differences at the terminal methine bridges A-B and C-D. These differences may mainly root in the interactions of the cofactor with the highly conserved Asp-194 that occur via its carboxylate function in bathy phytochromes. The weaker interactions via the carbonyl function in prototypical phytochromes may lead to a higher structural flexibility of the chromophore pocket opening a reaction channel for the thermal (ZZE --> ZZZ) Pfr to Pr back-conversion.

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Positive Feedback Regulation of Human Inducible Nitric-oxide Synthase Expression by Ras Protein S-Nitrosylation.

Publication Date: 2013 May 31 PMID: 23599434
Authors: Lee, M. - Choy, J. C.
Journal: J Biol Chem

The production of nitric oxide (NO) by inducible NO synthase (iNOS) regulates many aspects of physiology and pathology. The expression of iNOS needs to be tightly regulated to balance the broad ranging properties of NO. We have investigated the feedback regulation of cytokine-induced iNOS expression by NO in human cells. The pharmacological inhibition of iNOS activity reduced iNOS protein levels in response to cytokine stimulation in a human epithelial cell line (A549 cells) as well as in primary human astrocytes and bronchial epithelial cells. The addition of exogenous NO using a NO donor prevented the reduction in iNOS levels caused by blockade of iNOS activity. Examination of signaling pathways affected by iNOS indicated that NO S-nitrosylated Ras. Transfection of cells with a S-nitrosylation-resistant Ras mutant reduced iNOS protein levels, indicating a role for this Ras modification in the amplification of iNOS levels. Further, the induction of iNOS protein levels correlated with the late activation of the phosphatidylinositol 3-kinase/Akt and mammalian target of rapamycin (mTOR) pathways, and inhibition of these signaling molecules reduced iNOS levels. Altogether, our findings reveal a previously unknown regulatory pathway that amplifies iNOS expression in human cells.

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Heat Shock Protein 90 Functions to Stabilize and Activate the Testis-specific Serine/Threonine Kinases, a Family of Kinases Essential for Male Fertility.

Publication Date: 2013 Jun 7 PMID: 23599433
Authors: Jha, K. N. - Coleman, A. R. - Wong, L. - Salicioni, A. M. - Howcroft, E. - Johnson, G. R.
Journal: J Biol Chem

Spermiogenesis is characterized by a profound morphological differentiation of the haploid spermatid into spermatozoa. The testis-specific serine/threonine kinases (TSSKs) comprise a family of post-meiotic kinases expressed in spermatids, are critical to spermiogenesis, and are required for male fertility in mammals. To explore the role of heat shock protein 90 (HSP90) in regulation of TSSKs, the stability and catalytic activity of epitope-tagged murine TSSKs were assessed in 293T and COS-7 cells. TSSK1, -2, -4, and -6 (small serine/threonine kinase) were all found to associate with HSP90, and pharmacological inhibition of HSP90 function using the highly specific drugs 17-AAG, SNX-5422, or NVP-AUY922 reduced TSSK protein levels in cells. The attenuation of HSP90 function abolished the catalytic activities of TSSK4 and -6 but did not significantly alter the specific activities of TSSK1 and -2. Inhibition of HSP90 resulted in increased TSSK ubiquitination and proteasomal degradation, indicating that HSP90 acts to control ubiquitin-mediated catabolism of the TSSKs. To study HSP90 and TSSKs in germ cells, a mouse primary spermatid culture model was developed and characterized. Using specific antibodies against murine TSSK2 and -6, it was demonstrated that HSP90 inhibition resulted in a marked decrease of the endogenous kinases in spermatids. Together, our findings demonstrate that HSP90 plays a broad and critical role in stabilization and activation of the TSSK family of protein kinases.

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The Leucine-rich Pentatricopeptide Repeat-containing Protein (LRPPRC) Does Not Activate Transcription in Mammalian Mitochondria.

Publication Date: 2013 May 31 PMID: 23599432
Authors: Harmel, J. - Ruzzenente, B. - Terzioglu, M. - Spahr, H. - Falkenberg, M. - Larsson, N. G.
Journal: J Biol Chem

Regulation of mtDNA expression is critical for controlling oxidative phosphorylation capacity and has been reported to occur at several different levels in mammalian mitochondria. LRPPRC (leucine-rich pentatricopeptide repeat-containing protein) has a key role in this regulation and acts at the post-transcriptional level to stabilize mitochondrial mRNAs, to promote mitochondrial mRNA polyadenylation, and to coordinate mitochondrial translation. However, recent studies have suggested that LRPPRC may have an additional intramitochondrial role by directly interacting with the mitochondrial RNA polymerase POLRMT to stimulate mtDNA transcription. In this study, we have further examined the intramitochondrial roles for LRPPRC by creating bacterial artificial chromosome transgenic mice with moderately increased LRPPRC expression and heterozygous Lrpprc knock-out mice with moderately decreased LRPPRC expression. Variation of LRPPRC levels in mice in vivo, occurring within a predicted normal physiological range, strongly affected the levels of an unprocessed mitochondrial precursor transcript (ND5-cytochrome b) but had no effect on steady-state levels of mitochondrial transcripts or de novo transcription of mtDNA. We further assessed the role of LRPPRC in mitochondrial transcription by performing size exclusion chromatography and immunoprecipitation experiments in human cell lines and mice, but we found no interaction between LRPPRC and POLRMT. Furthermore, addition of purified LRPPRC to a recombinant human in vitro transcription system did not activate mtDNA transcription. On the basis of these data, we conclude that LRPPRC does not directly regulate mtDNA transcription but rather acts as a post-transcriptional regulator of mammalian mtDNA expression.

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Induction of C-X-C Chemokine Receptor Type 7 (CXCR7) Switches Stromal Cell-derived Factor-1 (SDF-1) Signaling and Phagocytic Activity in Macrophages Linked to Atherosclerosis.

Publication Date: 2013 May 31 PMID: 23599431
Authors: Ma, W. - Liu, Y. - Ellison, N. - Shen, J.
Journal: J Biol Chem

The discovery of CXCR7 as a new receptor for SDF-1 places many previously described SDF-1 functions attributed to CXCR4 in question, though whether CXCR7 acts as a signaling or "decoy" receptor has been in debate. It is known that CXCR7 is not expressed in normal blood leukocytes; however, the potential role of leukocyte CXCR7 in disease states has not been addressed. The aim of this study was to determine the expression and function of macrophage CXCR7 linked to atherosclerosis. Here, we show that CXCR7 was detected in macrophage-positive area of aortic atheroma of ApoE-null mice, but not in healthy aorta. During monocyte differentiation to macrophages, CXCR7 was up-regulated at mRNA and protein levels, with more expression in M1 than in M2 phenotype. In addition, CXCR7 induction was associated with a SDF-1 signaling switch from the pro-survival ERK and AKT pathways in monocytes to the pro-inflammatory JNK and p38 pathways in macrophages. The latter effect was mimicked by a CXCR7-selective agonist TC14012 and abolished by siRNA knockdown of CXCR7. Furthermore, CXCR7 activation increased macrophage phagocytic activity, which was suppressed by CXCR7 siRNA silencing or by inhibiting either the JNK or p38 pathways, but was not affected by blocking CXCR4. Finally, activation of CXCR7 by I-TAC showed a similar signaling and phagocytic activity in macrophages with no detectable CXCR3. We conclude that CXCR7 is induced during monocyte-to-macrophage differentiation, which is required for SDF-1 and I-TAC signaling to JNK and p38 pathways, leading to enhanced macrophage phagocytosis, thus possibly contributing to atherogenesis.

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Mild Electrical Stimulation at 0.1-ms Pulse Width Induces p53 Protein Phosphorylation and G2 Arrest in Human Epithelial Cells.

Publication Date: 2013 May 31 PMID: 23599430
Authors: Fukuda, R. - Suico, M. A. - Koyama, K. - Omachi, K. - Kai, Y. - Matsuyama, S. - Mitsutake, K. - Taura, M. - Morino-Koga, S. - Shuto, T. - Kai, H.
Journal: J Biol Chem

Exogenous low-intensity electrical stimulation has been used for treatment of various intractable diseases despite the dearth of information on the molecular underpinnings of its effects. Our work and that of others have demonstrated that applied electrical stimulation at physiological strength or mild electrical stimulation (MES) activates the PI3K-Akt pathway, but whether MES activates other molecules remains unknown. Considering that MES is a form of physiological stress, we hypothesized that it can activate the tumor suppressor p53, which is a key modulator of the cell cycle and apoptosis in response to cell stresses. The potential response of p53 to an applied electrical current of low intensity has not been investigated. Here, we show that p53 was transiently phosphorylated at Ser-15 in epithelial cells treated with an imperceptible voltage (1 V/cm) and a 0.1-ms pulse width. MES-induced p53 phosphorylation was inhibited by pretreatment with a p38 MAPK inhibitor and transfection of dominant-negative mutants of p38, MKK3b, and MKK6b, implying the involvement of the p38 MAPK signaling pathway. Furthermore, MES treatment enhanced p53 transcriptional function and increased the expression of p53 target genes p21, BAX, PUMA, NOXA, and IRF9. Importantly, MES treatment triggered G2 cell cycle arrest, but not cell apoptosis. MES treatment had no effect on the cell cycle in HCT116 p53(-/-) cells, suggesting a dependence on p53. These findings identify some molecular targets of electrical stimulation and incorporate the p38-p53 signaling pathway among the transduction pathways that MES affects.

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Mechanisms of mitochondrial damage in keratinocytes by pemphigus vulgaris antibodies.

Publication Date: 2013 Jun 7 PMID: 23599429
Authors: Kalantari-Dehaghi, M. - Chen, Y. - Deng, W. - Chernyavsky, A. - Marchenko, S. - Wang, P. H. - Grando, S. A.
Journal: J Biol Chem

The development of nonhormonal treatment of pemphigus vulgaris (PV) has been hampered by a lack of clear understanding of the mechanisms leading to keratinocyte (KC) detachment and death in pemphigus. In this study, we sought to identify changes in the vital mitochondrial functions in KCs treated with the sera from PV patients and healthy donors. PV sera significantly increased proton leakage from KCs, suggesting that PV IgGs increase production of reactive oxygen species. Indeed, measurement of intracellular reactive oxygen species production showed a drastic increase of cell staining in response to treatment by PV sera, which was confirmed by FACS analysis. Exposure of KCs to PV sera also caused dramatic changes in the mitochondrial membrane potential detected with the JC-1 dye. These changes can trigger the mitochondria-mediated intrinsic apoptosis. Although sera from different PV patients elicited unique patterns of mitochondrial damage, the mitochondria-protecting drugs nicotinamide (also called niacinamide), minocycline, and cyclosporine A exhibited a uniform protective effect. Their therapeutic activity was validated in the passive transfer model of PV in neonatal BALB/c mice. The highest efficacy of mitochondrial protection of the combination of these drugs found in mitochondrial assay was consistent with the ability of the same drug combination to abolish acantholysis in mouse skin. These findings provide a theoretical background for clinical reports of the efficacy of mitochondria-protecting drugs in PV patients. Pharmacological protection of mitochondria and/or compensation of an altered mitochondrial function may therefore become a novel approach to development of personalized nonhormonal therapies of patients with this potentially lethal autoimmune blistering disease.

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beta-Amyloid (Abeta) Oligomers Impair Brain-derived Neurotrophic Factor Retrograde Trafficking by Down-regulating Ubiquitin C-terminal Hydrolase, UCH-L1.

Publication Date: 2013 Jun 7 PMID: 23599427
Authors: Poon, W. W. - Carlos, A. J. - Aguilar, B. L. - Berchtold, N. C. - Kawano, C. K. - Zograbyan, V. - Yaopruke, T. - Shelanski, M. - Cotman, C. W.
Journal: J Biol Chem

We previously found that BDNF-dependent retrograde trafficking is impaired in AD transgenic mouse neurons. Utilizing a novel microfluidic culture chamber, we demonstrate that Abeta oligomers compromise BDNF-mediated retrograde transport by impairing endosomal vesicle velocities, resulting in impaired downstream signaling driven by BDNF/TrkB, including ERK5 activation, and CREB-dependent gene regulation. Our data suggest that a key mechanism mediating the deficit involves ubiquitin C-terminal hydrolase L1 (UCH-L1), a deubiquitinating enzyme that functions to regulate cellular ubiquitin. Abeta-induced deficits in BDNF trafficking and signaling are mimicked by LDN (an inhibitor of UCH-L1) and can be reversed by increasing cellular UCH-L1 levels, demonstrated here using a transducible TAT-UCH-L1 strategy. Finally, our data reveal that UCH-L1 mRNA levels are decreased in the hippocampi of AD brains. Taken together, our data implicate that UCH-L1 is important for regulating neurotrophin receptor sorting to signaling endosomes and supporting retrograde transport. Further, our results support the idea that in AD, Abeta may down-regulate UCH-L1 in the AD brain, which in turn impairs BDNF/TrkB-mediated retrograde signaling, compromising synaptic plasticity and neuronal survival.

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Innate Immunity in Pluripotent Human Cells: ATTENUATED RESPONSE TO INTERFERON-beta.

Publication Date: 2013 May 31 PMID: 23599426
Authors: Hong, X. X. - Carmichael, G. G.
Journal: J Biol Chem

Type I interferon (IFN-alpha/beta) binds to cell surface receptors IFNAR1 and IFNAR2 and triggers a signaling cascade that leads to the transcription of hundreds of IFN-stimulated genes. This response is a crucial component in innate immunity in that it establishes an "antiviral state" in cells and protects them against further damage. Previous work demonstrated that, compared with their differentiated counterparts, pluripotent human cells have a much weaker response to cytoplasmic double-stranded RNA (dsRNA) and are only able to produce a minimal amount of IFN-beta. We show here that human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) also exhibit an attenuated response to IFN-beta. Even though all known type I IFN signaling components are expressed in these cells, STAT1 phosphorylation is greatly diminished upon IFN-beta treatment. This attenuated response correlates with a high expression of suppressor of cytokine signaling 1 (SOCS1). Upon differentiation of hESCs into trophoblasts, cells acquire the ability to respond to IFN-beta, and this is accompanied by a significant induction of STAT1 phosphorylation as well as a decrease in SOCS1 expression. Furthermore, SOCS1 knockdown in hiPSCs enhances their ability to respond to IFN-beta. Taken together, our results suggest that an attenuated cellular response to type I IFNs may be a general feature of pluripotent human cells and that this is associated with high expression of SOCS1.

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Yeast Pif1 Helicase Exhibits a One-base-pair Stepping Mechanism for Unwinding Duplex DNA.

Publication Date: 2013 May 31 PMID: 23596008
Authors: Ramanagoudr-Bhojappa, R. - Chib, S. - Byrd, A. K. - Aarattuthodiyil, S. - Pandey, M. - Patel, S. S. - Raney, K. D.
Journal: J Biol Chem

Kinetic analysis of the DNA unwinding and translocation activities of helicases is necessary for characterization of the biochemical mechanism(s) for this class of enzymes. Saccharomyces cerevisiae Pif1 helicase was characterized using presteady state kinetics to determine rates of DNA unwinding, displacement of streptavidin from biotinylated DNA, translocation on single-stranded DNA (ssDNA), and ATP hydrolysis activities. Unwinding of substrates containing varying duplex lengths was fit globally to a model for stepwise unwinding and resulted in an unwinding rate of approximately 75 bp/s and a kinetic step size of 1 base pair. Pif1 is capable of displacing streptavidin from biotinylated oligonucleotides with a linear increase in the rates as the length of the oligonucleotides increased. The rate of translocation on ssDNA was determined by measuring dissociation from varying lengths of ssDNA and is essentially the same as the rate of unwinding of dsDNA, making Pif1 an active helicase. The ATPase activity of Pif1 on ssDNA was determined using fluorescently labeled phosphate-binding protein to measure the rate of phosphate release. The quantity of phosphate released corresponds to a chemical efficiency of 0.84 ATP/nucleotides translocated. Hence, when all of the kinetic data are considered, Pif1 appears to move along DNA in single nucleotide or base pair steps, powered by hydrolysis of 1 molecule of ATP.

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Examining the Role of the HIV-1 Reverse Transcriptase p51 Subunit in Positioning and Hydrolysis of RNA/DNA Hybrids.

Publication Date: 2013 May 31 PMID: 23595992
Authors: Chung, S. - Miller, J. T. - Lapkouski, M. - Tian, L. - Yang, W. - Le Grice, S. F.
Journal: J Biol Chem

Recent crystallographic analysis of p66/p51 human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT) complexed with a non-polypurine tract RNA/DNA hybrid has illuminated novel and important contacts between structural elements at the C terminus of the noncatalytic p51 subunit and the nucleic acid duplex in the vicinity of the ribonuclease H (RNase H) active site. In particular, a short peptide spanning residues Phe-416-Pro-421 was shown to interact with the DNA strand, cross the minor groove of the helix, and then form Van der Waals contacts with the RNA strand adjacent to the scissile phosphate. At the base of the adjoining alpha-helix M', Tyr-427 forms a hydrogen bond with Asn-348, the latter of which, when mutated to Ile, is implicated in resistance to both nucleoside and non-nucleoside RT inhibitors. Based on our structural data, we analyzed the role of the p51 C terminus by evaluating selectively mutated p66/p51 heterodimers carrying (i) p51 truncations that encroach on alpha-M', (ii) alterations that interrupt the Asn-348-Tyr-427 interaction, and (iii) alanine substitutions throughout the region Phe-416-Pro-421. Collectively, our data support the notion that the p51 C terminus makes an important contribution toward hybrid binding and orienting the RNA strand for catalysis at the RNase H active site.

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Macro histone variants are critical for the differentiation of human pluripotent cells.

Publication Date: 2013 May 31 PMID: 23595991
Authors: Barrero, M. J. - Sese, B. - Marti, M. - Izpisua Belmonte, J. C.
Journal: J Biol Chem

We have previously shown that macro histone variants (macroH2A) are expressed at low levels in stem cells and are up-regulated during differentiation. Here we show that the knockdown of macro histone variants impaired the in vitro and in vivo differentiation of human pluripotent cells, likely through defects in the silencing of pluripotency-related genes. ChIP experiments showed that during differentiation macro histone variants are recruited to the regulatory regions of pluripotency and developmental genes marked with H3K27me3 contributing to the silencing of these genes.

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Regulation of a Dynamic Interaction between Two Microtubule-binding Proteins, EB1 and TIP150, by the Mitotic p300/CBP-associated Factor (PCAF) Orchestrates Kinetochore Microtubule Plasticity and Chromosome Stability during Mitosis.

Publication Date: 2013 May 31 PMID: 23595990
Authors: Ward, T. - Wang, M. - Liu, X. - Wang, Z. - Xia, P. - Chu, Y. - Wang, X. - Liu, L. - Jiang, K. - Yu, H. - Yan, M. - Wang, J. - Hill, D. L. - Huang, Y. - Zhu, T. - Yao, X.
Journal: J Biol Chem

The microtubule cytoskeleton network orchestrates cellular dynamics and chromosome stability in mitosis. Although tubulin acetylation is essential for cellular plasticity, it has remained elusive how kinetochore microtubule plus-end dynamics are regulated by p300/CBP-associated factor (PCAF) acetylation in mitosis. Here, we demonstrate that the plus-end tracking protein, TIP150, regulates dynamic kinetochore-microtubule attachments by promoting the stability of spindle microtubule plus-ends. Suppression of TIP150 by siRNA results in metaphase alignment delays and perturbations in chromosome biorientation. TIP150 is a tetramer that binds an end-binding protein (EB1) dimer through the C-terminal domains, and overexpression of the C-terminal TIP150 or disruption of the TIP150-EB1 interface by a membrane-permeable peptide perturbs chromosome segregation. Acetylation of EB1-PCAF regulates the TIP150 interaction, and persistent acetylation perturbs EB1-TIP150 interaction and accurate metaphase alignment, resulting in spindle checkpoint activation. Suppression of the mitotic checkpoint serine/threonine protein kinase, BubR1, overrides mitotic arrest induced by impaired EB1-TIP150 interaction, but cells exhibit whole chromosome aneuploidy. Thus, the results identify a mechanism by which the TIP150-EB1 interaction governs kinetochore microtubule plus-end plasticity and establish that the temporal control of the TIP150-EB1 interaction by PCAF acetylation ensures chromosome stability in mitosis.

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Ligand-bound Thyroid Hormone Receptor Contributes to Reprogramming of Pancreatic Acinar Cells into Insulin-producing Cells.

Publication Date: 2013 May 31 PMID: 23595988
Authors: Furuya, F. - Shimura, H. - Asami, K. - Ichijo, S. - Takahashi, K. - Kaneshige, M. - Oikawa, Y. - Aida, K. - Endo, T. - Kobayashi, T.
Journal: J Biol Chem

One goal of diabetic regenerative medicine is to instructively convert mature pancreatic exocrine cells into insulin-producing cells. We recently reported that ligand-bound thyroid hormone receptor alpha (TRalpha) plays a critical role in expansion of the beta-cell mass during postnatal development. Here, we used an adenovirus vector that expresses TRalpha driven by the amylase 2 promoter (AdAmy2TRalpha) to induce the reprogramming of pancreatic acinar cells into insulin-producing cells. Treatment with l-3,5,3-triiodothyronine increases the association of TRalpha with the p85alpha subunit of phosphatidylinositol 3-kinase (PI3K), leading to the phosphorylation and activation of Akt and the expression of Pdx1, Ngn3, and MafA in purified acinar cells. Analyses performed with the lectin-associated cell lineage tracing system and the Cre/loxP-based direct cell lineage tracing system indicate that newly synthesized insulin-producing cells originate from elastase-expressing pancreatic acinar cells. Insulin-containing secretory granules were identified in these cells by electron microscopy. The inhibition of p85alpha expression by siRNA or the inhibition of PI3K by LY294002 prevents the expression of Pdx1, Ngn3, and MafA and the reprogramming to insulin-producing cells. In immunodeficient mice with streptozotocin-induced hyperglycemia, treatment with AdAmy2TRalpha leads to the reprogramming of pancreatic acinar cells to insulin-producing cells in vivo. Our findings suggest that ligand-bound TRalpha plays a critical role in beta-cell regeneration during postnatal development via activation of PI3K signaling.

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cAMP-responsive Element-binding Protein (CREB)-regulated Transcription Coactivator 2 (CRTC2) Promotes Glucagon Clearance and Hepatic Amino Acid Catabolism to Regulate Glucose Homeostasis.

Publication Date: 2013 May 31 PMID: 23595987
Authors: Erion, D. M. - Kotas, M. E. - McGlashon, J. - Yonemitsu, S. - Hsiao, J. J. - Nagai, Y. - Iwasaki, T. - Murray, S. F. - Bhanot, S. - Cline, G. W. - Samuel, V. T. - Shulman, G. I. - Gillum, M. P.
Journal: J Biol Chem

cAMP-responsive element-binding protein (CREB)-regulated transcription coactivator 2 (CRTC2) regulates transcription of gluconeogenic genes by specifying targets for the transcription factor CREB in response to glucagon. We used an antisense oligonucleotide directed against CRTC2 in both normal rodents and in rodent models of increased gluconeogenesis to better understand the role of CRTC2 in metabolic disease. In the context of severe hyperglycemia and elevated hepatic glucose production, CTRC2 knockdown (KD) improved glucose homeostasis by reducing endogenous glucose production. Interestingly, despite the known role of CRTC2 in coordinating gluconeogenic gene expression, CRTC2 KD in a rodent model of type 2 diabetes resulted in surprisingly little alteration of glucose production. However, CRTC2 KD animals had elevated circulating concentrations of glucagon and a approximately 80% reduction in glucagon clearance. When this phenomenon was prevented with somatostatin or a glucagon-neutralizing antibody, endogenous glucose production was reduced by CRTC2 KD. Additionally, CRTC2 inhibition resulted in reduced expression of several glucagon-induced pyridoxal 5'-phosphate-dependent enzymes that convert amino acids to gluconeogenic intermediates, suggesting that it may control substrate availability as well as gluconeogenic gene expression. CRTC2 is an important regulator of gluconeogenesis with tremendous impact in models of elevated hepatic glucose production. Surprisingly, it is also part of a previously unidentified negative feedback loop that degrades glucagon and regulates amino acid metabolism to coordinately control glucose homeostasis in vivo.

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Targeted Deletion of Growth Hormone (GH) Receptor in Macrophage Reveals Novel Osteopontin-mediated Effects of GH on Glucose Homeostasis and Insulin Sensitivity in Diet-induced Obesity.

Publication Date: 2013 May 31 PMID: 23595986
Authors: Lu, C. - Kumar, P. A. - Sun, J. - Aggarwal, A. - Fan, Y. - Sperling, M. A. - Lumeng, C. N. - Menon, R. K.
Journal: J Biol Chem

We investigated GH action on macrophage (MPhi) by creating a MPhi-specific GH receptor-null mouse model (MacGHR KO). On a normal diet (10% fat), MacGHR KO and littermate controls exhibited similar growth profiles and glucose excursions on intraperitoneal glucose (ipGTT) and insulin tolerance (ITT) tests. However, when challenged with high fat diet (HFD, 45% fat) for 18 weeks, MacGHR KO mice exhibited impaired ipGTT and ITT compared with controls. In MacGHR KO, adipose-tissue (AT) MPhi abundance was increased with skewing toward M1 polarization. Expression of pro-inflammatory cytokines (IL1beta, TNF-alpha, IL6, and osteopontin (OPN)) were increased in MacGHR KO AT stromal vascular fraction (SVF). In MacGHR KO AT, crown-like-structures were increased with decreased insulin-dependent Akt phosphorylation. The abundance of phosphorylated NF-kappaB and of OPN was increased in SVF and bone-marrow-derived MPhi in MacGHR KO. GH, acting via an NF-kappaB site in the distal OPN promoter, inhibited the OPN promoter. Thus in diet-induced obesity (DIO), lack of GH action on the MPhi exerts an unexpected deleterious effect on glucose homeostasis by accentuating AT inflammation and NF-kappaB-dependent activation of OPN expression. These novel results in mice support the possibility that administration of GH could have salutary effects on DIO-associated chronic inflammation and insulin resistance in humans.

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The Kindlin 3 Pleckstrin Homology Domain Has an Essential Role in Lymphocyte Function-associated Antigen 1 (LFA-1) Integrin-mediated B Cell Adhesion and Migration.

Publication Date: 2013 May 24 PMID: 23595985
Authors: Hart, R. - Stanley, P. - Chakravarty, P. - Hogg, N.
Journal: J Biol Chem

The protein kindlin 3 is mutated in the leukocyte adhesion deficiency III (LAD-III) disorder, leading to widespread infection due to the failure of leukocytes to migrate into infected tissue sites. To gain understanding of how kindlin 3 controls leukocyte function, we have focused on its pleckstrin homology (PH) domain and find that deletion of this domain eliminates the ability of kindlin 3 to participate in adhesion and migration of B cells mediated by the leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1). PH domains are often involved in membrane localization of proteins through binding to phosphoinositides. We show that the kindlin 3 PH domain has binding affinity for phosphoinositide PI(3,4,5)P3 over PI(4,5)P2. It has a major role in membrane association of kindlin 3 that is enhanced by the binding of LFA-1 to intercellular adhesion molecule 1 (ICAM-1). A splice variant, kindlin 3-IPRR, has a four-residue insert in the PH domain at a critical site that influences phosphoinositide binding by enhancing binding to PI(4,5)P2 as well as by binding to PI(3,4,5)P3. However kindlin 3-IPRR is unable to restore the ability of LAD-III B cells to adhere to and migrate on LFA-1 ligand ICAM-1, potentially by altering the dynamics or PI specificity of binding to the membrane. Thus, the correct functioning of the kindlin 3 PH domain is central to the role that kindlin 3 performs in guiding lymphocyte adhesion and motility behavior, which in turn is required for a successful immune response.

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Mutant Copper-Zinc Superoxide Dismutase (SOD1) Induces Protein Secretion Pathway Alterations and Exosome Release in Astrocytes: IMPLICATIONS FOR DISEASE SPREADING AND MOTOR NEURON PATHOLOGY IN AMYOTROPHIC LATERAL SCLEROSIS.

Publication Date: 2013 May 31 PMID: 23592792
Authors: Basso, M. - Pozzi, S. - Tortarolo, M. - Fiordaliso, F. - Bisighini, C. - Pasetto, L. - Spaltro, G. - Lidonnici, D. - Gensano, F. - Battaglia, E. - Bendotti, C. - Bonetto, V.
Journal: J Biol Chem

Amyotrophic lateral sclerosis is the most common motor neuron disease and is still incurable. The mechanisms leading to the selective motor neuron vulnerability are still not known. The interplay between motor neurons and astrocytes is crucial in the outcome of the disease. We show that mutant copper-zinc superoxide dismutase (SOD1) overexpression in primary astrocyte cultures is associated with decreased levels of proteins involved in secretory pathways. This is linked to a general reduction of total secreted proteins, except for specific enrichment in a number of proteins in the media, such as mutant SOD1 and valosin-containing protein (VCP)/p97. Because there was also an increase in exosome release, we can deduce that astrocytes expressing mutant SOD1 activate unconventional secretory pathways, possibly as a protective mechanism. This may help limit the formation of intracellular aggregates and overcome mutant SOD1 toxicity. We also found that astrocyte-derived exosomes efficiently transfer mutant SOD1 to spinal neurons and induce selective motor neuron death. We conclude that the expression of mutant SOD1 has a substantial impact on astrocyte protein secretion pathways, contributing to motor neuron pathology and disease spread.

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Rationally Designed Transmembrane Peptide Mimics of the Multidrug Transporter Protein Cdr1 Act as Antagonists to Selectively Block Drug Efflux and Chemosensitize Azole-resistant Clinical Isolates of Candida albicans.

Publication Date: 2013 Jun 7 PMID: 23592791
Authors: Maurya, I. K. - Thota, C. K. - Verma, S. D. - Sharma, J. - Rawal, M. K. - Ravikumar, B. - Sen, S. - Chauhan, N. - Lynn, A. M. - Chauhan, V. S. - Prasad, R.
Journal: J Biol Chem

Drug-resistant pathogenic fungi use several families of membrane-embedded transporters to efflux antifungal drugs from the cells. The efflux pump Cdr1 (Candida drug resistance 1) belongs to the ATP-binding cassette (ABC) superfamily of transporters. Cdr1 is one of the most predominant mechanisms of multidrug resistance in azole-resistant (AR) clinical isolates of Candida albicans. Blocking drug efflux represents an attractive approach to combat the multidrug resistance of this opportunistic human pathogen. In this study, we rationally designed and synthesized transmembrane peptide mimics (TMPMs) of Cdr1 protein (Cdr1p) that correspond to each of the 12 transmembrane helices (TMHs) of the two transmembrane domains of the protein to target the primary structure of the Cdr1p. Several FITC-tagged TMPMs specifically bound to Cdr1p and blocked the efflux of entrapped fluorescent dyes from the AR (Gu5) isolate. These TMPMs did not affect the efflux of entrapped fluorescent dye from cells expressing the Cdr1p homologue Cdr2p or from cells expressing a non-ABC transporter Mdr1p. Notably, the time correlation of single photon counting fluorescence measurements confirmed the specific interaction of FITC-tagged TMPMs with their respective TMH. By using mutant variants of Cdr1p, we show that these TMPM antagonists contain the structural information necessary to target their respective TMHs of Cdr1p and specific binding sites that mediate the interactions between the mimics and its respective helix. Additionally, TMPMs that were devoid of any demonstrable hemolytic, cytotoxic, and antifungal activities chemosensitize AR clinical isolates and demonstrate synergy with drugs that further improved the therapeutic potential of fluconazole in vivo.

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Diabetes-induced Central Neuritic Dystrophy and Cognitive Deficits Are Associated with the Formation of Oligomeric Reticulon-3 via Oxidative Stress.

Publication Date: 2013 May 31 PMID: 23592790
Authors: Zhao, B. - Pan, B. S. - Shen, S. W. - Sun, X. - Hou, Z. Z. - Yan, R. - Sun, F. Y.
Journal: J Biol Chem

Diabetes is a high risk factor to dementia. To investigate the molecular mechanism of diabetic dementia, we induced type 2 diabetes in rats and examined potential changes in their cognitive functions and the neural morphology of the brains. We found that the diabetic rats with an impairment of spatial learning and memory showed the occurrence of RTN3-immunoreactive dystrophic neurites in the cortex. Biochemical examinations revealed the increase of a high molecular weight form of RTN3 (HW-RTN3) in diabetic brains. The corresponding decrease of monomeric RTN3 was correlated with the reduction of its inhibitory effects on the activity of beta-secretase (BACE1), a key enzyme for generation of beta-amyloid peptides. The results from immunoprecipitation combined with protein carbonyl detection showed that carbonylated RTN3 was significantly higher in cortical tissues of diabetic rats compared with control rats, indicating that diabetes-induced oxidative stress led to RTN3 oxidative damage. In neuroblastoma SH-SY5Y cells, high glucose and/or H2O2 treatment significantly increased the amounts of carbonylated proteins and HW-RTN3, whereas monomeric RTN3 was reduced. Hence, we conclude that diabetes-induced cognitive deficits and central neuritic dystrophy are correlated with the formation of aggregated RTN3 via oxidative stress. We provided the first evidence that oxidative damage caused the formation of toxic RTN3 aggregates, which participated in the pathogenesis of central neuritic dystrophy in diabetic brain. Present findings may offer a new therapeutic strategy to prevent or reduce diabetic dementia.

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Mutation of Light-dependent Phosphorylation Sites of the Drosophila Transient Receptor Potential-like (TRPL) Ion Channel Affects Its Subcellular Localization and Stability.

Publication Date: 2013 May 31 PMID: 23592784
Authors: Cerny, A. C. - Oberacker, T. - Pfannstiel, J. - Weigold, S. - Will, C. - Huber, A.
Journal: J Biol Chem

The Drosophila phototransduction cascade terminates in the opening of the ion channel transient receptor potential (TRP) and TRP-like (TRPL). Contrary to TRP, TRPL undergoes light-dependent subcellular trafficking between rhabdomeric photoreceptor membranes and an intracellular storage compartment, resulting in long term light adaptation. Here, we identified in vivo phosphorylation sites of TRPL that affect TRPL stability and localization. Quantitative mass spectrometry revealed a light-dependent change in the TRPL phosphorylation pattern. Mutation of eight C-terminal phosphorylation sites neither affected multimerization of the channels nor the electrophysiological response of flies expressing the mutated channels. However, these mutations resulted in mislocalization and enhanced degradation of TRPL after prolonged dark-adaptation. Mutation of subsets of the eight C-terminal phosphorylation sites also led to a reduction of TRPL content and partial mislocalization in the dark. This suggests that a light-dependent switch in the phosphorylation pattern of the TRPL channel mediates stable expression of TRPL in the rhabdomeres upon prolonged dark-adaptation.

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A Molecular Switch Governs the Interaction between the Human Complement Protease C1s and Its Substrate, Complement C4.

Publication Date: 2013 May 31 PMID: 23592783
Authors: Perry, A. J. - Wijeyewickrema, L. C. - Wilmann, P. G. - Gunzburg, M. J. - D'Andrea, L. - Irving, J. A. - Pang, S. S. - Duncan, R. C. - Wilce, J. A. - Whisstock, J. C. - Pike, R. N.
Journal: J Biol Chem

The complement system is an ancient innate immune defense pathway that plays a front line role in eliminating microbial pathogens. Recognition of foreign targets by antibodies drives sequential activation of two serine proteases, C1r and C1s, which reside within the complement Component 1 (C1) complex. Active C1s propagates the immune response through its ability to bind and cleave the effector molecule complement Component 4 (C4). Currently, the precise structural and biochemical basis for the control of the interaction between C1s and C4 is unclear. Here, using surface plasmon resonance, we show that the transition of the C1s zymogen to the active form is essential for C1s binding to C4. To understand this, we determined the crystal structure of a zymogen C1s construct (comprising two complement control protein (CCP) domains and the serine protease (SP) domain). These data reveal that two loops (492-499 and 573-580) in the zymogen serine protease domain adopt a conformation that would be predicted to sterically abrogate C4 binding. The transition from zymogen to active C1s repositions both loops such that they would be able to interact with sulfotyrosine residues on C4. The structure also shows the junction of the CCP1 and CCP2 domains of C1s for the first time, yielding valuable information about the exosite for C4 binding located at this position. Together, these data provide a structural explanation for the control of the interaction with C1s and C4 and, furthermore, point to alternative strategies for developing therapeutic approaches for controlling activation of the complement cascade.

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Ataxia Telangiectasia-mutated- and Rad3-related Protein Regulates the DNA Damage-induced G2/M Checkpoint through the Aurora A Cofactor Bora Protein.

Publication Date: 2013 May 31 PMID: 23592782
Authors: Qin, B. - Gao, B. - Yu, J. - Yuan, J. - Lou, Z.
Journal: J Biol Chem

Polo-like kinase1 (Plk1) activation is inhibited in response to DNA damage, and this inhibition contributes to the activation of the G2/M checkpoint, although the molecular mechanism by which Plk1 is inhibited is not clear. Here we report that the DNA damage signaling pathway inhibits Plk1 activity through Bora. Following UV irradiation, ataxia telangiectasia-mutated- and Rad3-related protein phosphorylates Bora at Thr-501. The phosphorylated Thr-501 is subsequently recognized by the E3 ubiquitin ligase SCF-beta-TRCP, which targets Bora for degradation. The degradation of Bora compromises Plk1 activation and contributes to DNA damage-induced G2 arrest. These findings shed new light on Plk1 regulation by the DNA damage response pathway.

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AP-1/Fos-TGase2 Axis Mediates Wounding-induced Plasmodium falciparum Killing in Anopheles gambiae.

Publication Date: 2013 May 31 PMID: 23592781
Authors: Nsango, S. E. - Pompon, J. - Xie, T. - Rademacher, A. - Fraiture, M. - Thoma, M. - Awono-Ambene, P. H. - Moyou, R. S. - Morlais, I. - Levashina, E. A.
Journal: J Biol Chem

Anopheline mosquitoes are the only vectors of human malaria worldwide. It is now widely accepted that mosquito immune responses play a crucial role in restricting Plasmodium development within the vector; therefore, further dissection of the molecular mechanisms underlying these processes should inform new vector control strategies urgently needed to roll back the disease. Here, using genome-wide transcriptional profiling, bioinformatics, and functional gene analysis, we identify a new axis of mosquito resistance to monoclonal Plasmodium falciparum infections that includes the AP-1 transcription factor Fos and the transglutaminase 2 (TGase2), a cross-linking enzyme with known roles in wound responses. We demonstrate that Fos regulates induction of TGase2 expression after wounding but does not affect expression of the components of the well characterized complement-like system. Silencing of Fos or of TGase2 aborts the wounding-induced mosquito killing of P. falciparum. These results reveal multiple signaling pathways that are required for efficient Plasmodium killing in Anopheles gambiae.

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Structural Mechanism of Ring-opening Reaction of Glucose by Human Serum Albumin.

Publication Date: 2013 May 31 PMID: 23592780
Authors: Wang, Y. - Yu, H. - Shi, X. - Luo, Z. - Lin, D. - Huang, M.
Journal: J Biol Chem

Glucose reacts with proteins nonenzymatically under physiological conditions. Such glycation is exacerbated in diabetic patients with high levels of blood sugar and induces various complications. Human albumin serum (HSA) is the most abundant protein in plasma and is glycated by glucose. The glycation sites on HSA remain controversial among different studies. Here, we report two protein crystal structures of HSA in complex with either glucose or fructose. These crystal structures reveal the presence of linear forms of sugar for both monosaccharides. The linear form of glucose forms a covalent bond to Lys-195 of HSA, but this is not the case for fructose. Based on these structures, we propose a mechanism for glucose ring opening involving both residues Lys-195 and Lys-199. These results provide mechanistic insights to understand the glucose ring-opening reaction and the glycation of proteins by monosaccharides.

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Cubilin, a high affinity receptor for fibroblast growth factor 8, is required for cell survival in the developing vertebrate head.

Publication Date: 2013 Jun 7 PMID: 23592779
Authors: Cases, O. - Perea-Gomez, A. - Aguiar, D. P. - Nykjaer, A. - Amsellem, S. - Chandellier, J. - Umbhauer, M. - Cereghini, S. - Madsen, M. - Collignon, J. - Verroust, P. - Riou, J. F. - Creuzet, S. E. - Kozyraki, R.
Journal: J Biol Chem

Cubilin (Cubn) is a multiligand endocytic receptor critical for the intestinal absorption of vitamin B12 and renal protein reabsorption. During mouse development, Cubn is expressed in both embryonic and extra-embryonic tissues, and Cubn gene inactivation results in early embryo lethality most likely due to the impairment of the function of extra-embryonic Cubn. Here, we focus on the developmental role of Cubn expressed in the embryonic head. We report that Cubn is a novel, interspecies-conserved Fgf receptor. Epiblast-specific inactivation of Cubn in the mouse embryo as well as Cubn silencing in the anterior head of frog or the cephalic neural crest of chick embryos show that Cubn is required during early somite stages to convey survival signals in the developing vertebrate head. Surface plasmon resonance analysis reveals that fibroblast growth factor 8 (Fgf8), a key mediator of cell survival, migration, proliferation, and patterning in the developing head, is a high affinity ligand for Cubn. Cell uptake studies show that binding to Cubn is necessary for the phosphorylation of the Fgf signaling mediators MAPK and Smad1. Although Cubn may not form stable ternary complexes with Fgf receptors (FgfRs), it acts together with and/or is necessary for optimal FgfR activity. We propose that plasma membrane binding of Fgf8, and most likely of the Fgf8 family members Fgf17 and Fgf18, to Cubn improves Fgf ligand endocytosis and availability to FgfRs, thus modulating Fgf signaling activity.

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A Novel Transcription Mechanism Activated by Ethanol: INDUCTION OF Slc7a11 GENE EXPRESSION VIA INHIBITION OF THE DNA-BINDING ACTIVITY OF TRANSCRIPTIONAL REPRESSOR OCTAMER-BINDING TRANSCRIPTION FACTOR 1 (OCT-1).

Publication Date: 2013 May 24 PMID: 23592778
Authors: Lin, X. - Yang, H. - Zhang, H. - Zhou, L. - Guo, Z.
Journal: J Biol Chem

Solute carrier family 7, member 11 (Slc7a11) is a plasma membrane cystine/glutamate exchanger that provides intracellular cystine to produce glutathione, a major cellular antioxidant. Oxidative and endoplasmic reticulum stresses up-regulate Slc7a11 expression by activation of nuclear factor erythroid 2-related factor 2 and transcription factor 4. This study examined the effect of ethanol on Slc7a11 expression and the underlying mechanism involved. Treatment of mouse hepatic stellate cells with ethanol significantly increased Slc7a11 mRNA and protein levels. Deletion of a 20-bp DNA sequence between -2044 to -2024 upstream of the transcription start site significantly increased basal activity and completely abolished the ethanol-induced activity of the Slc7a11 promoter. This deletion did not affect Slc7a11 promoter activity induced by oxidative or endoplasmic reticulum stress. DNA sequence analysis revealed a binding motif for octamer-binding transcription factor 1 (OCT-1) in the deleted fragment. Mutation of this OCT-1 binding motif resulted in a similar effect as the deletion experiment, i.e. it increased the basal promoter activity and abolished the response to ethanol. Ethanol exposure significantly inhibited OCT-1 binding to the Slc7a11 promoter region, although it did not alter OCT-1 mRNA and protein levels. OCT-1 reportedly functions as either a transcriptional enhancer or repressor, depending on the target genes. Results from this study suggest that OCT-1 functions as a repressor on the Slc7a11 promoter and that ethanol inhibits OCT-1 binding to the Slc7a11 promoter, thereby increasing Slc7a11 expression. Taken together, inhibition of the DNA binding activity of transcriptional repressor OCT-1 is a mechanism by which ethanol up-regulates Slc711 expression.

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Facilitated Hyperpolarization Signaling in Vascular Smooth Muscle-overexpressing TRIC-A Channels.

Publication Date: 2013 May 31 PMID: 23592776
Authors: Tao, S. - Yamazaki, D. - Komazaki, S. - Zhao, C. - Iida, T. - Kakizawa, S. - Imaizumi, Y. - Takeshima, H.
Journal: J Biol Chem

The TRIC channel subtypes, namely TRIC-A and TRIC-B, are intracellular monovalent cation-specific channels and likely mediate counterion movements to support efficient Ca(2+) release from the sarco/endoplasmic reticulum. Vascular smooth muscle cells (VSMCs) contain both TRIC subtypes and two Ca(2+) release mechanisms; incidental opening of ryanodine receptors (RyRs) generates local Ca(2+) sparks to induce hyperpolarization and relaxation, whereas agonist-induced activation of inositol trisphosphate receptors produces global Ca(2+) transients causing contraction. Tric-a knock-out mice develop hypertension due to insufficient RyR-mediated Ca(2+) sparks in VSMCs. Here we describe transgenic mice overexpressing TRIC-A channels under the control of a smooth muscle cell-specific promoter. The transgenic mice developed congenital hypotension. In Tric-a-overexpressing VSMCs from the transgenic mice, the resting membrane potential decreased because RyR-mediated Ca(2+) sparks were facilitated and cell surface Ca(2+)-dependent K(+) channels were hyperactivated. Under such hyperpolarized conditions, L-type Ca(2+) channels were inactivated, and thus, the resting intracellular Ca(2+) levels were reduced in Tric-a-overexpressing VSMCs. Moreover, Tric-a overexpression impaired inositol trisphosphate-sensitive stores to diminish agonist-induced Ca(2+) signaling in VSMCs. These altered features likely reduced vascular tonus leading to the hypotensive phenotype. Our Tric-a-transgenic mice together with Tric-a knock-out mice indicate that TRIC-A channel density in VSMCs is responsible for controlling basal blood pressure at the whole-animal level.

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Molecularly Distinct Routes of Mitochondrial Ca2+ Uptake Are Activated Depending on the Activity of the Sarco/Endoplasmic Reticulum Ca2+ ATPase (SERCA).

Publication Date: 2013 May 24 PMID: 23592775
Authors: Waldeck-Weiermair, M. - Deak, A. T. - Groschner, L. N. - Alam, M. R. - Jean-Quartier, C. - Malli, R. - Graier, W. F.
Journal: J Biol Chem

The transfer of Ca(2+) across the inner mitochondrial membrane is an important physiological process linked to the regulation of metabolism, signal transduction, and cell death. While the definite molecular composition of mitochondrial Ca(2+) uptake sites remains unknown, several proteins of the inner mitochondrial membrane, that are likely to accomplish mitochondrial Ca(2+) fluxes, have been described: the novel uncoupling proteins 2 and 3, the leucine zipper-EF-hand containing transmembrane protein 1 and the mitochondrial calcium uniporter. It is unclear whether these proteins contribute to one unique mitochondrial Ca(2+) uptake pathway or establish distinct routes for mitochondrial Ca(2+) sequestration. In this study, we show that a modulation of Ca(2+) release from the endoplasmic reticulum by inhibition of the sarco/endoplasmatic reticulum ATPase modifies cytosolic Ca(2+) signals and consequently switches mitochondrial Ca(2+) uptake from an uncoupling protein 3- and mitochondrial calcium uniporter-dependent, but leucine zipper-EF-hand containing transmembrane protein 1-independent to a leucine zipper-EF-hand containing transmembrane protein 1- and mitochondrial calcium uniporter-mediated, but uncoupling protein 3-independent pathway. Thus, the activity of sarco/endoplasmatic reticulum ATPase is significant for the mode of mitochondrial Ca(2+) sequestration and determines which mitochondrial proteins might actually accomplish the transfer of Ca(2+) across the inner mitochondrial membrane. Moreover, our findings herein support the existence of distinct mitochondrial Ca(2+) uptake routes that might be essential to ensure an efficient ion transfer into mitochondria despite heterogeneous cytosolic Ca(2+) rises.

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Control of Adipogenesis by the Autocrine Interplays between Angiotensin 1-7/Mas Receptor and Angiotensin II/AT1 Receptor Signaling Pathways.

Publication Date: 2013 May 31 PMID: 23592774
Authors: Than, A. - Leow, M. K. - Chen, P.
Journal: J Biol Chem

Angiotensin II (AngII), a peptide hormone released by adipocytes, can be catabolized by adipose angiotensin-converting enzyme 2 (ACE2) to form Ang(1-7). Co-expression of AngII receptors (AT1 and AT2) and Ang(1-7) receptors (Mas) in adipocytes implies the autocrine regulation of the local angiotensin system upon adipocyte functions, through yet unknown interactive mechanisms. In the present study, we reveal the adipogenic effects of Ang(1-7) through activation of Mas receptor and its subtle interplays with the antiadipogenic AngII-AT1 signaling pathways. Specifically, in human and 3T3-L1 preadipocytes, Ang(1-7)-Mas signaling promotes adipogenesis via activation of PI3K/Akt and inhibition of MAPK kinase/ERK pathways, and Ang(1-7)-Mas antagonizes the antiadipogenic effect of AngII-AT1 by inhibiting the AngII-AT1-triggered MAPK kinase/ERK pathway. The autocrine regulation of the AngII/AT1-ACE2-Ang(1-7)/Mas axis upon adipogenesis has also been revealed. This study suggests the importance of the local regulation of the delicately balanced angiotensin system upon adipogenesis and its potential as a novel therapeutic target for obesity and related metabolic disorders.

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G-protein Receptor Kinase 5 Regulates the Cannabinoid Receptor 2-induced Up-regulation of Serotonin 2A Receptors.

Publication Date: 2013 May 31 PMID: 23592773
Authors: Franklin, J. M. - Carrasco, G. A.
Journal: J Biol Chem

We have recently reported that cannabinoid agonists can up-regulate and enhance the activity of serotonin 2A (5-HT2A) receptors in the prefrontal cortex (PFCx). Increased expression and activity of cortical 5-HT2A receptors has been associated with neuropsychiatric disorders, such as anxiety and schizophrenia. Here we report that repeated CP55940 exposure selectively up-regulates GRK5 proteins in rat PFCx and in a neuronal cell culture model. We sought to examine the mechanism underlying the regulation of GRK5 and to identify the role of GRK5 in the cannabinoid agonist-induced up-regulation and enhanced activity of 5-HT2A receptors. Interestingly, we found that cannabinoid agonist-induced up-regulation of GRK5 involves CB2 receptors, beta-arrestin 2, and ERK1/2 signaling because treatment with CB2 shRNA lentiviral particles, beta-arrestin 2 shRNA lentiviral particles, or ERK1/2 inhibitor prevented the cannabinoid agonist-induced up-regulation of GRK5. Most importantly, we found that GRK5 shRNA lentiviral particle treatment prevented the cannabinoid agonist-induced up-regulation and enhanced 5-HT2A receptor-mediated calcium release. Repeated cannabinoid exposure was also associated with enhanced phosphorylation of CB2 receptors and increased interaction between beta-arrestin 2 and ERK1/2. These latter phenomena were also significantly inhibited by GRK5 shRNA lentiviral treatment. Our results suggest that sustained activation of CB2 receptors, which up-regulates 5-HT2A receptor signaling, enhances GRK5 expression; the phosphorylation of CB2 receptors; and the beta-arrestin 2/ERK interactions. These data could provide a rationale for some of the adverse effects associated with repeated cannabinoid agonist exposure.

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Hyper-O-GlcNAcylation Is Anti-apoptotic and Maintains Constitutive NF-kappaB Activity in Pancreatic Cancer Cells.

Publication Date: 2013 May 24 PMID: 23592772
Authors: Ma, Z. - Vocadlo, D. J. - Vosseller, K.
Journal: J Biol Chem

Cancer cell metabolic reprogramming includes a shift in energy production from oxidative phosphorylation to less efficient glycolysis even in the presence of oxygen (Warburg effect) and use of glutamine for increased biosynthetic needs. This necessitates greatly increased glucose and glutamine uptake, both of which enter the hexosamine biosynthetic pathway (HBP). The HBP end product UDP-N-acetylglucosamine (UDP-GlcNAc) is used in enzymatic post-translational modification of many cytosolic and nuclear proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc). Here, we observed increased HBP flux and hyper-O-GlcNAcylation in human pancreatic ductal adenocarcinoma (PDAC). PDAC hyper-O-GlcNAcylation was associated with elevation of OGT and reduction of the enzyme that removes O-GlcNAc (OGA). Reducing hyper-O-GlcNAcylation had no effect on non-transformed pancreatic epithelial cell growth, but inhibited PDAC cell proliferation, anchorage-independent growth, orthotopic tumor growth, and triggered apoptosis. PDAC is supported by oncogenic NF-kappaB transcriptional activity. The NF-kappaB p65 subunit and upstream kinases IKKalpha/IKKbeta were O-GlcNAcylated in PDAC. Reducing hyper-O-GlcNAcylation decreased PDAC cell p65 activating phosphorylation (S536), nuclear translocation, NF-kappaB transcriptional activity, and target gene expression. Conversely, mimicking PDAC hyper-O-GlcNAcylation through pharmacological inhibition of OGA suppressed suspension culture-induced apoptosis and increased IKKalpha and p65 O-GlcNAcylation, accompanied by activation of NF-kappaB signaling. Finally, reducing p65 O-GlcNAcylation specifically by mutating two p65 O-GlcNAc sites (T322A and T352A) attenuated the induction of PDAC cell anchorage-independent growth. Our data indicate that hyper-O-GlcNAcylation is anti-apoptotic and contributes to NF-kappaB oncogenic activation in PDAC.

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Crystal and Solution Studies Reveal That the Transcriptional Regulator AcnR of Corynebacterium glutamicum Is Regulated by Citrate-Mg2+ Binding to a Non-canonical Pocket.

Publication Date: 2013 May 31 PMID: 23589369
Authors: Garcia-Nafria, J. - Baumgart, M. - Turkenburg, J. P. - Wilkinson, A. J. - Bott, M. - Wilson, K. S.
Journal: J Biol Chem

Corynebacterium glutamicum is an important industrial bacterium as well as a model organism for the order Corynebacteriales, whose citric acid cycle occupies a central position in energy and precursor supply. Expression of aconitase, which isomerizes citrate into isocitrate, is controlled by several transcriptional regulators, including the dimeric aconitase repressor AcnR, assigned by sequence identity to the TetR family. We report the structures of AcnR in two crystal forms together with ligand binding experiments and in vivo studies. First, there is a citrate-Mg(2+) moiety bound in both forms, not in the canonical TetR ligand binding site but rather in a second pocket more distant from the DNA binding domain. Second, the citrate-Mg(2+) binds with a KD of 6 mm, within the range of physiological significance. Third, citrate-Mg(2+) lowers the affinity of AcnR for its target DNA in vitro. Fourth, analyses of several AcnR point mutations provide evidence for the possible involvement of the corresponding residues in ligand binding, DNA binding, and signal transfer. AcnR derivatives defective in citrate-Mg(2+) binding severely inhibit growth of C. glutamicum on citrate. Finally, the structures do have a pocket corresponding to the canonical tetracycline site, and although we have not identified a ligand that binds there, comparison of the two crystal forms suggests differences in the region of the canonical pocket that may indicate a biological significance.

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Structural Basis for Phosphorylated Autoinducer-2 Modulation of the Oligomerization State of the Global Transcription Regulator LsrR from Escherichia coli.

Publication Date: 2013 May 31 PMID: 23589368
Authors: Wu, M. - Tao, Y. - Liu, X. - Zang, J.
Journal: J Biol Chem

Quorum-sensing systems are widely used by bacteria to control behavior in response to fluctuations in cell density. Several small diffusible molecules called autoinducers act as signaling molecules in quorum-sensing processes through interplay with sensors. Autoinducers modulate vital physiological functions such as nutrient acquisition, gene transcription, and virulence factor production. In Escherichia coli, LsrR serves as a global transcription regulator that responds to autoinducer-2 to regulate the expression of a variety of genes, including the lsr operon and the lsrR gene. Here, we report the crystal structure of full-length LsrR from E. coli, which has an N-terminal DNA-binding domain and a C-terminal ligand-binding domain connected by a beta-strand. Although only two molecules are found in one asymmetric unit, two neighboring dimers pack to form a tetramer that is consistent with the oligomerization state of LsrR in solution. Mutagenesis experiments and gel shift assays indicated that Gln-33 and Tyr-26 might be involved in interactions between LsrR and DNA. The LsrR-binding site for phosphorylated autoinducer-2 was predicted by structural comparisons of LsrR with CggR and SorC. Cross-linking, size exclusion chromatography, and gel shift assays determined that phosphorylated autoinducer-2 triggered the disassembly of the LsrR tetramer into dimers and reduced the DNA binding ability of LsrR. Our findings reveal a mechanism for the change in the oligomerization state of LsrR in the presence of phosphorylated autoinducer-2. Based on these observations, we propose that phosphorylated autoinducer-2 triggers the disassembly of the LsrR tetramer to activate the transcription of its target genes.

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The Hyaluronic Acid Receptor CD44 Coordinates Normal and Metaplastic Gastric Epithelial Progenitor Cell Proliferation.

Publication Date: 2013 May 31 PMID: 23589310
Authors: Khurana, S. S. - Riehl, T. E. - Moore, B. D. - Fassan, M. - Rugge, M. - Romero-Gallo, J. - Noto, J. - Peek, R. M. Jr - Stenson, W. F. - Mills, J. C.
Journal: J Biol Chem

The stem cell in the isthmus of gastric units continually replenishes the epithelium. Atrophy of acid-secreting parietal cells (PCs) frequently occurs during infection with Helicobacter pylori, predisposing patients to cancer. Atrophy causes increased proliferation of stem cells, yet little is known about how this process is regulated. Here we show that CD44 labels a population of small, undifferentiated cells in the gastric unit isthmus where stem cells are known to reside. Loss of CD44 in vivo results in decreased proliferation of the gastric epithelium. When we induce PC atrophy by Helicobacter infection or tamoxifen treatment, this CD44(+) population expands from the isthmus toward the base of the unit. CD44 blockade during PC atrophy abrogates the expansion. We find that CD44 binds STAT3, and inhibition of either CD44 or STAT3 signaling causes decreased proliferation. Atrophy-induced CD44 expansion depends on pERK, which labels isthmal cells in mice and humans. Our studies delineate an in vivo signaling pathway, ERK --> CD44 --> STAT3, that regulates normal and atrophy-induced gastric stem/progenitor-cell proliferation. We further show that we can intervene pharmacologically at each signaling step in vivo to modulate proliferation.

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Substrate Selectivity of the Acid-activated Glutamate/gamma-Aminobutyric acid (GABA) Antiporter GadC from Escherichia coli.

Publication Date: 2013 May 24 PMID: 23589309
Authors: Ma, D. - Lu, P. - Shi, Y.
Journal: J Biol Chem

GadC, a central component of the Escherichia coli acid resistance system, is a Glu/GABA antiporter. A previous structural study and biochemical characterization showed that GadC exhibits a stringent pH dependence for substrate transport, with no detectable activity at pH values above 6.5. However, the substrate selectivity and the mechanism of pH-dependent transport activity of GadC remain enigmatic. In this study, we demonstrate that GadC selectively transports Glu with no net charge and GABA with a positive charge. A C-plug-truncated variant of GadC (residues 1-470) transported Gln (a mimic of Glu with no net charge), but not Glu, even at pH 8.0. The pH-dependent transport of Gln by this GadC variant was shifted approximately 1 unit toward a higher pH compared with Glu transport. Taken together, the results identify the substrate selectivity for GadC and show that the protonation states of substrates are crucial for transport.

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An Autoinhibited Structure of alpha-Catenin and Its Implications for Vinculin Recruitment to Adherens Junctions.

Publication Date: 2013 May 31 PMID: 23589308
Authors: Ishiyama, N. - Tanaka, N. - Abe, K. - Yang, Y. J. - Abbas, Y. M. - Umitsu, M. - Nagar, B. - Bueler, S. A. - Rubinstein, J. L. - Takeichi, M. - Ikura, M.
Journal: J Biol Chem

alpha-Catenin is an actin- and vinculin-binding protein that regulates cell-cell adhesion by interacting with cadherin adhesion receptors through beta-catenin, but the mechanisms by which it anchors the cadherin-catenin complex to the actin cytoskeleton at adherens junctions remain unclear. Here we determined crystal structures of alphaE-catenin in the autoinhibited state and the actin-binding domain of alphaN-catenin. Together with the small-angle x-ray scattering analysis of full-length alphaN-catenin, we deduced an elongated multidomain assembly of monomeric alpha-catenin that structurally and functionally couples the vinculin- and actin-binding mechanisms. Cellular and biochemical studies of alphaE- and alphaN-catenins show that alphaE-catenin recruits vinculin to adherens junctions more effectively than alphaN-catenin, partly because of its higher affinity for actin filaments. We propose a molecular switch mechanism involving multistate conformational changes of alpha-catenin. This would be driven by actomyosin-generated tension to dynamically regulate the vinculin-assisted linkage between adherens junctions and the actin cytoskeleton.

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12/15-Lipoxygenase Mediates High-fat Diet-induced Endothelial Tight Junction Disruption and Monocyte Transmigration: A NEW ROLE FOR 15(S)-HYDROXYEICOSATETRAENOIC ACID IN ENDOTHELIAL CELL DYSFUNCTION.

Publication Date: 2013 May 31 PMID: 23589307
Authors: Kundumani-Sridharan, V. - Dyukova, E. - Hansen, D. E. 3rd - Rao, G. N.
Journal: J Biol Chem

A convincing body of evidence suggests that 12/15-lipoxygenase (12/15-LO) plays a role in atherosclerosis. However, the mechanisms of its involvement in the pathogenesis of this disease are not clear. Therefore, the purpose of this study is to understand the mechanisms by which 12/15-LO mediates endothelial dysfunction. 15(S)-Hydroxyeicosatetraenoic acid (15(S)-HETE), the major 12/15-LO metabolite of arachidonic acid (AA), induced endothelial barrier permeability via Src and Pyk2-dependent zonula occluden (ZO)-2 tyrosine phosphorylation and its dissociation from the tight junction complexes. 15(S)-HETE also stimulated macrophage adhesion to the endothelial monolayer in Src and Pyk2-dependent manner. Ex vivo studies revealed that exposure of arteries from WT mice to AA or 15(S)-HETE led to Src-Pyk2-dependent ZO-2 tyrosine phosphorylation, tight junction disruption, and macrophage adhesion, whereas the arteries from 12/15-LO knock-out mice are protected from these effects of AA. Feeding WT mice with a high-fat diet induced the expression of 12/15-LO in the arteries leading to tight junction disruption and macrophage adhesion and deletion of the 12/15-LO gene disallowed these effects. Thus, the findings of this study provide the first evidence of the role of 12/15-LO and its AA metabolite, 15(S)-HETE, in high-fat diet-induced endothelial tight junction disruption and macrophage adhesion, the crucial events underlying the pathogenesis of atherosclerosis.

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The Cyclomodulin Cycle Inhibiting Factor (CIF) Alters Cullin Neddylation Dynamics.

Publication Date: 2013 May 24 PMID: 23589306
Authors: Toro, T. B. - Toth, J. I. - Petroski, M. D.
Journal: J Biol Chem

The bacterial effector protein cycle inhibiting factor (CIF) converts glutamine 40 of NEDD8 to glutamate (Q40E), causing cytopathic effects and inhibiting cell proliferation. Although these have been attributed to blocking the functions of cullin-RING ubiquitin ligases, how CIF modulates NEDD8-dependent signaling is unclear. Here we use conditional NEDD8-dependent yeast to explore the effects of CIF on cullin neddylation. Although CIF causes cullin deneddylation and the generation of free NEDD8 Q40E, inhibiting the COP9 signalosome (CSN) allows Q40E to form only on NEDD8 attached to cullins. In the presence of the CSN, NEDD8 Q40E is removed from cullins more rapidly than NEDD8, leading to a decrease in steady-state cullin neddylation. As NEDD8 Q40E is competent for cullin conjugation in the absence of functional CSN and with overexpression of the NEDD8 ligase Dcn1, our data are consistent with NEDD8 deamidation causing enhanced deneddylation of cullins by the CSN. This leads to a dramatic change in the extent of activated cullin-RING ubiquitin ligases.

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Heat Shock Protein 90 (Hsp90) Selectively Regulates the Stability of KDM4B/JMJD2B Histone Demethylase.

Publication Date: 2013 May 24 PMID: 23589305
Authors: Ipenberg, I. - Guttmann-Raviv, N. - Khoury, H. P. - Kupershmit, I. - Ayoub, N.
Journal: J Biol Chem

The family of KDM4A-D histone demethylases selectively demethylates H3K9 and H3K36 and is implicated in key cellular processes including DNA damage response, transcription, cell cycle regulation, cellular differentiation, senescence, and carcinogenesis. Various human cancers exhibit elevated protein levels of KDM4A-D members, and their depletion impairs tumor formation, suggesting that their enhanced activity promotes carcinogenesis. However, the mechanisms regulating the KDM4 protein stability remain largely unknown. Here, we show that the molecular chaperon Hsp90 interacts with and stabilizes KDM4B protein. Pharmacological inhibition of Hsp90 with geldanamycin resulted in ubiquitin-dependent proteasomal degradation of KDM4B, but not of KDM4C, suggesting that the turnover of these demethylases is regulated by distinct mechanisms. This degradation was accompanied by increased methylation of H3K9. We further show that KDM4B is ubiquitinated on lysines 337 and 562; simultaneous substitution of these residues to arginine suppressed the geldanamycin-induced degradation of KDM4B, suggesting that the ubiquitination of Lys-337 and Lys-562 targets KDM4B for proteasomal degradation upon Hsp90 inhibition. These findings constitute a novel pathway by which Hsp90 activity alters the histone code via regulation of KDM4B stability. This pathway may prove a druggable target for the treatment of tumors driven by enhanced KDM4B activity.

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Lgr4/Gpr48 Negatively Regulates TLR2/4-associated Pattern Recognition and Innate Immunity by Targeting CD14 Expression.

Publication Date: 2013 May 24 PMID: 23589304
Authors: Du, B. - Luo, W. - Li, R. - Tan, B. - Han, H. - Lu, X. - Li, D. - Qian, M. - Zhang, D. - Zhao, Y. - Liu, M.
Journal: J Biol Chem

The recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs) is pivotal in both innate and adaptive immune responses. Here we demonstrate that deletion of Lgr4/Gpr48 (G-protein-coupled receptor 48), a seven-transmembrane glycoprotein hormone receptor, potentiates TLR2/4-associated cytokine production and attenuates mouse resistance to septic shock. The expression of CD14, a co-receptor for TLR2/4-associated pathogen-associated molecular patterns, is increased significantly in Lgr4-deficient macrophages, which is consistent with the increased immune response, whereas the binding activity of cAMP-response element-binding protein is decreased significantly in Lgr4-deficient macrophages, which up-regulate the expression of CD14 at the transcriptional level. Together, our data demonstrate that Lgr4/Gpr48 plays a critical role in modulating the TLR2/4 signaling pathway and represents a useful therapeutic approach of targeting Lgr4/Gpr48 in TLR2/4-associated septic shock and autoimmune diseases.

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Dissecting the Roles of Tyrosines 490 and 785 of TrkA Protein in the Induction of Downstream Protein Phosphorylation Using Chimeric Receptors.

Publication Date: 2013 Jun 7 PMID: 23589303
Authors: Biarc, J. - Chalkley, R. J. - Burlingame, A. L. - Bradshaw, R. A.
Journal: J Biol Chem

Receptor tyrosine kinases generally act by forming phosphotyrosine-docking sites on their own endodomains that propagate signals through cascades of post-translational modifications driven by the binding of adaptor/effector proteins. The pathways that are stimulated in any given receptor tyrosine kinase are a function of the initial docking sites that are activated and the availability of downstream participants. In the case of the Trk receptors, which are activated by nerve growth factor, there are only two established phosphotyrosine-docking sites (Tyr-490 and Tyr-785 on TrkA) that are known to be directly involved in signal transduction. Taking advantage of this limited repertoire of docking sites and the availability of PC12 cell lines stably transfected with chimeric receptors composed of the extracellular domain of the PDGF receptor and the transmembrane and intracellular domains of TrkA, the downstream TrkA-induced phosphoproteome was assessed for the "native" receptor and mutants lacking Tyr-490 or both Tyr-490 and Tyr-785. Basal phosphorylation levels were compared with those formed after 20 min of stimulation with PDGF. Several thousand phosphopeptides were identified after TiO2 enrichment, and many were up- or down-regulated by receptor activation. The modified proteins in the native sample contained many of the well established participants in TrkA signaling. The results from the mutant receptors allowed grouping of these downstream targets by their dependence on the two characterized docking site(s). A clear subset that was not dependent on either Tyr-490 or Tyr-785 emerged, providing direct evidence that there are other sites on TrkA that are involved in downstream signaling.

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The Fer Tyrosine Kinase Is Important for Platelet-derived Growth Factor-BB-induced Signal Transducer and Activator of Transcription 3 (STAT3) Protein Phosphorylation, Colony Formation in Soft Agar, and Tumor Growth in Vivo.

Publication Date: 2013 May 31 PMID: 23589302
Authors: Lennartsson, J. - Ma, H. - Wardega, P. - Pelka, K. - Engstrom, U. - Hellberg, C. - Heldin, C. H.
Journal: J Biol Chem

Fer is a cytoplasmic tyrosine kinase that is activated in response to platelet-derived growth factor (PDGF) stimulation. In the present report, we show that Fer associates with the activated PDGF beta-receptor (PDGFRbeta) through multiple autophosphorylation sites, i.e. Tyr-579, Tyr-581, Tyr-740, and Tyr-1021. Using low molecular weight inhibitors, we found that PDGF-BB-induced Fer activation is dependent on PDGFRbeta kinase activity, but not on the enzymatic activity of Src or Jak kinases. In cells in which Fer was down-regulated using siRNA, PDGF-BB was unable to induce phosphorylation of STAT3, whereas phosphorylations of STAT5, ERK1/2, and Akt were unaffected. PDGF-BB-induced activation of STAT3 occurred also in cells expressing kinase-dead Fer, suggesting a kinase-independent adaptor role of Fer. Expression of Fer was dispensable for PDGF-BB-induced proliferation and migration but essential for colony formation in soft agar. Tumor growth in vivo was delayed in cells depleted of Fer expression. Our data suggest a critical role of Fer in PDGF-BB-induced STAT3 activation and cell transformation.

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Interactome Analyses of Mature gamma-Secretase Complexes Reveal Distinct Molecular Environments of Presenilin (PS) Paralogs and Preferential Binding of Signal Peptide Peptidase to PS2.

Publication Date: 2013 May 24 PMID: 23589300
Authors: Jeon, A. H. - Bohm, C. - Chen, F. - Huo, H. - Ruan, X. - Ren, C. H. - Ho, K. - Qamar, S. - Mathews, P. M. - Fraser, P. E. - Mount, H. T. - St George-Hyslop, P. - Schmitt-Ulms, G.
Journal: J Biol Chem

gamma-Secretase plays a pivotal role in the production of neurotoxic amyloid beta-peptides (Abeta) in Alzheimer disease (AD) and consists of a heterotetrameric core complex that includes the aspartyl intramembrane protease presenilin (PS). The human genome codes for two presenilin paralogs. To understand the causes for distinct phenotypes of PS paralog-deficient mice and elucidate whether PS mutations associated with early-onset AD affect the molecular environment of mature gamma-secretase complexes, quantitative interactome comparisons were undertaken. Brains of mice engineered to express wild-type or mutant PS1, or HEK293 cells stably expressing PS paralogs with N-terminal tandem-affinity purification tags served as biological source materials. The analyses revealed novel interactions of the gamma-secretase core complex with a molecular machinery that targets and fuses synaptic vesicles to cellular membranes and with the H(+)-transporting lysosomal ATPase macrocomplex but uncovered no differences in the interactomes of wild-type and mutant PS1. The catenin/cadherin network was almost exclusively found associated with PS1. Another intramembrane protease, signal peptide peptidase, predominantly co-purified with PS2-containing gamma-secretase complexes and was observed to influence Abeta production.

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High error rates in selenocysteine insertion in Mammalian cells treated with the antibiotic doxycycline, chloramphenicol, or geneticin.

Publication Date: 2013 May 24 PMID: 23589299
Authors: Tobe, R. - Naranjo-Suarez, S. - Everley, R. A. - Carlson, B. A. - Turanov, A. A. - Tsuji, P. A. - Yoo, M. H. - Gygi, S. P. - Gladyshev, V. N. - Hatfield, D. L.
Journal: J Biol Chem

Antibiotics target bacteria by interfering with essential processes such as translation, but their effects on translation in mammalian cells are less well characterized. We found that doxycycline, chloramphenicol, and Geneticin (G418) interfered with insertion of selenocysteine (Sec), which is encoded by the stop codon, UGA, into selenoproteins in murine EMT6 cells. Treatment of EMT6 cells with these antibiotics reduced enzymatic activities and Sec insertion into thioredoxin reductase 1 (TR1) and glutathione peroxidase 1 (GPx1). However, these proteins were differentially affected due to varying errors in Sec insertion at UGA. In the presence of doxycycline, chloramphenicol, or G418, the Sec-containing form of TR1 decreased, whereas the arginine-containing and truncated forms of this protein increased. We also detected antibiotic-specific misinsertion of cysteine and tryptophan. Furthermore, misinsertion of arginine in place of Sec was commonly observed in GPx1 and glutathione peroxidase 4. TR1 was the most affected and GPx1 was the least affected by these translation errors. These observations were consistent with the differential use of two Sec tRNA isoforms and their distinct roles in supporting accuracy of Sec insertion into selenoproteins. The data reveal widespread errors in inserting Sec into proteins and in dysregulation of selenoprotein expression and function upon antibiotic treatment.

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Caspase 1 Activation Is Protective against Hepatocyte Cell Death by Up-regulating Beclin 1 Protein and Mitochondrial Autophagy in the Setting of Redox Stress.

Publication Date: 2013 May 31 PMID: 23589298
Authors: Sun, Q. - Gao, W. - Loughran, P. - Shapiro, R. - Fan, J. - Billiar, T. R. - Scott, M. J.
Journal: J Biol Chem

Caspase 1 activation can be induced by oxidative stress, which leads to the release of the proinflammatory cytokines IL1beta and IL18 in myeloid cells and a potentially damaging inflammatory response. However, little is known about the role of caspase 1 in non-immune cells, such as hepatocytes, that express and activate the inflammasome but do not produce a significant amount of IL1beta/IL18. Here we demonstrate that caspase 1 activation protects against cell death after redox stress induced by hypoxia/reoxygenation in hepatocytes. Mechanistically, we show that caspase 1 reduces mitochondrial respiration and reactive oxygen species by increasing mitochondrial autophagy and subsequent clearance of mitochondria in hepatocytes after hypoxia/reoxygenation. Caspase 1 increases autophagic flux through up-regulating autophagy initiator beclin 1 during redox stress and is an important cell survival factor in hepatocytes. We find that during hemorrhagic shock with resuscitation, an in vivo mouse model associated with severe hepatic redox stress, caspase 1 activation is also protective against liver injury and excessive oxidative stress through the up-regulation of beclin 1. Our findings suggest an alternative role for caspase 1 activation in promoting adaptive responses to oxidative stress and, more specifically, in limiting reactive oxygen species production and damage in cells and tissues where IL1beta/IL18 are not highly expressed.

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Cysteine Proteinase from Streptococcus pyogenes Enables Evasion of Innate Immunity via Degradation of Complement Factors.

Publication Date: 2013 May 31 PMID: 23589297
Authors: Honda-Ogawa, M. - Ogawa, T. - Terao, Y. - Sumitomo, T. - Nakata, M. - Ikebe, K. - Maeda, Y. - Kawabata, S.
Journal: J Biol Chem

Streptococcus pyogenes is an important human pathogen that causes invasive diseases such as necrotizing fasciitis, sepsis, and streptococcal toxic shock syndrome. We investigated the function of a major cysteine protease from S. pyogenes that affects the amount of C1-esterase inhibitor (C1-INH) and other complement factors and aimed to elucidate the mechanism involved in occurrence of streptococcal toxic shock syndrome from the aspect of the complement system. First, we revealed that culture supernatant of a given S. pyogenes strain and recombinant SpeB degraded the C1-INH. Then, we determined the N-terminal sequence of the C1-INH fragment degraded by recombinant SpeB. Interestingly, the region containing one of the identified cleavage sites is not present in patients with C1-INH deficiency. Scanning electron microscopy of the speB mutant incubated in human serum showed the abnormal superficial architecture and irregular oval structure. Furthermore, unlike the wild-type strain, that mutant strain showed lower survival capacity than normal as compared with heat-inactivated serum, whereas it had a significantly higher survival rate in serum without the C1-INH than in normal serum. Also, SpeB degraded multiple complement factors and the membrane attack complex. Flow cytometric analyses revealed deposition of C9, one of the components of membrane the attack complex, in greater amounts on the surface of the speB mutant, whereas lower amounts of C9 were bound to the wild-type strain surface. These results suggest that SpeB can interrupt the human complement system via degrading the C1-INH, thus enabling S. pyogenes to evade eradication in a hostile environment.

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Regulation of Matrix Assembly through Rigidity-dependent Fibronectin Conformational Changes.

Publication Date: 2013 May 24 PMID: 23589296
Authors: Carraher, C. L. - Schwarzbauer, J. E.
Journal: J Biol Chem

Cells sense and respond to the mechanical properties of their microenvironment. We investigated whether these properties affect the ability of cells to assemble a fibrillar fibronectin (FN) matrix. Analysis of matrix assembled by cells grown on FN-coated polyacrylamide gels of varying stiffnesses showed that rigid substrates stimulate FN matrix assembly and activation of focal adhesion kinase (FAK) compared with the level of assembly and FAK signaling on softer substrates. Stimulating integrins with Mn(2+) treatment increased FN assembly on softer gels, suggesting that integrin binding is deficient on soft substrates. Guanidine hydrochloride-induced extension of the substrate-bound FN rescued assembly on soft substrates to a degree similar to that of Mn(2+) treatment and increased activation of FAK along with the initiation of assembly at FN matrix assembly sites. In contrast, increasing actin-mediated cell contractility did not rescue FN matrix assembly on soft substrates. Thus, rigidity-dependent FN matrix assembly is determined by extracellular events, namely the engagement of FN by cells and the induction of FN conformational changes. Extensibility of FN in response to substrate stiffness may serve as a mechanosensing mechanism whereby cells use pericellular FN to probe the stiffness of their environment.

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Insulin Directly Regulates Steroidogenesis via Induction of the Orphan Nuclear Receptor DAX-1 in Testicular Leydig Cells.

Publication Date: 2013 May 31 PMID: 23589295
Authors: Ahn, S. W. - Gang, G. T. - Kim, Y. D. - Ahn, R. S. - Harris, R. A. - Lee, C. H. - Choi, H. S.
Journal: J Biol Chem

Testosterone level is low in insulin-resistant type 2 diabetes. Whether this is due to negative effects of high level of insulin on the testes caused by insulin resistance has not been studied in detail. In this study, we found that insulin directly binds to insulin receptors in Leydig cell membranes and activates phospho-insulin receptor-beta (phospho-IR-beta), phospho-IRS1, and phospho-AKT, leading to up-regulation of DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1) gene expression in the MA-10 mouse Leydig cell line. Insulin also inhibits cAMP-induced and liver receptor homolog-1 (LRH-1)-induced steroidogenic enzyme gene expression and steroidogenesis. In contrast, knockdown of DAX-1 reversed insulin-mediated inhibition of steroidogenesis. Whether insulin directly represses steroidogenesis through regulation of steroidogenic enzyme gene expression was assessed in insulin-injected mouse models and high fat diet-induced obesity. In insulin-injected mouse models, insulin receptor signal pathway was activated and subsequently inhibited steroidogenesis via induction of DAX-1 without significant change of luteinizing hormone or FSH levels. Likewise, the levels of steroidogenic enzyme gene expression and steroidogenesis were low, but interestingly, the level of DAX-1 was high in the testes of high fat diet-fed mice. These results represent a novel regulatory mechanism of steroidogenesis in Leydig cells. Insulin-mediated induction of DAX-1 in Leydig cells of testis may be a key regulatory step of serum sex hormone level in insulin-resistant states.

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Acidic Domain in Dentin Phosphophoryn Facilitates Cellular Uptake: IMPLICATIONS IN TARGETED PROTEIN DELIVERY.

Publication Date: 2013 May 31 PMID: 23589294
Authors: Ravindran, S. - Snee, P. T. - Ramachandran, A. - George, A.
Journal: J Biol Chem

Dentin phosphophoryn is nature's most acidic protein found predominantly in the dentin extracellular matrix. Its unique amino acid composition containing Asp-Ser (DS)-rich repeats makes it highly anionic. It has a low isoelectric point (pI 1.1) and, therefore, tends to be negatively charged at physiological pH. Phosphophoryn is normally associated with matrix mineralization as it can bind avidly to Ca(2+). It is well known that several macromolecules present in the extracellular matrix can be internalized and localized to specific intracellular compartments. In this study we demonstrate that dentin phosphophoryn (DPP) is internalized by several cell types via a non-conventional endocytic process. Utilizing a DSS polypeptide derived from DPP, we demonstrate the repetitive DSS-rich domain facilitates that endocytosis. As a proof-of-concept, we further demonstrate the use of this polypeptide as a protein delivery vehicle by delivering the osteoblast transcription factor Runx2 to the nucleus of mesenchymal cells. The functionality of the endocytosed Runx2 protein was demonstrated by performing gene expression analysis of Runx2 target genes. Nuclear localization was also demonstrated with the fusion protein DSS-Runx2 conjugated to quantum dots in two- and three-dimensional culture models in vitro and in vivo. Overall, we demonstrate that the DSS domain of DPP functions as a novel cell-penetrating peptide, and these findings demonstrate new opportunities for intracellular delivery of therapeutic proteins and cell tracking in vivo.

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Antigen-specific Immunotherapy Regulates B Cell Activities in the Intestine.

Publication Date: 2013 Jun 7 PMID: 23589293
Authors: Yang, Q. - Liang, Y. - Si, L. - Ji, Q. - Xu, Q. - Zhang, Y. - Li, X.
Journal: J Biol Chem

Mature B cells (BCs) express CD23 and B cell receptors. Whether activation of CD23 and B cell receptors has different effects on BC activities is unclear. This study aims to investigate the mechanism by which the specific antigen immunotherapy regulates the activation of BCs in the skewed Th2 responses. Mice were sensitized to ovalbumin. The specific antigen vaccination (SAV) at graded doses was employed to modulate the activities of BCs in which the expression of IL-10, IgE, matrix metalloproteinase-9 (MMP9), CD23, and serum soluble CD23 by BCs was evaluated. The immune regulatory effect of BCs primed by lower or higher SAV doses was observed with an adoptive transfer mouse experiment. SAV activated CD23 to produce IL-10 in BCs at lower doses. The higher doses of SAV increased the expression of MMP9 in BCs that reduced the amounts of CD23 in BCs and increased the serum levels of soluble CD23, which was abrogated by the pretreatment with MMP9 inhibitor. Adoptively transfer with BCs primed by lower doses of SAV inhibited the ongoing antigen-specific Th2 responses whereas the BCs primed by higher doses of SAV exacerbated the ongoing Th2 responses. Exposure to specific antigens at optimal doses can activate BCs to produce IL-10 to suppress the skewed antigen-specific Th2 responses. The antigen doses of SAV higher than the optimal doses may promote the production of soluble CD23 to exacerbate the ongoing immune responses.

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NADPH Oxidase 4 Regulates Cardiomyocyte Differentiation via Redox Activation of c-Jun Protein and the cis-Regulation of GATA-4 Gene Transcription.

Publication Date: 2013 May 31 PMID: 23589292
Authors: Murray, T. V. - Smyrnias, I. - Shah, A. M. - Brewer, A. C.
Journal: J Biol Chem

NADPH oxidase 4 (Nox4) generates reactive oxygen species (ROS) that can modulate cellular phenotype and function in part through the redox modulation of the activity of transcription factors. We demonstrate here the potential of Nox4 to drive cardiomyocyte differentiation in pluripotent embryonal carcinoma cells, and we show that this involves the redox activation of c-Jun. This in turn acts to up-regulate GATA-4 expression, one of the earliest markers of cardiotypic differentiation, through a defined and highly conserved cis-acting motif within the GATA-4 promoter. These data therefore suggest a mechanism whereby ROS act in pluripotential cells in vivo to regulate the initial transcription of critical tissue-restricted determinant(s) of the cardiomyocyte phenotype, including GATA-4. The ROS-dependent activation, mediated by Nox4, of widely expressed redox-regulated transcription factors, such as c-Jun, is fundamental to this process.

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The Serum- and Glucocorticoid-inducible Kinases SGK1 and SGK3 Regulate hERG Channel Expression via Ubiquitin Ligase Nedd4-2 and GTPase Rab11.

Publication Date: 2013 May 24 PMID: 23589291
Authors: Lamothe, S. M. - Zhang, S.
Journal: J Biol Chem

The hERG (human ether-a-go-go-related gene) encodes the alpha subunit of the rapidly activating delayed rectifier potassium channel (IKr). Dysfunction of hERG channels due to mutations or certain medications causes long QT syndrome, which can lead to fatal ventricular arrhythmias or sudden death. Although the abundance of hERG in the plasma membrane is a key determinant of hERG functionality, the mechanisms underlying its regulation are not well understood. In the present study, we demonstrated that overexpression of the stress-responsive serum- and glucocorticoid-inducible kinase (SGK) isoforms SGK1 and SGK3 increased the current and expression level of the membrane-localized mature proteins of hERG channels stably expressed in HEK 293 (hERG-HEK) cells. Furthermore, the synthetic glucocorticoid, dexamethasone, increased the current and abundance of mature ERG proteins in both hERG-HEK cells and neonatal cardiac myocytes through the enhancement of SGK1 but not SGK3 expression. We have previously shown that mature hERG channels are degraded by ubiquitin ligase Nedd4-2 via enhanced channel ubiquitination. Here, we showed that SGK1 or SGK3 overexpression increased Nedd4-2 phosphorylation, which is known to inhibit Nedd4-2 activity. Nonetheless, disruption of the Nedd4-2 binding site in hERG channels did not eliminate the SGK-induced increase in hERG expression. Additional disruption of Rab11 proteins led to a complete elimination of SGK-mediated increase in hERG expression. These results show that SGK enhances the expression level of mature hERG channels by inhibiting Nedd4-2 as well as by promoting Rab11-mediated hERG recycling.

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Glucosylceramide Transferase Activity Is Critical for Encystation and Viable Cyst Production by an Intestinal Protozoan, Giardia lamblia.

Publication Date: 2013 Jun 7 PMID: 23589290
Authors: Mendez, T. L. - De Chatterjee, A. - Duarte, T. T. - Gazos-Lopes, F. - Robles-Martinez, L. - Roy, D. - Sun, J. - Maldonado, R. A. - Roychowdhury, S. - Almeida, I. C. - Das, S.
Journal: J Biol Chem

The production of viable cysts by Giardia is essential for its survival in the environment and for spreading the infection via contaminated food and water. The hallmark of cyst production (also known as encystation) is the biogenesis of encystation-specific vesicles (ESVs) that transport cyst wall proteins to the plasma membrane of the trophozoite before laying down the protective cyst wall. However, the molecules that regulate ESV biogenesis and maintain cyst viability have never before been identified. Here, we report that giardial glucosylceramide transferase-1 (gGlcT1), an enzyme of sphingolipid biosynthesis, plays a key role in ESV biogenesis and maintaining cyst viability. We find that overexpression of this enzyme induced the formation of aggregated/enlarged ESVs and generated clustered cysts with reduced viability. The silencing of gGlcT1 synthesis by antisense morpholino oligonucleotide abolished ESV production and generated mostly nonviable cysts. Interestingly, when gGlcT1-overexpressed Giardia was transfected with anti-gGlcT1 morpholino, the enzyme activity, vesicle biogenesis, and cyst viability returned to normal, suggesting that the regulated expression of gGlcT1 is important for encystation and viable cyst production. Furthermore, the overexpression of gGlcT1 increased the influx of membrane lipids and fatty acids without altering the fluidity of plasma membranes, indicating that the expression of gGlcT1 activity is linked to lipid internalization and maintaining the overall lipid balance in this parasite. Taken together, our results suggest that gGlcT1 is a key player of ESV biogenesis and cyst viability and therefore could be targeted for developing new anti-giardial therapies.

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Electrostatic and hydrophobic interactions differentially tune membrane binding kinetics of the c2 domain of protein kinase Calpha.

Publication Date: 2013 Jun 7 PMID: 23589289
Authors: Scott, A. M. - Antal, C. E. - Newton, A. C.
Journal: J Biol Chem

The cellular activation of conventional protein kinase C (PKC) isozymes is initiated by the binding of their C2 domains to membranes in response to elevations in intracellular Ca(2+). Following this C2 domain-mediated membrane recruitment, the C1 domain binds its membrane-embedded ligand diacylglycerol, resulting in activation of PKC. Here we explore the molecular mechanisms by which the C2 domain controls the initial step in the activation of PKC. Using stopped-flow fluorescence spectroscopy to measure association and dissociation rate constants, we show that hydrophobic interactions are the major driving force in the binding of the C2 domain to anionic membranes, whereas electrostatic interactions dominate in membrane retention. Specifically, mutation of select hydrophobic or select basic residues in the Ca(2+)-binding loops reduces membrane affinity by distinct mechanisms; mutation of hydrophobic residues primarily alters association rate constants, whereas mutation of charged residues affects dissociation rate constants. Live cell imaging reveals that introduction of these mutations into full-length PKCalpha not only reduces the Ca(2+)-dependent translocation to plasma membrane but, by impairing the plasma membrane-sensing role of the C2 domain, causes phorbol ester-triggered redistribution of PKCalpha to other membranes, such as the Golgi. These data underscore the key role of the C2 domain in driving conventional PKC isozymes to the plasma membrane and reveal that not only the amplitude but also the subcellular location of conventional PKC signaling can be tuned by altering the affinity of this module for membranes.

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Molecular Determinants of the Substrate Specificity of the Complement-initiating Protease, C1r.

Publication Date: 2013 May 31 PMID: 23589288
Authors: Wijeyewickrema, L. C. - Yongqing, T. - Tran, T. P. - Thompson, P. E. - Viljoen, J. E. - Coetzer, T. H. - Duncan, R. C. - Kass, I. - Buckle, A. M. - Pike, R. N.
Journal: J Biol Chem

The serine protease, C1r, initiates activation of the classical pathway of complement, which is a crucial innate defense mechanism against pathogens and altered-self cells. C1r both autoactivates and subsequently cleaves and activates C1s. Because complement is implicated in many inflammatory diseases, an understanding of the interaction between C1r and its target substrates is required for the design of effective inhibitors of complement activation. Examination of the active site specificity of C1r using phage library technology revealed clear specificity for Gln at P2 and Ile at P1', which are found in these positions in physiological substrates of C1r. Removal of one or both of the Gln at P2 and Ile at P1' in the C1s substrate reduced the rate of C1r activation. Substituting a Gln residue into the P2 of the activation site of MASP-3, a protein with similar domain structure to C1s that is not normally cleaved by C1r, enabled efficient activation of this enzyme. Molecular dynamics simulations and structural modeling of the interaction of the C1s activation peptide with the active site of C1r revealed the molecular mechanisms that particularly underpin the specificity of the enzyme for the P2 Gln residue. The complement control protein domains of C1r also made important contributions to efficient activation of C1s by this enzyme, indicating that exosite interactions were also important. These data show that C1r specificity is well suited to its cleavage targets and that efficient cleavage of C1s is achieved through both active site and exosite contributions.

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Transforming Growth Factor-beta1 (TGF-beta1)-stimulated Fibroblast to Myofibroblast Differentiation Is Mediated by Hyaluronan (HA)-facilitated Epidermal Growth Factor Receptor (EGFR) and CD44 Co-localization in Lipid Rafts.

Publication Date: 2013 May 24 PMID: 23589287
Authors: Midgley, A. C. - Rogers, M. - Hallett, M. B. - Clayton, A. - Bowen, T. - Phillips, A. O. - Steadman, R.
Journal: J Biol Chem

Fibroblast to myofibroblast differentiation drives effective wound healing and is largely regulated by the cytokine transforming growth factor-beta1 (TGF-beta1). Myofibroblasts express alpha-smooth muscle actin and are present in granulation tissue, where they are responsible for wound contraction. Our previous studies show that fibroblast differentiation in response to TGF-beta1 is dependent on and mediated by the linear polysaccharide hyaluronan (HA). Both the HA receptor, CD44, and the epidermal growth factor receptor (EGFR) are involved in this differentiation response. The aim of this study was to understand the mechanisms linking HA-, CD44-, and EGFR-regulated TGF-beta1-dependent differentiation. CD44 and EGFR co-localization within membrane-bound lipid rafts was necessary for differentiation, and this triggered downstream mitogen-activated protein kinase (MAPK/ERK) and Ca(2+)/calmodulin kinase II (CaMKII) activation. We also found that ERK phosphorylation was upstream of CaMKII phosphorylation, that ERK activation was necessary for CaMKII signaling, and that both kinases were essential for differentiation. In addition, HA synthase-2 (HAS2) siRNA attenuated both ERK and CaMKII signaling and sequestration of CD44 into lipid rafts, preventing differentiation. In summary, the data suggest that HAS2-dependent production of HA facilitates TGF-beta1-dependent fibroblast differentiation through promoting CD44 interaction with EGFR held within membrane-bound lipid rafts. This induces MAPK/ERK, followed by CaMKII activation, leading to differentiation. This pathway is synergistic with the classical TGF-beta1-dependent SMAD-signaling pathway and may provide a novel opportunity for intervention in wound healing.

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Inflammatory Cytokines Induce NOTCH Signaling in Nucleus Pulposus Cells: IMPLICATIONS IN INTERVERTEBRAL DISC DEGENERATION.

Publication Date: 2013 Jun 7 PMID: 23589286
Authors: Wang, H. - Tian, Y. - Wang, J. - Phillips, K. L. - Binch, A. L. - Dunn, S. - Cross, A. - Chiverton, N. - Zheng, Z. - Shapiro, I. M. - Le Maitre, C. L. - Risbud, M. V.
Journal: J Biol Chem

The objective of the study was to investigate how inflammatory cytokines, IL-1beta, and TNF-alpha control NOTCH signaling activity in nucleus pulposus (NP) cells. An increase in expression of selective NOTCH receptors (NOTCH1 and -2), ligand (JAGGED2), and target genes (HES1, HEY1, and HEY2) was observed in NP cells following cytokine treatment. A concomitant increase in NOTCH signaling as evidenced by induction in activity of target gene HES1 and HEY1 promoters and reporter 12xCSL was seen. Moreover, treatment increased activity of a 2-kb NOTCH2 promoter. Treatment of cells with NF-kappaB and MAPK inhibitors abolished the inductive effect of cytokines on NOTCH2 promoter and its expression. Gain and loss-of-function studies confirmed the inductive effect of p65 on NOTCH2 promoter activity. In contrast, p50 blocked the cytokine induction of promoter activity. Supporting promoter studies, lentiviral delivery of sh-p65, and sh-IKKbeta significantly decreased cytokine dependent change in NOTCH2 expression. Interestingly, MAPK signaling showed an isoform-specific control of NOTCH2 promoter; p38alpha/beta2/delta, ERK1, and ERK2 contributed to cytokine dependent induction, whereas p38gamma played no role. Analysis of human NP tissues showed that NOTCH1 and -2 and HEY2 expression correlated with each other. Moreover, expression of NOTCH2 and IL-1beta as well as the number of cells immunopositive for NOTCH2 significantly increased in histologically degenerate discs compared with non-degenerate discs. Taken together, these results explain the observed dysregulated expression of NOTCH genes in degenerative disc disease. Thus, controlling IL-1beta and TNF-alpha activities during disc disease may restore NOTCH signaling and nucleus pulposus cell function.

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Sphingosine 1-Phosphate (S1P) Receptor Agonists Mediate Pro-fibrotic Responses in Normal Human Lung Fibroblasts via S1P2 and S1P3 Receptors and Smad-independent Signaling.

Publication Date: 2013 May 24 PMID: 23589284
Authors: Sobel, K. - Menyhart, K. - Killer, N. - Renault, B. - Bauer, Y. - Studer, R. - Steiner, B. - Bolli, M. H. - Nayler, O. - Gatfield, J.
Journal: J Biol Chem

Synthetic sphingosine 1-phosphate receptor 1 modulators constitute a new class of drugs for the treatment of autoimmune diseases. Sphingosine 1-phosphate (S1P) signaling, however, is also involved in the development of fibrosis. Using normal human lung fibroblasts, we investigated the induction of fibrotic responses by the S1P receptor (S1PR) agonists S1P, FTY720-P, ponesimod, and SEW2871 and compared them with the responses induced by the known fibrotic mediator TGF-beta1. In contrast to TGF-beta1, S1PR agonists did not induce expression of the myofibroblast marker alpha-smooth muscle actin. However, TGF-beta1, S1P, and FTY720-P caused robust stimulation of extracellular matrix (ECM) synthesis and increased pro-fibrotic marker gene expression including connective tissue growth factor. Ponesimod showed limited and SEW2871 showed no pro-fibrotic potential in these readouts. Analysis of pro-fibrotic signaling pathways showed that in contrast to TGF-beta1, S1PR agonists did not activate Smad2/3 signaling but rather activated PI3K/Akt and ERK1/2 signaling to induce ECM synthesis. The strong induction of ECM synthesis by the nonselective agonists S1P and FTY720-P was due to the stimulation of S1P2 and S1P3 receptors, whereas the weaker induction of ECM synthesis at high concentrations of ponesimod was due to a low potency activation of S1P3 receptors. Finally, in normal human lung fibroblast-derived myofibroblasts that were generated by TGF-beta1 pretreatment, S1P and FTY720-P were effective stimulators of ECM synthesis, whereas ponesimod was inactive, because of the down-regulation of S1P3R expression in myofibroblasts. These data demonstrate that S1PR agonists are pro-fibrotic via S1P2R and S1P3R stimulation using Smad-independent pathways.

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Structural Basis for the Enzymatic Formation of the Key Strawberry Flavor Compound 4-Hydroxy-2,5-dimethyl-3(2H)-furanone.

Publication Date: 2013 Jun 7 PMID: 23589283
Authors: Schiefner, A. - Sinz, Q. - Neumaier, I. - Schwab, W. - Skerra, A.
Journal: J Biol Chem

The last step in the biosynthetic route to the key strawberry flavor compound 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) is catalyzed by Fragaria x ananassa enone oxidoreductase (FaEO), earlier putatively assigned as quinone oxidoreductase (FaQR). The ripening-induced enzyme catalyzes the reduction of the exocyclic double bond of the highly reactive precursor 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone (HMMF) in a NAD(P)H-dependent manner. To elucidate the molecular mechanism of this peculiar reaction, we determined the crystal structure of FaEO in six different states or complexes at resolutions of
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Identification of an Active Site-bound Nitrile Hydratase Intermediate through Single Turnover Stopped-flow Spectroscopy.

Publication Date: 2013 May 31 PMID: 23589282
Authors: Gumataotao, N. - Kuhn, M. L. - Hajnas, N. - Holz, R. C.
Journal: J Biol Chem

Stopped-flow kinetic data were obtained for the iron-type nitrile hydratase from Rhodococcus equi TG328-2 (ReNHase) using methacrylonitrile as the substrate. Multiple turnover experiments suggest a three-step kinetic model that allows for the reversible binding of substrate, the presence of an intermediate, and the formation of product. Microscopic rate constants determined from these data are in good agreement with steady state data confirming that the stopped-flow method used was appropriate for the reaction. Single turnover stopped-flow experiments were used to identify catalytic intermediates. These data were globally fit confirming a three-step kinetic model. Independent absorption spectra acquired between 0.005 and 0.5 s of the reaction reveal a significant increase in absorbance at 375, 460, and 550 nm along with the hypsochromic shift of an Fe(3+)<--S ligand-to-metal charge transfer band from 700 to 650 nm. The observed UV-visible absorption bands for the Fe(3+)-nitrile intermediate species are similar to low spin Fe(3+)-enzyme and model complexes bound by NO or N3((-)). These data provide spectroscopic evidence for the direct coordination of the nitrile substrate to the nitrile hydratase active site low spin Fe(3+) center.

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Structural Basis for a Cofactor-dependent Oxidation Protection and Catalysis of Cyanobacterial Succinic Semialdehyde Dehydrogenase.

Publication Date: 2013 May 31 PMID: 23589281
Authors: Park, J. - Rhee, S.
Journal: J Biol Chem

Succinic semialdehyde dehydrogenase (SSADH) from cyanobacterium Synechococcus differs from other SSADHs in the gamma-aminobutyrate shunt. Synechococcus SSADH (SySSADH) is a TCA cycle enzyme and completes a 2-oxoglutarate dehydrogenase-deficient cyanobacterial TCA cycle through a detour metabolic pathway. SySSADH produces succinate in an NADP(+)-dependent manner with a single cysteine acting as the catalytic residue in the catalytic loop. Crystal structures of SySSADH were determined in their apo form, as a binary complex with NADP(+) and as a ternary complex with succinic semialdehyde and NADPH, providing details about the catalytic mechanism by revealing a covalent adduct of a cofactor with the catalytic cysteine in the binary complex and a proposed thiohemiacetal intermediate in the ternary complex. Further analyses showed that SySSADH is an oxidation-sensitive enzyme and that the formation of the NADP-cysteine adduct is a kinetically preferred event that protects the catalytic cysteine from H2O2-dependent oxidative stress. These structural and functional features of SySSADH provide a molecular basis for cofactor-dependent oxidation protection in 1-Cys SSADH, which is unique relative to other 2-Cys SSADHs employing a redox-dependent formation of a disulfide bridge.

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N-terminal and Central Segments of the Type 1 Ryanodine Receptor Mediate Its Interaction with FK506-binding Proteins.

Publication Date: 2013 May 31 PMID: 23585572
Authors: Girgenrath, T. - Mahalingam, M. - Svensson, B. - Nitu, F. R. - Cornea, R. L. - Fessenden, J. D.
Journal: J Biol Chem

We used site-directed labeling of the type 1 ryanodine receptor (RyR1) and fluorescence resonance energy transfer (FRET) measurements to map RyR1 sequence elements forming the binding site of the 12-kDa binding protein for the immunosuppressant drug, FK506. This protein, FKBP12, promotes the RyR1 closed state, thereby inhibiting Ca(2+) leakage in resting muscle. Although FKBP12 function is well established, its binding determinants within the RyR1 protein sequence remain unresolved. To identify these sequence determinants using FRET, we created five single-Cys FKBP variants labeled with Alexa Fluor 488 (denoted D-FKBP) and then targeted these D-FKBPs to full-length RyR1 constructs containing decahistidine (His10) "tags" placed within N-terminal (amino acid residues 76-619) or central (residues 2157-2777) regions of RyR1. The FRET acceptor Cy3NTA bound specifically and saturably to these His tags, allowing distance analysis of FRET measured from each D-FKBP variant to Cy3NTA bound to each His tag. Results indicate that D-FKBP binds proximal to both N-terminal and central domains of RyR1, thus suggesting that the FKBP binding site is composed of determinants from both regions. These findings further imply that the RyR1 N-terminal and central domains are proximal to one another, a core premise of the domain-switch hypothesis of RyR function. We observed FRET from GFP fused at position 620 within the N-terminal domain to central domain His-tagged sites, thus further supporting this hypothesis. Taken together, these results support the conclusion that N-terminal and central domain elements are closely apposed near the FKBP binding site within the RyR1 three-dimensional structure.

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Water-soluble LYNX1 Residues Important for Interaction with Muscle-type and/or Neuronal Nicotinic Receptors.

Publication Date: 2013 May 31 PMID: 23585571
Authors: Lyukmanova, E. N. - Shulepko, M. A. - Buldakova, S. L. - Kasheverov, I. E. - Shenkarev, Z. O. - Reshetnikov, R. V. - Filkin, S. Y. - Kudryavtsev, D. S. - Ojomoko, L. O. - Kryukova, E. V. - Dolgikh, D. A. - Kirpichnikov, M. P. - Bregestovski, P. D. - Tsetlin, V. I.
Journal: J Biol Chem

Human LYNX1, belonging to the Ly6/neurotoxin family of three-finger proteins, is membrane-tethered with a glycosylphosphatidylinositol anchor and modulates the activity of nicotinic acetylcholine receptors (nAChR). Recent preparation of LYNX1 as an individual protein in the form of water-soluble domain lacking glycosylphosphatidylinositol anchor (ws-LYNX1; Lyukmanova, E. N., Shenkarev, Z. O., Shulepko, M. A., Mineev, K. S., D'Hoedt, D., Kasheverov, I. E., Filkin, S. Y., Krivolapova, A. P., Janickova, H., Dolezal, V., Dolgikh, D. A., Arseniev, A. S., Bertrand, D., Tsetlin, V. I., and Kirpichnikov, M. P. (2011) NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1. J. Biol. Chem. 286, 10618-10627) revealed the attachment at the agonist-binding site in the acetylcholine-binding protein (AChBP) and muscle nAChR but outside it, in the neuronal nAChRs. Here, we obtained a series of ws-LYNX1 mutants (T35A, P36A, T37A, R38A, K40A, Y54A, Y57A, K59A) and examined by radioligand analysis or patch clamp technique their interaction with the AChBP, Torpedo californica nAChR and chimeric receptor composed of the alpha7 nAChR extracellular ligand-binding domain and the transmembrane domain of alpha1 glycine receptor (alpha7-GlyR). Against AChBP, there was either no change in activity (T35A, T37A), slight decrease (K40A, K59A), and even enhancement for the rest mutants (most pronounced for P36A and R38A). With both receptors, many mutants lost inhibitory activity, but the increased inhibition was observed for P36A at alpha7-GlyR. Thus, there are subtype-specific and common ws-LYNX1 residues recognizing distinct targets. Because ws-LYNX1 was inactive against glycine receptor, its "non-classical" binding sites on alpha7 nAChR should be within the extracellular domain. Micromolar affinities and fast washout rates measured for ws-LYNX1 and its mutants are in contrast to nanomolar affinities and irreversibility of binding for alpha-bungarotoxin and similar snake alpha-neurotoxins also targeting alpha7 nAChR. This distinction may underlie their different actions, i.e. nAChRs modulation versus irreversible inhibition, for these two types of three-finger proteins.

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The Oncogene HER2/neu (ERBB2) Requires the Hypoxia-inducible Factor HIF-1 for Mammary Tumor Growth and Anoikis Resistance.

Publication Date: 2013 May 31 PMID: 23585570
Authors: Whelan, K. A. - Schwab, L. P. - Karakashev, S. V. - Franchetti, L. - Johannes, G. J. - Seagroves, T. N. - Reginato, M. J.
Journal: J Biol Chem

ERBB2, a receptor tyrosine kinase amplified in breast cancer, is a well established regulator of tumor growth in vivo and anoikis resistance leading to disruption of architecture in three-dimensional mammary epithelial acinar structures in vitro. ERBB2 promotes anoikis resistance by maintaining signaling pathways and by rescuing metabolic defects and thus inhibiting accumulation of deleterious reactive oxygen species. Recent evidence suggests that hypoxia, via hypoxia-inducible factors (HIFs), can inhibit anoikis; thus, we hypothesized that HIF-1 may play a role in ERBB2-mediated anoikis resistance and oncogenesis. Indeed, tumors isolated from MMTV-Neu mice contain elevated HIF-1alpha levels and tumor cells created from MMTV-Neu mice harboring deletion of Hif1alpha alleles reduced primary tumor growth in vivo. ERBB2 overexpressing cancer cells stabilize HIF under normoxic conditions and require HIF-1 for ERBB2-mediated anchorage-independence, three-dimensional culture growth and anoikis resistance. HIF-1 reduction in ERBB2 cells was associated with induction of the pro-anoikis protein BIM and decreased ERK and AKT signaling during cell detachment. ERBB2-mediated inhibition of metabolic defects, including decreased reactive oxygen species generation in suspension, required HIF-1 expression that was critical for ERBB2-mediated oncogenesis. Gene expression profiling of hypoxic three-dimensional acinar structures identified a number of genes elevated in response to hypoxia that are known ERBB2 targets, suggesting that hypoxic conditions and ERBB2 overexpression share both phenotypic and genetic components via HIF-1 regulation. Thus, our data demonstrate that ERBB2 requires HIF-1 for tumor growth and suggest that HIF is a major downstream regulator of ERBB2 that protects cells from anoikis and metabolic stress caused by decreased matrix adhesion.

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Structure and mechanism of rhomboid protease.

Publication Date: 2013 May 31 PMID: 23585569
Authors: Ha, Y. - Akiyama, Y. - Xue, Y.
Journal: J Biol Chem

Rhomboid protease was first discovered in Drosophila. Mutation of the fly gene interfered with growth factor signaling and produced a characteristic phenotype of a pointed head skeleton. The name rhomboid has since been widely used to describe a large family of related membrane proteins that have diverse biological functions but share a common catalytic core domain composed of six membrane-spanning segments. Most rhomboid proteases cleave membrane protein substrates near the N terminus of their transmembrane domains. How these proteases function within the confines of the membrane is not completely understood. Recent progress in crystallographic analysis of the Escherichia coli rhomboid protease GlpG in complex with inhibitors has provided new insights into the catalytic mechanism of the protease and its conformational change. Improved biochemical assays have also identified a substrate sequence motif that is specifically recognized by many rhomboid proteases.

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Structural biology of presenilins and signal Peptide peptidases.

Publication Date: 2013 May 24 PMID: 23585568
Authors: Tomita, T. - Iwatsubo, T.
Journal: J Biol Chem

Presenilin and signal peptide peptidase are multispanning intramembrane-cleaving proteases with a conserved catalytic GxGD motif. Presenilin comprises the catalytic subunit of gamma-secretase, a protease responsible for the generation of amyloid-beta peptides causative of Alzheimer disease. Signal peptide peptidase proteins are implicated in the regulation of the immune system. Both protease family proteins have been recognized as druggable targets for several human diseases, but their detailed structure still remains unknown. Recently, the x-ray structures of some archaeal GxGD proteases have been determined. We review the recent progress in biochemical and biophysical probing of the structure of these atypical proteases.

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The Spontaneously Adhesive Leukocyte Function-associated Antigen-1 (LFA-1) Integrin in Effector T Cells Mediates Rapid Actin- and Calmodulin-dependent Adhesion Strengthening to Ligand under Shear Flow.

Publication Date: 2013 May 24 PMID: 23585567
Authors: Lek, H. S. - Morrison, V. L. - Conneely, M. - Campbell, P. A. - McGloin, D. - Kliche, S. - Watts, C. - Prescott, A. - Fagerholm, S. C.
Journal: J Biol Chem

Integrins in effector T cells are highly expressed and important for trafficking of these cells and for their effector functions. However, how integrins are regulated in effector T cells remains poorly characterized. Here, we have investigated effector T cell leukocyte function-associated antigen-1 (LFA-1) regulation in primary murine effector T cells. These cells have high LFA-1 integrin expression and display high spontaneous binding to intercellular adhesion molecule-1 (ICAM-1) ligand under static conditions. In addition, these cells are able to migrate spontaneously on ICAM-1. Atomic force microscopy measurements showed that the force required for unbinding of integrin-ligand interactions increases over time (0.5-20-s contact time). The maximum unbinding force for this interaction was approximately 140 piconewtons at 0.5-s contact time, increasing to 580 piconewtons at 20-s contact time. Also, the total work required to disrupt the interaction increased over the 20-s contact time, indicating LFA-1-mediated adhesion strengthening in primary effector T cells over a very quick time frame. Effector T cells adhered spontaneously to ICAM-1 under conditions of shear flow, in the absence of chemokine stimulation, and this binding was independent of protein kinase B/Akt and protein kinase C kinase activity, but dependent on calcium/calmodulin signaling and an intact actin cytoskeleton. These results indicate that effector T cell integrins are highly expressed and spontaneously adhesive in the absence of inside-out integrin signaling but that LFA-1-mediated firm adhesion under conditions of shear flow requires downstream integrin signaling, which is dependent on calcium/calmodulin and the actin cytoskeleton.

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Mammalian Target of Rapamycin (mTor) Mediates Tau Protein Dyshomeostasis: IMPLICATION FOR ALZHEIMER DISEASE.

Publication Date: 2013 May 31 PMID: 23585566
Authors: Tang, Z. - Bereczki, E. - Zhang, H. - Wang, S. - Li, C. - Ji, X. - Branca, R. M. - Lehtio, J. - Guan, Z. - Filipcik, P. - Xu, S. - Winblad, B. - Pei, J. J.
Journal: J Biol Chem

Previous evidence from post-mortem Alzheimer disease (AD) brains and drug (especially rapamycin)-oriented in vitro and in vivo models implicated an aberrant accumulation of the mammalian target of rapamycin (mTor) in tangle-bearing neurons in AD brains and its role in the formation of abnormally hyperphosphorylated tau. Compelling evidence indicated that the sequential molecular events such as the synthesis and phosphorylation of tau can be regulated through p70 S6 kinase, the well characterized immediate downstream target of mTor. In the present study, we further identified that the active form of mTor per se accumulates in tangle-bearing neurons, particularly those at early stages in AD brains. By using mass spectrometry and Western blotting, we identified three phosphoepitopes of tau directly phosphorylated by mTor. We have developed a variety of stable cell lines with genetic modification of mTor activity using SH-SY5Y neuroblastoma cells as background. In these cellular systems, we not only confirmed the tau phosphorylation sites found in vitro but also found that mTor mediates the synthesis and aggregation of tau, resulting in compromised microtubule stability. Changes of mTor activity cause fluctuation of the level of a battery of tau kinases such as protein kinase A, v-Akt murine thymoma viral oncogene homolog-1, glycogen synthase kinase 3beta, cyclin-dependent kinase 5, and tau protein phosphatase 2A. These results implicate mTor in promoting an imbalance of tau homeostasis, a condition required for neurons to maintain physiological function.

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Mechanisms of Neuroprotection from Hypoxia-Ischemia (HI) Brain Injury by Up-regulation of Cytoglobin (CYGB) in a Neonatal Rat Model.

Publication Date: 2013 May 31 PMID: 23585565
Authors: Tian, S. F. - Yang, H. H. - Xiao, D. P. - Huang, Y. J. - He, G. Y. - Ma, H. R. - Xia, F. - Shi, X. C.
Journal: J Biol Chem

This study was designed to investigate the expression profile of CYGB, its potential neuroprotective function, and underlying molecular mechanisms using a model of neonatal hypoxia-ischemia (HI) brain injury. Cygb mRNA and protein expression were evaluated within the first 36 h after the HI model was induced using RT-PCR and Western blotting. Cygb mRNA expression was increased at 18 h in a time-dependent manner, and its level of protein expression increased progressively in 24 h. To verify the neuroprotective effect of CYGB, a gene transfection technique was employed. Cygb cDNA and shRNA delivery adenovirus systems were established (Cygb-cDNA-ADV and Cygb-shRNA-ADV, respectively) and injected into the brains of 3-day-old rats 4 days before they were induced with HI treatment. Rats from different groups were euthanized 24 h post-HI, and brain samples were harvested. 2,3,5-Triphenyltetrazolium chloride, TUNEL, and Nissl staining indicated that an up-regulation of CYGB resulted in reduced acute brain injury. The superoxide dismutase level was found to be dependent on expression of CYGB. The Morris water maze test in 28-day-old rats demonstrated that CYGB expression was associated with improvement of long term cognitive impairment. Studies also demonstrated that CYGB can up-regulate mRNA and protein levels of VEGF and increase both the density and diameter of the microvessels but inhibits activation of caspase-2 and -3. Thus, this is the first in vivo study focusing on the neuroprotective role of CYGB. The reduction of neonatal HI injury by CYGB may be due in part to antioxidant and antiapoptotic mechanisms and by promoting angiogenesis.

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Coordinated Processing of 3' Slipped (CAG)n/(CTG)n Hairpins by DNA Polymerases beta and delta Preferentially Induces Repeat Expansions.

Publication Date: 2013 May 24 PMID: 23585564
Authors: Chan, N. L. - Guo, J. - Zhang, T. - Mao, G. - Hou, C. - Yuan, F. - Huang, J. - Zhang, Y. - Wu, J. - Gu, L. - Li, G. M.
Journal: J Biol Chem

Expansion of CAG/CTG trinucleotide repeats causes certain familial neurological disorders. Hairpin formation in the nascent strand during DNA synthesis is considered a major path for CAG/CTG repeat expansion. However, the underlying mechanism is unclear. We show here that removal or retention of a nascent strand hairpin during DNA synthesis depends on hairpin structures and types of DNA polymerases. Polymerase (pol) delta alone removes the 3'-slipped hairpin using its 3'-5' proofreading activity when the hairpin contains no immediate 3' complementary sequences. However, in the presence of pol beta, pol delta preferentially facilitates hairpin retention regardless of hairpin structures. In this reaction, pol beta incorporates several nucleotides to the hairpin 3'-end, which serves as an effective primer for the continuous DNA synthesis by pol delta, thereby leading to hairpin retention and repeat expansion. These findings strongly suggest that coordinated processing of 3'-slipped (CAG)n/(CTG)n hairpins by polymerases delta and beta on during DNA synthesis induces CAG/CTG repeat expansions.

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IOP1 Protein Is an External Component of the Human Cytosolic Iron-Sulfur Cluster Assembly (CIA) Machinery and Functions in the MMS19 Protein-dependent CIA Pathway.

Publication Date: 2013 Jun 7 PMID: 23585563
Authors: Seki, M. - Takeda, Y. - Iwai, K. - Tanaka, K.
Journal: J Biol Chem

The emerging link between iron metabolism and genome integrity is increasingly clear. Recent studies have revealed that MMS19 and cytosolic iron-sulfur cluster assembly (CIA) factors form a complex and have central roles in CIA pathway. However, the composition of the CIA complex, particularly the involvement of the Fe-S protein IOP1, is still unclear. The roles of each component are also largely unknown. Here, we show that MMS19, MIP18, and CIAO1 form a tight "core" complex and that IOP1 is an "external" component of this complex. Although IOP1 and the core complex form a complex both in vivo and in vitro, IOP1 behaves differently in vivo. A deficiency in any core component leads to down-regulation of all of the components. In contrast, IOP1 knockdown does not affect the level of any core component. In MMS19-overproducing cells, other core components are also up-regulated, but the protein level of IOP1 remains unchanged. IOP1 behaves like a target protein in the CIA reaction, like other Fe-S helicases, and the core complex may participate in the maturation process of IOP1. Alternatively, the core complex may catch and hold IOP1 when it becomes mature to prevent its degradation. In any case, IOP1 functions in the MMS19-dependent CIA pathway. We also reveal that MMS19 interacts with target proteins. MIP18 has a role to bridge MMS19 and CIAO1. CIAO1 also binds IOP1. Based on our in vivo and in vitro data, new models of the CIA machinery are proposed.

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Prostate-derived Sterile 20-like Kinases (PSKs/TAOKs) Phosphorylate Tau Protein and Are Activated in Tangle-bearing Neurons in Alzheimer Disease.

Publication Date: 2013 May 24 PMID: 23585562
Authors: Tavares, I. A. - Touma, D. - Lynham, S. - Troakes, C. - Schober, M. - Causevic, M. - Garg, R. - Noble, W. - Killick, R. - Bodi, I. - Hanger, D. P. - Morris, J. D.
Journal: J Biol Chem

In Alzheimer disease (AD), the microtubule-associated protein tau is highly phosphorylated and aggregates into characteristic neurofibrillary tangles. Prostate-derived sterile 20-like kinases (PSKs/TAOKs) 1 and 2, members of the sterile 20 family of kinases, have been shown to regulate microtubule stability and organization. Here we show that tau is a good substrate for PSK1 and PSK2 phosphorylation with mass spectrometric analysis of phosphorylated tau revealing more than 40 tau residues as targets of these kinases. Notably, phosphorylated residues include motifs located within the microtubule-binding repeat domain on tau (Ser-262, Ser-324, and Ser-356), sites that are known to regulate tau-microtubule interactions. PSK catalytic activity is enhanced in the entorhinal cortex and hippocampus, areas of the brain that are most susceptible to Alzheimer pathology, in comparison with the cerebellum, which is relatively spared. Activated PSK is associated with neurofibrillary tangles, dystrophic neurites surrounding neuritic plaques, neuropil threads, and granulovacuolar degeneration bodies in AD brain. By contrast, activated PSKs and phosphorylated tau are rarely detectible in immunostained control human brain. Our results demonstrate that tau is a substrate for PSK and suggest that this family of kinases could contribute to the development of AD pathology and dementia.

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Differential Role for Transcription Factor Oct4 Nucleocytoplasmic Dynamics in Somatic Cell Reprogramming and Self-renewal of Embryonic Stem Cells.

Publication Date: 2013 May 24 PMID: 23580657
Authors: Oka, M. - Moriyama, T. - Asally, M. - Kawakami, K. - Yoneda, Y.
Journal: J Biol Chem

Oct4 is a member of the POU family of transcription factors and plays a critical role in both maintenance of the undifferentiated state of embryonic stem (ES) cells and in the reprogramming of somatic cells to induced pluripotent stem cells. Oct4 is imported into the nucleus where it functions as a transcription factor; however, the spatiotemporal dynamic behavior of Oct4 remains largely unknown. In the present study we show that Oct4 is a nucleocytoplasmic shuttling protein. Furthermore, although Oct4 mutants with altered nuclear import/export activity were able to maintain the self-renewal of ES cells, they displayed limited potential for cellular reprogramming. These results indicate that the intracellular localization of Oct4, which is dependent on nucleocytoplasmic shuttling, must be more strictly regulated for cellular reprogramming, suggesting that Oct4 plays differential roles in the self-renewal of ES cells and in somatic cell reprogramming.

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Transcriptional Regulation of Serine/Threonine Protein Kinase (AKT) Genes by Glioma-associated Oncogene Homolog 1.

Publication Date: 2013 May 24 PMID: 23580656
Authors: Agarwal, N. K. - Qu, C. - Kunkulla, K. - Liu, Y. - Vega, F.
Journal: J Biol Chem

Aberrant activation of Hedgehog signaling has been described in a growing number of cancers, including malignant lymphomas. Here, we report that canonical Hedgehog signaling modulates the transcriptional expression of AKT genes and that AKT1 is a direct transcriptional target of GLI1. We identified two putative binding sites for GLI1 in the AKT1 promoter region and confirmed their functionality using chromatin immunoprecipitation, luciferase reporter, and site-directed mutagenesis assays. Moreover, we provide evidence that GLI1 contributes to the survival of diffuse large B-cell lymphoma (DLBCL) cells and that this effect occurs in part through promotion of the transcription of AKT genes. This finding is of interest as constitutive activation of AKT has been described in DLBCL, but causative factors that explain AKT expression in this lymphoma type are not completely known. In summary, we demonstrated the existence of a novel cross-talk at the transcriptional level between Hedgehog signaling and AKT with biological significance in DLBCL.

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Disruption of the Dapper3 Gene Aggravates Ureteral Obstruction-mediated Renal Fibrosis by Amplifying Wnt/beta-catenin Signaling.

Publication Date: 2013 May 24 PMID: 23580654
Authors: Xue, H. - Xiao, Z. - Zhang, J. - Wen, J. - Wang, Y. - Chang, Z. - Zhao, J. - Gao, X. - Du, J. - Chen, Y. G.
Journal: J Biol Chem

Wnt/beta-catenin signaling plays key roles in embryonic development and tissue homeostasis. Dapper3/Dact3, one of the three members of the Dapper gene family, is transcriptionally repressed in colorectal cancer and may function as a negative regulator of Wnt/beta-catenin signaling. To investigate its physiological functions, we generated a mouse strain harboring conditional null alleles of Dapper3 (Dapper3(flox/flox)), and homozygous Dapper3-deficient (Dapper3(-/-)) mice were produced after crossing with EIIa-cre transgenic mice. We found that Dapper3 is not essential for mouse embryogenesis, postnatal survival, and reproduction. However, adult Dapper3(-/-) mice exhibited a mild reduction in body weight compared with their wild-type littermates, suggesting a functional role of Dapper3 in postnatal growth. To investigate the role of Dapper3 in renal fibrosis, we employed the unilateral ureteral obstruction model. Dapper3 mRNA expression was up-regulated in kidney after unilateral ureteral obstruction. Loss of the Dapper3 gene enhanced myofibroblast activation and extracellular matrix overproduction in the obstructed kidney. Moreover, this aggravated fibrotic phenotype was accompanied with accumulation of Dishevelled2 and beta-catenin proteins and activation of Wnt-targeted fibrotic genes. In primary renal tubular cells, Dapper3 inhibits Wnt-induced epithelial-to-mesenchymal transition. Consistently, Dapper3 interacted with and down-regulated Dishevelled2 protein and attenuated the Wnt-responsive Topflash reporter expression. These findings together suggest that Dapper3 antagonizes the fibrotic actions of Wnt signaling in kidney.

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Mouse Embryonic Stem Cells Are Deficient in Type I Interferon Expression in Response to Viral Infections and Double-stranded RNA.

Publication Date: 2013 May 31 PMID: 23580653
Authors: Wang, R. - Wang, J. - Paul, A. M. - Acharya, D. - Bai, F. - Huang, F. - Guo, Y. L.
Journal: J Biol Chem

Embryonic stem cells (ESCs) are considered to be a promising cell source for regenerative medicine because of their unlimited capacity for self-renewal and differentiation. However, little is known about the innate immunity in ESCs and ESC-derived cells. We investigated the responses of mouse (m)ESCs to three types of live viruses as follows: La Crosse virus, West Nile virus, and Sendai virus. Our results demonstrated mESCs were susceptible to viral infection, but they were unable to express type I interferons (IFNalpha and IFNbeta, IFNalpha/beta), which differ from fibroblasts (10T1/2 cells) that robustly express IFNalpha/beta upon viral infections. The failure of mESCs to express IFNalpha/beta was further demonstrated by treatment with polyIC, a synthetic viral dsRNA analog that strongly induced IFNalpha/beta in 10T1/2 cells. Although polyIC transiently inhibited the transcription of pluripotency markers, the stem cell morphology was not significantly affected. However, polyIC can induce dsRNA-activated protein kinase in mESCs, and this activation resulted in a strong inhibition of cell proliferation. We conclude that the cytosolic receptor dsRNA-activated protein kinase is functional, but the mechanisms that mediate type I IFN expression are deficient in mESCs. This conclusion is further supported by the findings that the major viral RNA receptors are either expressed at very low levels (TLR3 and MDA5) or may not be active (retinoic acid-inducible gene I) in mESCs.

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A Lipoprotein Receptor Cluster IV Mutant Preferentially Binds Amyloid-beta and Regulates Its Clearance from the Mouse Brain.

Publication Date: 2013 May 24 PMID: 23580652
Authors: Sagare, A. P. - Bell, R. D. - Srivastava, A. - Sengillo, J. D. - Singh, I. - Nishida, Y. - Chow, N. - Zlokovic, B. V.
Journal: J Biol Chem

Soluble low density lipoprotein receptor-related protein-1 (sLRP1) binds approximately 70% of amyloid beta-peptide (Abeta) in human plasma. In Alzheimer disease (AD) and individuals with mild cognitive impairment converting to AD, plasma sLRP1 levels are reduced and sLRP1 is oxidized, which results in diminished Abeta peripheral binding and higher levels of free Abeta in plasma. Experimental studies have shown that free circulating Abeta re-enters the brain and that sLRP1 and/or its recombinant wild type cluster IV (WT-LRPIV) prevent Abeta from entering the brain. Treatment of Alzheimer APPsw(+/0) mice with WT-LRPIV has been shown to reduce brain Abeta pathology. In addition to Abeta, LRPIV binds multiple ligands. To enhance LRPIV binding for Abeta relative to other LRP1 ligands, we generated a library of LRPIV-derived fragments and full-length LRPIV variants with glycine replacing aspartic acid residues 3394, 3556, and 3674 in the calcium binding sites. Compared with WT-LRPIV, a lead LRPIV-D3674G mutant had 1.6- and 2.7-fold higher binding affinity for Abeta40 and Abeta42 in vitro, respectively, and a lower binding affinity for other LRP1 ligands (e.g. apolipoprotein E2, E3, and E4 (1.3-1.8-fold), tissue plasminogen activator (2.7-fold), matrix metalloproteinase-9 (4.1-fold), and Factor Xa (3.8-fold)). LRPIV-D3674G cleared mouse endogenous brain Abeta40 and Abeta42 25-27% better than WT-LRPIV. A 3-month subcutaneous treatment of APPsw(+/0) mice with LRPIV-D3674G (40 mug/kg/day) reduced Abeta40 and Alphabeta42 levels in the hippocampus, cortex, and cerebrospinal fluid by 60-80% and improved cerebral blood flow responses and hippocampal function at 9 months of age. Thus, LRPIV-D3674G is an efficient new Abeta clearance therapy.

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HDAC6 Regulates Mutant SOD1 Aggregation through Two SMIR Motifs and Tubulin Acetylation.

Publication Date: 2013 May 24 PMID: 23580651
Authors: Gal, J. - Chen, J. - Barnett, K. R. - Yang, L. - Brumley, E. - Zhu, H.
Journal: J Biol Chem

Histone deacetylase 6 (HDAC6) is a tubulin deacetylase that regulates protein aggregation and turnover. Mutations in Cu/Zn superoxide dismutase (SOD1) linked to familial amyotrophic lateral sclerosis (ALS) make the mutant protein prone to aggregation. However, the role of HDAC6 in mutant SOD1 aggregation and the ALS etiology is unclear. Here we report that HDAC6 knockdown increased mutant SOD1 aggregation in cultured cells. Different from its known role in mediating the degradation of poly-ubiquitinated proteins, HDAC6 selectively interacted with mutant SOD1 via two motifs similar to the SOD1 mutant interaction region (SMIR) that we identified previously in p62/sequestosome 1. Expression of the aggregation-prone mutant SOD1 increased alpha-tubulin acetylation, and the acetylation-mimicking K40Q alpha-tubulin mutant promoted mutant SOD1 aggregation. Our results suggest that ALS-linked mutant SOD1 can modulate HDAC6 activity and increase tubulin acetylation, which, in turn, facilitates the microtubule- and retrograde transport-dependent mutant SOD1 aggregation. HDAC6 impairment might be a common feature in various subtypes of ALS.

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Insight to the Interaction of the Dihydrolipoamide Acetyltransferase (E2) Core with the Peripheral Components in the Escherichia coli Pyruvate Dehydrogenase Complex via Multifaceted Structural Approaches.

Publication Date: 2013 May 24 PMID: 23580650
Authors: Chandrasekhar, K. - Wang, J. - Arjunan, P. - Sax, M. - Park, Y. H. - Nemeria, N. S. - Kumaran, S. - Song, J. - Jordan, F. - Furey, W.
Journal: J Biol Chem

Multifaceted structural approaches were undertaken to investigate interaction of the E2 component with E3 and E1 components from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), as a representative of the PDHc from Gram-negative bacteria. The crystal structure of E3 at 2.5 A resolution reveals similarity to other E3 structures and was an important starting point for understanding interaction surfaces between E3 and E2. Biochemical studies revealed that R129E-E2 and R150E-E2 substitutions in the peripheral subunit-binding domain (PSBD) of E2 greatly diminished PDHc activity, affected interactions with E3 and E1 components, and affected reductive acetylation of E2. Because crystal structures are unavailable for any complete E2-containing complexes, peptide-specific hydrogen/deuterium exchange mass spectrometry was used to identify loci of interactions between 3-lipoyl E2 and E3. Two peptides from the PSBD, including Arg-129, and three peptides from E3 displayed statistically significant reductions in deuterium uptake resulting from interaction between E3 and E2. Of the peptides identified on E3, two were from the catalytic site, and the third was from the interface domain, which for all known E3 structures is believed to interact with the PSBD. NMR clearly demonstrates that there is no change in the lipoyl domain structure on complexation with E3. This is the first instance where the entire wild-type E2 component was employed to understand interactions with E3. A model for PSBD-E3 binding was independently constructed and found to be consistent with the importance of Arg-129, as well as revealing other electrostatic interactions likely stabilizing this complex.

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The sld Genetic Defect: TWO INTRONIC CA REPEATS PROMOTE INSERTION OF THE SUBSEQUENT INTRON AND mRNA DECAY.

Publication Date: 2013 May 24 PMID: 23580649
Authors: Das, B. - Cash, M. N. - Robinson, B. - Kuhns, C. S. - Latchney, L. R. - Fallon, M. A. - Elliott, R. W. - Hand, A. R. - Culp, D. J.
Journal: J Biol Chem

The autosomal recessive mutation, sld, attenuates mucous cell expression in murine sublingual glands with corresponding effects on mucin 19 (Muc19). We conducted a systematic study including genetic mapping, sequencing, and functional analyses to elucidate a mutation to explain the sld phenotype in neonatal mice. Genetic mapping and gene expression analyses localized the sld mutation within the gene Muc19/Smgc, specifically attenuating Muc19 transcripts, and Muc19 knock-out mice mimic the sld phenotype in neonates. Muc19 transcription is unaffected in sld mice, whereas mRNA stability is markedly decreased. Decreased mRNA stability is not due to a defect in 3'-end processing nor to sequence differences in Muc19 transcripts. Comparative sequencing of the Muc19/Smgc gene identified four candidate intronic mutations within the Muc19 coding region. Minigene splicing assays revealed a novel splicing event in which insertion of two additional repeats within a CA repeat region of intron 53 of the sld genome enhances retention of intron 54, decreasing the levels of correctly spliced transcripts. Moreover, pateamine A, an inhibitor of nonsense-mediated mRNA decay, inhibits degradation of aberrant Muc19 transcripts. The mutation in intron 53 thus enhances aberrant splicing leading to degradation of aberrant transcripts and decreased Muc19 message stability, consistent with the sld phenotype. We propose a working model of the unique splicing event enhanced by the mutation, as well as putative explanations for the gradual but limited increase in Muc19 glycoprotein expression and its restricted localization to subpopulations of mucous cells in sld mice during postnatal gland development.

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Atypical Cohesin-Dockerin Complex Responsible for Cell Surface Attachment of Cellulosomal Components: BINDING FIDELITY, PROMISCUITY, AND STRUCTURAL BUTTRESSES.

Publication Date: 2013 Jun 7 PMID: 23580648
Authors: Salama-Alber, O. - Jobby, M. K. - Chitayat, S. - Smith, S. P. - White, B. A. - Shimon, L. J. - Lamed, R. - Frolow, F. - Bayer, E. A.
Journal: J Biol Chem

The rumen bacterium Ruminococcus flavefaciens produces a highly organized multienzyme cellulosome complex that plays a key role in the degradation of plant cell wall polysaccharides, notably cellulose. The R. flavefaciens cellulosomal system is anchored to the bacterial cell wall through a relatively small ScaE scaffoldin subunit, which bears a single type IIIe cohesin responsible for the attachment of two major dockerin-containing scaffoldin proteins, ScaB and the cellulose-binding protein CttA. Although ScaB recruits the catalytic machinery onto the complex, CttA mediates attachment of the bacterial substrate via its two putative carbohydrate-binding modules. In an effort to understand the structural basis for assembly and cell surface attachment of the cellulosome in R. flavefaciens, we determined the crystal structure of the high affinity complex (Kd = 20.83 nm) between the cohesin module of ScaE (CohE) and its cognate X-dockerin (XDoc) modular dyad from CttA at 1.97-A resolution. The structure reveals an atypical calcium-binding loop containing a 13-residue insert. The results further pinpoint two charged specificity-related residues on the surface of the cohesin module that are responsible for specific versus promiscuous cross-strain binding of the dockerin module. In addition, a combined functional role for the three enigmatic dockerin inserts was established whereby these extraneous segments serve as structural buttresses that reinforce the stalklike conformation of the X-module, thus segregating its tethered complement of cellulosomal components from the cell surface. The novel structure of the RfCohE-XDoc complex sheds light on divergent dockerin structure and function and provides insight into the specificity features of the type IIIe cohesin-dockerin interaction.

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From Soil to Structure, a Novel Dimeric beta-Glucosidase Belonging to Glycoside Hydrolase Family 3 Isolated from Compost Using Metagenomic Analysis.

Publication Date: 2013 May 24 PMID: 23580647
Authors: McAndrew, R. P. - Park, J. I. - Heins, R. A. - Reindl, W. - Friedland, G. D. - D'haeseleer, P. - Northen, T. - Sale, K. L. - Simmons, B. A. - Adams, P. D.
Journal: J Biol Chem

A recent metagenomic analysis sequenced a switchgrass-adapted compost community to identify enzymes from microorganisms that were specifically adapted to switchgrass under thermophilic conditions. These enzymes are being examined as part of the pretreatment process for the production of "second-generation" biofuels. Among the enzymes discovered was JMB19063, a novel three-domain beta-glucosidase that belongs to the GH3 (glycoside hydrolase 3) family. Here, we report the structure of JMB19063 in complex with glucose and the catalytic variant D261N crystallized in the presence of cellopentaose. JMB19063 is first structure of a dimeric member of the GH3 family, and we demonstrate that dimerization is required for catalytic activity. Arg-587 and Phe-598 from the C-terminal domain of the opposing monomer are shown to interact with bound ligands in the D261N structure. Enzyme assays confirmed that these residues are absolutely essential for full catalytic activity.

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Structural Insights into the Substrate Specificity of a 6-Phospho-beta-glucosidase BglA-2 from Streptococcus pneumoniae TIGR4.

Publication Date: 2013 May 24 PMID: 23580646
Authors: Yu, W. L. - Jiang, Y. L. - Pikis, A. - Cheng, W. - Bai, X. H. - Ren, Y. M. - Thompson, J. - Zhou, C. Z. - Chen, Y.
Journal: J Biol Chem

The 6-phospho-beta-glucosidase BglA-2 (EC 3.2.1.86) from glycoside hydrolase family 1 (GH-1) catalyzes the hydrolysis of beta-1,4-linked cellobiose 6-phosphate (cellobiose-6'P) to yield glucose and glucose 6-phosphate. Both reaction products are further metabolized by the energy-generating glycolytic pathway. Here, we present the first crystal structures of the apo and complex forms of BglA-2 with thiocellobiose-6'P (a non-metabolizable analog of cellobiose-6'P) at 2.0 and 2.4 A resolution, respectively. Similar to other GH-1 enzymes, the overall structure of BglA-2 from Streptococcus pneumoniae adopts a typical (beta/alpha)8 TIM-barrel, with the active site located at the center of the convex surface of the beta-barrel. Structural analyses, in combination with enzymatic data obtained from site-directed mutant proteins, suggest that three aromatic residues, Tyr(126), Tyr(303), and Trp(338), at subsite +1 of BglA-2 determine substrate specificity with respect to 1,4-linked 6-phospho-beta-glucosides. Moreover, three additional residues, Ser(424), Lys(430), and Tyr(432) of BglA-2, were found to play important roles in the hydrolytic selectivity toward phosphorylated rather than non-phosphorylated compounds. Comparative structural analysis suggests that a tryptophan versus a methionine/alanine residue at subsite -1 may contribute to the catalytic and substrate selectivity with respect to structurally similar 6-phospho-beta-galactosidases and 6-phospho-beta-glucosidases assigned to the GH-1 family.

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Control of Myofibroblast Differentiation by Microtubule Dynamics through a Regulated Localization of mDia2.

Publication Date: 2013 May 31 PMID: 23580645
Authors: Sandbo, N. - Ngam, C. - Torr, E. - Kregel, S. - Kach, J. - Dulin, N.
Journal: J Biol Chem

Myofibroblast differentiation plays a critical role in wound healing and in the pathogenesis of fibrosis. We have previously shown that myofibroblast differentiation is mediated by the activity of serum response factor (SRF), which is tightly controlled by the actin polymerization state. In this study, we investigated the role of the microtubule cytoskeleton in modulating myofibroblast phenotype. Treatment of human lung fibroblasts with the microtubule-destabilizing agent, colchicine, resulted in a formation of numerous stress fibers and expression of myofibroblast differentiation marker proteins. These effects of colchicine were independent of Smad signaling but were mediated by Rho signaling and SRF, as they were attenuated by the Rho kinase inhibitor, Y27632, or by the SRF inhibitor, CCG-1423. TGF-beta-induced myofibroblast differentiation was not accompanied by gross changes in the microtubule polymerization state. However, microtubule stabilization by paclitaxel attenuated TGF-beta-induced myofibroblast differentiation. Paclitaxel had no effect on TGF-beta-induced Smad activation and Smad-dependent gene transcription but inhibited actin polymerization, nuclear accumulation of megakaryoblastic leukemia-1 protein, and SRF activation. The microtubule-associated formin, mDIA2, localized to actin stress fibers upon treatment with TGF-beta, and paclitaxel prevented this localization. Treatment with the formin inhibitor, SMI formin homology 2 domain, inhibited stress fiber formation and myofibroblast differentiation induced by TGF-beta, without affecting Smad-phosphorylation or microtubule polymerization. Together, these data suggest that (a) TGF-beta promotes association of mDia2 with actin stress fibers, which further drives stress fiber formation and myofibroblast differentiation, and (b) microtubule polymerization state controls myofibroblast differentiation through the regulation of mDia2 localization.

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Transgenic Mouse alpha- and beta-Cardiac Myosins Containing the R403Q Mutation Show Isoform-dependent Transient Kinetic Differences.

Publication Date: 2013 May 24 PMID: 23580644
Authors: Lowey, S. - Bretton, V. - Gulick, J. - Robbins, J. - Trybus, K. M.
Journal: J Biol Chem

Familial hypertrophic cardiomyopathy (FHC) is a major cause of sudden cardiac death in young athletes. The discovery in 1990 that a point mutation at residue 403 (R403Q) in the beta-myosin heavy chain (MHC) caused a severe form of FHC was the first of many demonstrations linking FHC to mutations in muscle proteins. A mouse model for FHC has been widely used to study the mechanochemical properties of mutated cardiac myosin, but mouse hearts express alpha-MHC, whereas the ventricles of larger mammals express predominantly beta-MHC. To address the role of the isoform backbone on function, we generated a transgenic mouse in which the endogenous alpha-MHC was partially replaced with transgenically encoded beta-MHC or alpha-MHC. A His6 tag was cloned at the N terminus, along with R403Q, to facilitate isolation of myosin subfragment 1 (S1). Stopped flow kinetics were used to measure the equilibrium constants and rates of nucleotide binding and release for the mouse S1 isoforms bound to actin. For the wild-type isoforms, we found that the affinity of MgADP for alpha-S1 (100 mum) is approximately 4-fold weaker than for beta-S1 (25 mum). Correspondingly, the MgADP release rate for alpha-S1 (350 s(-1)) is approximately 3-fold greater than for beta-S1 (120 s(-1)). Introducing the R403Q mutation caused only a minor reduction in kinetics for beta-S1, but R403Q in alpha-S1 caused the ADP release rate to increase by 20% (430 s(-1)). These transient kinetic studies on mouse cardiac myosins provide strong evidence that the functional impact of an FHC mutation on myosin depends on the isoform backbone.

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Palmitoylation of Protease-activated Receptor-1 Regulates Adaptor Protein Complex-2 and -3 Interaction with Tyrosine-based Motifs and Endocytic Sorting.

Publication Date: 2013 May 31 PMID: 23580642
Authors: Canto, I. - Trejo, J.
Journal: J Biol Chem

Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor for the coagulant protease thrombin. Thrombin binds to and cleaves the N terminus of PAR1, generating a new N terminus that functions as a tethered ligand that cannot diffuse away. In addition to rapid desensitization, PAR1 trafficking is critical for the regulation of cellular responses. PAR1 displays constitutive and agonist-induced internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), which binds to a distal tyrosine-based motif localized within the C-terminal tail (C-tail) domain. Once internalized, PAR1 is sorted from endosomes to lysosomes via AP-3 interaction with a second C-tail tyrosine motif proximal to the transmembrane domain. However, the regulatory processes that control adaptor protein recognition of PAR1 C-tail tyrosine-based motifs are not known. Here, we report that palmitoylation of PAR1 is critical for regulating proper utilization of tyrosine-based motifs and endocytic sorting. We show that PAR1 is basally palmitoylated at highly conserved C-tail cysteines. A palmitoylation-deficient PAR1 mutant is competent to signal and exhibits a marked increase in constitutive internalization and lysosomal degradation compared with wild type receptor. Intriguingly, enhanced constitutive internalization of PAR1 is mediated by AP-2 and requires the proximal tyrosine-based motif rather than the distal tyrosine motif used by wild type receptor. Moreover, palmitoylation-deficient PAR1 displays increased degradation that is mediated by AP-3. These findings suggest that palmitoylation of PAR1 regulates appropriate utilization of tyrosine-based motifs by adaptor proteins and endocytic trafficking, processes that are critical for maintaining appropriate expression of PAR1 at the cell surface.

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Structural Insights into the Chaperone Activity of the 40-kDa Heat Shock Protein DnaJ: BINDING AND REMODELING OF A NATIVE SUBSTRATE.

Publication Date: 2013 May 24 PMID: 23580641
Authors: Cuellar, J. - Perales-Calvo, J. - Muga, A. - Valpuesta, J. M. - Moro, F.
Journal: J Biol Chem

Hsp40 chaperones bind and transfer substrate proteins to Hsp70s and regulate their ATPase activity. The interaction of Hsp40s with native proteins modifies their structure and function. A good model for this function is DnaJ, the bacterial Hsp40 that interacts with RepE, the repressor/activator of plasmid F replication, and together with DnaK regulates its function. We characterize here the structure of the DnaJ-RepE complex by electron microscopy, the first described structure of a complex between an Hsp40 and a client protein. The comparison of the complexes of DnaJ with two RepE mutants reveals an intrinsic plasticity of the DnaJ dimer that allows the chaperone to adapt to different substrates. We also show that DnaJ induces conformational changes in dimeric RepE, which increase the intermonomeric distance and remodel both RepE domains enhancing its affinity for DNA.

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Assembly of the yeast exoribonuclease rrp6 with its associated cofactor rrp47 occurs in the nucleus and is critical for the controlled expression of rrp47.

Publication Date: 2013 May 31 PMID: 23580640
Authors: Feigenbutz, M. - Jones, R. - Besong, T. M. - Harding, S. E. - Mitchell, P.
Journal: J Biol Chem

Rrp6 is a key catalytic subunit of the nuclear RNA exosome that plays a pivotal role in the processing, degradation, and quality control of a wide range of cellular RNAs. Here we report our findings on the assembly of the complex involving Rrp6 and its associated protein Rrp47, which is required for many Rrp6-mediated RNA processes. Recombinant Rrp47 is expressed as a non-globular homodimer. Analysis of the purified recombinant Rrp6.Rrp47 complex revealed a heterodimer, suggesting that Rrp47 undergoes a structural reconfiguration upon interaction with Rrp6. Studies using GFP fusion proteins show that Rrp6 and Rrp47 are localized to the yeast cell nucleus independently of one another. Consistent with this data, Rrp6, but not Rrp47, is found associated with the nuclear import adaptor protein Srp1. We show that the interaction with Rrp6 is critical for Rrp47 stability in vivo; in the absence of Rrp6, newly synthesized Rrp47 is rapidly degraded in a proteasome-dependent manner. These data resolve independent nuclear import routes for Rrp6 and Rrp47, reveal a structural reorganization of Rrp47 upon its interaction with Rrp6, and demonstrate a proteasome-dependent mechanism that efficiently suppresses the expression of Rrp47 in the absence of Rrp6.

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Factor VIIIa A2 Subunit Shows a High Affinity Interaction with Factor IXa: CONTRIBUTION OF A2 SUBUNIT RESIDUES 707-714 TO THE INTERACTION WITH FACTOR IXa.

Publication Date: 2013 May 24 PMID: 23580639
Authors: Griffiths, A. E. - Rydkin, I. - Fay, P. J.
Journal: J Biol Chem

Factor (F) VIIIa forms a number of contacts with FIXa in assembling the FXase enzyme complex. Surface plasmon resonance was used to examine the interaction between immobilized biotinylated active site-modified FIXa, and FVIII and FVIIIa subunits. The FVIIIa A2 subunit bound FIXa with high affinity (Kd = 3.9 +/- 1.6 nm) that was similar to the A3C1C2 subunit (Kd = 3.6 +/- 0.6 nm). This approach was used to evaluate a series of baculovirus-expressed, isolated A2 domain (bA2) variants where alanine substitutions were made for individual residues within the sequence 707-714, the C-terminal region of A2 thought to be FIXa interactive. Three of six bA2 variants examined displayed 2- to 4-fold decreased affinity for FIXa as compared with WT bA2. The variant bA2 proteins were also tested in two reconstitution systems to determine activity and affinity parameters in forming FXase and FVIIIa. Vmax values for all variants were similar to the WT values, indicating that these residues do not affect cofactor function. All variants showed substantially greater increases in apparent Kd relative to WT in reconstituting the FXase complex (8- to 26-fold) compared with reconstituting FVIIIa (1.3- to 6-fold) suggesting that the mutations altered interaction with FIXa. bA2 domain variants with Ala replacing Lys(707), Asp(712), and Lys(713) demonstrated the greatest increases in apparent Kd (17- to 26-fold). These results indicate a high affinity interaction between the FVIIIa A2 subunit and FIXa and show a contribution of several residues within the 707-714 sequence to this binding.

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Regulatory Coordination between Two Major Intracellular Homeostatic Systems: HEAT SHOCK RESPONSE AND AUTOPHAGY.

Publication Date: 2013 May 24 PMID: 23576438
Authors: Dokladny, K. - Zuhl, M. N. - Mandell, M. - Bhattacharya, D. - Schneider, S. - Deretic, V. - Moseley, P. L.
Journal: J Biol Chem

The eukaryotic cell depends on multitiered homeostatic systems ensuring maintenance of proteostasis, organellar integrity, function and turnover, and overall cellular viability. At the two opposite ends of the homeostatic system spectrum are heat shock response and autophagy. Here, we tested whether there are interactions between these homeostatic systems, one universally operational in all prokaryotic and eukaryotic cells, and the other one (autophagy) is limited to eukaryotes. We found that heat shock response regulates autophagy. The interaction between the two systems was demonstrated by testing the role of HSF-1, the central regulator of heat shock gene expression. Knockdown of HSF-1 increased the LC3 lipidation associated with formation of autophagosomal organelles, whereas depletion of HSF-1 potentiated both starvation- and rapamycin-induced autophagy. HSP70 expression but not expression of its ATPase mutant inhibited starvation or rapamycin-induced autophagy. We also show that exercise induces autophagy in humans. As predicted by our in vitro studies, glutamine supplementation as a conditioning stimulus prior to exercise significantly increased HSP70 protein expression and prevented the expected exercise induction of autophagy. Our data demonstrate for the first time that heat shock response, from the top of its regulatory cascade (HSF-1) down to the execution stages delivered by HSP70, controls autophagy thus connecting and coordinating the two extreme ends of the homeostatic systems in the eukaryotic cell.

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Reciprocal regulation of protein kinase and pyruvate kinase activities of pyruvate kinase m2 by growth signals.

Publication Date: 2013 May 31 PMID: 23576436
Authors: Gao, X. - Wang, H. - Yang, J. J. - Chen, J. - Jie, J. - Li, L. - Zhang, Y. - Liu, Z. R.
Journal: J Biol Chem

Pyruvate kinase isoform M2 (PKM2) is an enzyme-catalyzing conversion of phosphoenolpyruvate to pyruvate in the glycolysis pathway. It was demonstrated that PKM2 interacts with tyrosine phosphopeptide, and the interaction with the tyrosine phosphopeptide affects the pyruvate kinase activity of PKM2. Our experiments suggest that PKM2 is also an active protein kinase (Gao, X., Wang, H., Yang, J. J., Liu, X., and Liu, Z. R. (2012) Mol. Cell 45, 598-609). We report here that growth signals reciprocally regulate the pyruvate kinase and protein kinase activities of PKM2 by different mechanisms. On the one hand, growth signals induce protein tyrosine phosphorylations. The tyrosine-phosphorylated protein(s) regulates the conversion of pyruvate kinase and protein kinase of PKM2 by directly interacting with PKM2. Binding of the tyrosyl-phosphorylated proteins at the fructose 1,6-bisphosphate-binding site converts the tetrameric PKM2 to a dimer. On the other hand, growth stimulations also lead to PKM2 phosphorylation, which consequently regulates the conversion of protein kinase and pyruvate kinase activities. Growth factor stimulations significantly increase the dimer/tetramer PKM2 ratio in cells and consequently activate the protein kinase activity of PKM2. Our study suggests that the conversion between the pyruvate kinase and protein kinase activities of PKM2 may be an important mechanism mediating the effects of growth signals in promoting cell proliferation.

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Auxiliary KChIP4a Suppresses A-type K+ Current through Endoplasmic Reticulum (ER) Retention and Promoting Closed-state Inactivation of Kv4 Channels.

Publication Date: 2013 May 24 PMID: 23576435
Authors: Tang, Y. Q. - Liang, P. - Zhou, J. - Lu, Y. - Lei, L. - Bian, X. - Wang, K.
Journal: J Biol Chem

In the brain and heart, auxiliary Kv channel-interacting proteins (KChIPs) co-assemble with pore-forming Kv4 alpha-subunits to form a native K(+) channel complex and regulate the expression and gating properties of Kv4 currents. Among the KChIP1-4 members, KChIP4a exhibits a unique N terminus that is known to suppress Kv4 function, but the underlying mechanism of Kv4 inhibition remains unknown. Using a combination of confocal imaging, surface biotinylation, and electrophysiological recordings, we identified a novel endoplasmic reticulum (ER) retention motif, consisting of six hydrophobic and aliphatic residues, 12-17 (LIVIVL), within the KChIP4a N-terminal KID, that functions to reduce surface expression of Kv4-KChIP complexes. This ER retention capacity is transferable and depends on its flanking location. In addition, adjacent to the ER retention motif, the residues 19-21 (VKL motif) directly promote closed-state inactivation of Kv4.3, thus leading to an inhibition of channel current. Taken together, our findings demonstrate that KChIP4a suppresses A-type Kv4 current via ER retention and enhancement of Kv4 closed-state inactivation.

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Role of SAP97 Protein in the Regulation of Corticotropin-releasing Factor Receptor 1 Endocytosis and Extracellular Signal-regulated Kinase 1/2 Signaling.

Publication Date: 2013 May 24 PMID: 23576434
Authors: Dunn, H. A. - Walther, C. - Godin, C. M. - Hall, R. A. - Ferguson, S. S.
Journal: J Biol Chem

The corticotropin-releasing factor (CRF) receptor 1 (CRFR1) is a target for the treatment of psychiatric diseases such as depression, schizophrenia, anxiety disorder, and bipolar disorder. The carboxyl-terminal tail of the CRFR1 terminates in a PDZ-binding motif that provides a potential site for the interaction of PSD-95/Discs Large/Zona Occludens 1 (PDZ) domain-containing proteins. In this study, we found that CRFR1 interacts with synapse-associated protein 97 (SAP97; also known as DLG1) by co-immunoprecipitation in human embryonic 293 (HEK 293) cells and cortical brain lysates and that this interaction is dependent upon an intact PDZ-binding motif at the end of the CRFR1 carboxyl-terminal tail. Similarly, we demonstrated that SAP97 is recruited to the plasma membrane in HEK 293 cells expressing CRFR1 and that mutation of the CRFR1 PDZ-binding motif results in the redistribution of SAP97 into the cytoplasm. Overexpression of SAP97 antagonized agonist-stimulated CRFR1 internalization, whereas single hairpin (shRNA) knockdown of endogenous SAP97 in HEK 293 cells resulted in increased agonist-stimulated CRFR1 endocytosis. CRFR1 was internalized as a complex with SAP97 resulting in the redistribution of SAP97 to endocytic vesicles. Overexpression or shRNA knockdown of SAP97 did not significantly affect CRFR1-mediated cAMP formation, but SAP97 knockdown did significantly attenuate CRFR1-stimulated ERK1/2 phosphorylation in a PDZ interaction-independent manner. Taken together, our studies show that SAP97 interactions with CRFR1 attenuate CRFR1 endocytosis and that SAP97 is involved in coupling G protein-coupled receptors to the activation of the ERK1/2 signaling pathway.

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Structure of the Mouse Sex Peptide Pheromone ESP1 Reveals a Molecular Basis for Specific Binding to the Class C G-protein-coupled Vomeronasal Receptor.

Publication Date: 2013 May 31 PMID: 23576433
Authors: Yoshinaga, S. - Sato, T. - Hirakane, M. - Esaki, K. - Hamaguchi, T. - Haga-Yamanaka, S. - Tsunoda, M. - Kimoto, H. - Shimada, I. - Touhara, K. - Terasawa, H.
Journal: J Biol Chem

Exocrine gland-secreting peptide 1 (ESP1) is a sex pheromone that is released in male mouse tear fluids and enhances female sexual receptive behavior. ESP1 is selectively recognized by a specific class C G-protein-coupled receptor (GPCR), V2Rp5, among the hundreds of receptors expressed in vomeronasal sensory neurons (VSNs). The specific sensing mechanism of the mammalian peptide pheromone by the class C GPCR remains to be elucidated. Here we identified the minimal functional region needed to retain VSN-stimulating activity in ESP1 and determined its three-dimensional structure, which adopts a helical fold stabilized by an intramolecular disulfide bridge with extensive charged patches. We then identified the amino acids involved in the activation of VSNs by a structure-based mutational analysis, revealing that the highly charged surface is crucial for the ESP1 activity. We also demonstrated that ESP1 specifically bound to an extracellular region of V2Rp5 by an in vitro pulldown assay. Based on homology modeling of V2Rp5 using the structure of the metabotropic glutamate receptor, we constructed a docking model of the ESP1-V2Rp5 complex in which the binding interface exhibited good electrostatic complementarity. These experimental results, supported by the molecular docking simulations, reveal that charge-charge interactions determine the specificity of ESP1 binding to V2Rp5 in the large extracellular region characteristic of class C GPCRs. The present study provides insights into the structural basis for the narrowly tuned sensing of mammalian peptide pheromones by class C GPCRs.

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The Cation-pi Box Is a Specific Phosphatidylcholine Membrane Targeting Motif.

Publication Date: 2013 May 24 PMID: 23576432
Authors: Cheng, J. - Goldstein, R. - Gershenson, A. - Stec, B. - Roberts, M. F.
Journal: J Biol Chem

Peripheral membrane proteins can be targeted to specific organelles or the plasma membrane by differential recognition of phospholipid headgroups. Although molecular determinants of specificity for several headgroups, including phosphatidylserine and phosphoinositides are well defined, specific recognition of the headgroup of the zwitterionic phosphatidylcholine (PC) is less well understood. In cytosolic proteins the cation-pi box provides a suitable receptor for choline recognition and binding through the trimethylammonium moiety. In PC, this moiety might provide a sufficient handle to bind to peripheral proteins via a cation-pi cage, where the pi systems of two or more aromatic residues are within 4-5 A of the quaternary amine. We prove this hypothesis by engineering the cation-pi box into secreted phosphatidylinositol-specific phospholipase C from Staphylococcus aureus, which lacks specific PC recognition. The N254Y/H258Y variant selectively binds PC-enriched vesicles, and x-ray crystallography reveals N254Y/H258Y binds choline and dibutyroylphosphatidylcholine within the cation-pi motif. Such simple PC recognition motifs could be engineered into a wide variety of secondary structures providing a generally applicable method for specific recognition of PC.

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Photoreceptor Proteins Initiate Microglial Activation via Toll-like Receptor 4 in Retinal Degeneration Mediated by All-trans-retinal.

Publication Date: 2013 May 24 PMID: 23572532
Authors: Kohno, H. - Chen, Y. - Kevany, B. M. - Pearlman, E. - Miyagi, M. - Maeda, T. - Palczewski, K. - Maeda, A.
Journal: J Biol Chem

Although several genetic and biochemical factors are associated with the pathogenesis of retinal degeneration, it has yet to be determined how these different impairments can cause similar degenerative phenotypes. Here, we report microglial/macrophage activation in both a Stargardt disease and age-related macular degeneration mouse model caused by delayed clearance of all-trans-retinal from the retina, and in a retinitis pigmentosa mouse model with impaired retinal pigment epithelium (RPE) phagocytosis. Mouse microglia displayed RPE cytotoxicity and increased production of inflammatory chemokines/cytokines, Ccl2, Il1b, and Tnf, after coincubation with ligands that activate innate immunity. Notably, phagocytosis of photoreceptor proteins increased the activation of microglia/macrophages and RPE cells isolated from model mice as well as wild-type mice. The mRNA levels of Tlr2 and Tlr4, which can recognize proteins as their ligands, were elevated in mice with retinal degeneration. Bone marrow-derived macrophages from Tlr4-deficient mice did not increase Ccl2 after coincubation with photoreceptor proteins. Tlr4(-/-)Abca4(-/-)Rdh8(-/-) mice displayed milder retinal degenerative phenotypes than Abca4(-/-)Rdh8(-/-) mice. Additionally, inactivation of microglia/macrophages by pharmacological approaches attenuated mouse retinal degeneration. This study demonstrates an important contribution of TLR4-mediated microglial activation by endogenous photoreceptor proteins in retinal inflammation that aggravates retinal cell death. This pathway is likely to represent an underlying common pathology in degenerative retinal disorders.

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Putrescine Importer PlaP Contributes to Swarming Motility and Urothelial Cell Invasion in Proteus mirabilis.

Publication Date: 2013 May 31 PMID: 23572531
Authors: Kurihara, S. - Sakai, Y. - Suzuki, H. - Muth, A. - Phanstiel, O. 4th - Rather, P. N.
Journal: J Biol Chem

Previously, we reported that the speA gene, encoding arginine decarboxylase, is required for swarming in the urinary tract pathogen Proteus mirabilis. In addition, this previous study suggested that putrescine may act as a cell-to-cell signaling molecule (Sturgill, G., and Rather, P. N. (2004) Mol. Microbiol. 51, 437-446). In this new study, PlaP, a putative putrescine importer, was characterized in P. mirabilis. In a wild-type background, a plaP null mutation resulted in a modest swarming defect and slightly decreased levels of intracellular putrescine. In a P. mirabilis speA mutant with greatly reduced levels of intracellular putrescine, plaP was required for the putrescine-dependent rescue of swarming motility. When a speA/plaP double mutant was grown in the presence of extracellular putrescine, the intracellular levels of putrescine were greatly reduced compared with the speA mutant alone, indicating that PlaP functioned as the primary putrescine importer. In urothelial cell invasion assays, a speA mutant exhibited a 50% reduction in invasion when compared with wild type, and this defect could be restored by putrescine in a PlaP-dependent manner. The putrescine analog Triamide-44 partially inhibited the uptake of putrescine by PlaP and decreased both putrescine stimulated swarming and urothelial cell invasion in a speA mutant.

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The Structure of CcmP, a Tandem Bacterial Microcompartment Domain Protein from the beta-Carboxysome, Forms a Subcompartment Within a Microcompartment.

Publication Date: 2013 May 31 PMID: 23572529
Authors: Cai, F. - Sutter, M. - Cameron, J. C. - Stanley, D. N. - Kinney, J. N. - Kerfeld, C. A.
Journal: J Biol Chem

The carboxysome is a bacterial organelle found in all cyanobacteria; it encapsulates CO2 fixation enzymes within a protein shell. The most abundant carboxysome shell protein contains a single bacterial microcompartment (BMC) domain. We present in vivo evidence that a hypothetical protein (dubbed CcmP) encoded in all beta-cyanobacterial genomes is part of the carboxysome. We show that CcmP is a tandem BMC domain protein, the first to be structurally characterized from a beta-carboxysome. CcmP forms a dimer of tightly stacked trimers, resulting in a nanocompartment-containing shell protein that may weakly bind 3-phosphoglycerate, the product of CO2 fixation. The trimers have a large central pore through which metabolites presumably pass into the carboxysome. Conserved residues surrounding the pore have alternate side-chain conformations suggesting that it can be open or closed. Furthermore, CcmP and its orthologs in alpha-cyanobacterial genomes form a distinct clade of shell proteins. Members of this subgroup are also found in numerous heterotrophic BMC-associated gene clusters encoding functionally diverse bacterial organelles, suggesting that the potential to form a nanocompartment within a microcompartment shell is widespread. Given that carboxysomes and architecturally related bacterial organelles are the subject of intense interest for applications in synthetic biology/metabolic engineering, our results describe a new type of building block with which to functionalize BMC shells.

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A Role for Cargo in Arf-dependent Adaptor Recruitment.

Publication Date: 2013 May 24 PMID: 23572528
Authors: Caster, A. H. - Sztul, E. - Kahn, R. A.
Journal: J Biol Chem

Membrane traffic requires the specific concentration of protein cargos and exclusion of other proteins into nascent carriers. Critical components of this selectivity are the protein adaptors that bind to short, linear motifs in the cytoplasmic tails of transmembrane protein cargos and sequester them into nascent carriers. The recruitment of the adaptors is mediated by activated Arf GTPases, and the Arf-adaptor complexes mark sites of carrier formation. However, the nature of the signal(s) that initiates carrier biogenesis remains unknown. We examined the specificity and initial sites of recruitment of Arf-dependent adaptors (AP-1 and GGAs) in response to the Golgi or endosomal localization of specific cargo proteins (furin, mannose-6-phosphate receptor (M6PR), and M6PR lacking a C-terminal domain M6PRDeltaC). We find that cargo promotes the recruitment of specific adaptors, suggesting that it is part of an upstream signaling event. Cargos do not promote adaptor recruitment to all compartments in which they reside, and thus additional factors regulate the cargo's ability to promote Arf activation and adaptor recruitment. We document that within a given compartment different cargos recruit different adaptors, suggesting that there is little or no free, activated Arf at the membrane and that Arf activation is spatially and temporally coupled to the cargo and the adaptor. Using temperature block, brefeldin A, and recovery from each, we found that the cytoplasmic tail of M6PR causes the recruitment of AP-1 and GGAs to recycling endosomes and not at the Golgi, as predicted by steady state staining profiles. These results are discussed with respect to the generation of novel models for cargo-dependent regulation of membrane traffic.

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Structure and Function of the DUF2233 Domain in Bacteria and in the Human Mannose 6-Phosphate Uncovering Enzyme.

Publication Date: 2013 Jun 7 PMID: 23572527
Authors: Das, D. - Lee, W. S. - Grant, J. C. - Chiu, H. J. - Farr, C. L. - Vance, J. - Klock, H. E. - Knuth, M. W. - Miller, M. D. - Elsliger, M. A. - Deacon, A. M. - Godzik, A. - Lesley, S. A. - Kornfeld, S. - Wilson, I. A.
Journal: J Biol Chem

DUF2233, a domain of unknown function (DUF), is present in many bacterial and several viral proteins and was also identified in the mammalian transmembrane glycoprotein N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase ("uncovering enzyme" (UCE)). We report the crystal structure of BACOVA_00430, a 315-residue protein from the human gut bacterium Bacteroides ovatus that is the first structural representative of the DUF2233 protein family. A notable feature of this structure is the presence of a surface cavity that is populated by residues that are highly conserved across the entire family. The crystal structure was used to model the luminal portion of human UCE (hUCE), which is involved in targeting of lysosomal enzymes. Mutational analysis of several residues in a highly conserved surface cavity of hUCE revealed that they are essential for function. The bacterial enzyme (BACOVA_00430) has approximately 1% of the catalytic activity of hUCE toward the substrate GlcNAc-P-mannose, the precursor of the Man-6-P lysosomal targeting signal. GlcNAc-1-P is a poor substrate for both enzymes. We conclude that, for at least a subset of proteins in this family, DUF2233 functions as a phosphodiester glycosidase.

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The interaction of polyglutamine peptides with lipid membranes is regulated by flanking sequences associated with huntingtin.

Publication Date: 2013 May 24 PMID: 23572526
Authors: Burke, K. A. - Kauffman, K. J. - Umbaugh, C. S. - Frey, S. L. - Legleiter, J.
Journal: J Biol Chem

Huntington disease (HD) is caused by an expanded polyglutamine (poly(Q)) repeat near the N terminus of the huntingtin (htt) protein. Expanded poly(Q) facilitates formation of htt aggregates, eventually leading to deposition of cytoplasmic and intranuclear inclusion bodies containing htt. Flanking sequences directly adjacent to the poly(Q) domain, such as the first 17 amino acids on the N terminus (Nt17) and the polyproline (poly(P)) domain on the C-terminal side of the poly(Q) domain, heavily influence aggregation. Additionally, htt interacts with a variety of membraneous structures within the cell, and Nt17 is implicated in lipid binding. To investigate the interaction between htt exon1 and lipid membranes, a combination of in situ atomic force microscopy, Langmuir trough techniques, and vesicle permeability assays were used to directly monitor the interaction of a variety of synthetic poly(Q) peptides with different combinations of flanking sequences (KK-Q35-KK, KK-Q35-P10-KK, Nt17-Q35-KK, and Nt17-Q35-P10-KK) on model membranes and surfaces. Each peptide aggregated on mica, predominately forming extended, fibrillar aggregates. In contrast, poly(Q) peptides that lacked the Nt17 domain did not appreciably aggregate on or insert into lipid membranes. Nt17 facilitated the interaction of peptides with lipid surfaces, whereas the poly(P) region enhanced this interaction. The aggregation of Nt17-Q35-P10-KK on the lipid bilayer closely resembled that of a htt exon1 construct containing 35 repeat glutamines. Collectively, this data suggests that the Nt17 domain plays a critical role in htt binding and aggregation on lipid membranes, and this lipid/htt interaction can be further modulated by the presence of the poly(P) domain.

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Integrin-binding Protein Nischarin Interacts with Tumor Suppressor Liver Kinase B1 (LKB1) to Regulate Cell Migration of Breast Epithelial Cells.

Publication Date: 2013 May 31 PMID: 23572524
Authors: Jain, P. - Baranwal, S. - Dong, S. - Struckhoff, A. P. - Worthylake, R. A. - Alahari, S. K.
Journal: J Biol Chem

Biallelic inactivation of LKB1, a serine/threonine kinase, has been detected in 30% of lung adenocarcinomas, and inhibition of breast tumor growth has been demonstrated. We have identified the tumor suppressor, Nischarin, as a novel binding partner of LKB1. Our mapping analysis shows that the N terminus of Nischarin interacts with amino acids 44-436 of LKB1. Time lapse microscopy and Transwell migration data show that the absence of both Nischarin and LKB1 from an invasive breast cancer cell line (MDA-MB-231) enhances migration as measured by increased distance and speed of migrating cells. Our data suggest that this is a result of elevated PAK1 and LIMK1 phosphorylation. Moreover, the absence of Nischarin and LKB1 increased tumor growth in vivo. Consistent with this, the percentage of S phase cells was increased, as demonstrated by flow cytometry and enhanced cyclin D1. The absence of Nischarin and LKB1 also led to a dramatic increase in the formation of lung metastases. Our studies, for the first time, demonstrate functional interaction between LKB1 and Nischarin to inhibit cell migration and breast tumor progression. Mechanistically, we show that these two proteins together regulate PAK-LIMK-Cofilin and cyclin D1/CDK4 pathways.

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Proteolytic Processing of the Caspase-9 Zymogen Is Required for Apoptosome-mediated Activation of Caspase-9.

Publication Date: 2013 May 24 PMID: 23572523
Authors: Hu, Q. - Wu, D. - Chen, W. - Yan, Z. - Shi, Y.
Journal: J Biol Chem

Maturation of the single-chain caspase-9 zymogen through autoproteolytic processing is mediated by the Apaf-1 apoptosome at the onset of apoptosis. Processed caspase-9 and the apoptosome form a holoenzyme with robust proteolytic activity that is 2-3 orders of magnitude higher than that of free processed caspase-9. An unresolved important question is the role of caspase-9 processing, with some experimental data suggesting its dispensability. In this study, we demonstrate that, in contrast to wild-type caspase-9, the unprocessed single-chain caspase-9 triple mutant E306A/D315A/D330A (Casp9-TM) could no longer be adequately activated by the apoptosome. Compared with the protease activity of wild-type caspase-9, that of Casp9-TM in the presence of the apoptosome was drastically reduced. The crippled protease activity of Casp9-TM in the presence of the apoptosome is likely attributable to a markedly reduced ability of Casp9-TM to form homodimers. These data identify an essential role for the autoproteolytic processing of caspase-9 in its activation.

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The Fish Pathogen Yersinia ruckeri Produces Holomycin and Uses an RNA Methyltransferase for Self-resistance.

Publication Date: 2013 May 24 PMID: 23572522
Authors: Qin, Z. - Baker, A. T. - Raab, A. - Huang, S. - Wang, T. - Yu, Y. - Jaspars, M. - Secombes, C. J. - Deng, H.
Journal: J Biol Chem

Holomycin and its derivatives belong to a class of broad-spectrum antibacterial natural products containing a rare dithiolopyrrolone heterobicyclic scaffold. The antibacterial mechanism of dithiolopyrrolone compounds has been attributed to the inhibition of bacterial RNA polymerase activities, although the exact mode of action has not been established in vitro. Some dithiopyrrolone derivatives display potent anticancer activities. Recently the biosynthetic gene cluster of holomycin has been identified and characterized in Streptomyces clavuligerus. Here we report that the fish pathogen Yersinia ruckeri is a holomycin producer, as evidenced through genome mining, chemical isolation, and structural elucidation as well as genetic manipulation. We also identified a unique regulatory gene hom15 at one end of the gene cluster encoding a cold-shock-like protein that likely regulates the production of holomycin in low cultivation temperatures. Inactivation of hom15 resulted in a significant loss of holomycin production. Finally, gene disruption of an RNA methyltransferase gene hom12 resulted in the sensitivity of the mutant toward holomycin. A complementation experiment of hom12 restored the resistance against holomycin. Although the wild-type Escherichia coli BL21(DE3) Gold is susceptible to holomycin, the mutant harboring hom12 showed tolerance toward holomycin. High resolution liquid chromatography (LC)-ESI/MS analysis of digested RNA fragments demonstrated that the wild-type Y. ruckeri and E. coli harboring hom12 contain a methylated RNA fragment, whereas the mutated Y. ruckeri and the wild-type E. coli only contain normal non-methylated RNA fragments. Taken together, our results strongly suggest that this putative RNA methyltransferase Hom12 is the self-resistance protein that methylates the RNA of Y. ruckeri to reduce the cytotoxic effect of holomycin during holomycin production.

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Structure-Function Analysis of a Broad Specificity Populus trichocarpa Endo-beta-glucanase Reveals an Evolutionary Link between Bacterial Licheninases and Plant XTH Gene Products.

Publication Date: 2013 May 31 PMID: 23572521
Authors: Eklof, J. M. - Shojania, S. - Okon, M. - McIntosh, L. P. - Brumer, H.
Journal: J Biol Chem

The large xyloglucan endotransglycosylase/hydrolase (XTH) gene family continues to be the focus of much attention in studies of plant cell wall morphogenesis due to the unique catalytic functions of the enzymes it encodes. The XTH gene products compose a subfamily of glycoside hydrolase family 16 (GH16), which also comprises a broad range of microbial endoglucanases and endogalactanases, as well as yeast cell wall chitin/beta-glucan transglycosylases. Previous whole-family phylogenetic analyses have suggested that the closest relatives to the XTH gene products are the bacterial licheninases (EC 3.2.1.73), which specifically hydrolyze linear mixed linkage beta(1-->3)/beta(1-->4)-glucans. In addition to their specificity for the highly branched xyloglucan polysaccharide, XTH gene products are distinguished from the licheninases and other GH16 enzyme subfamilies by significant active site loop alterations and a large C-terminal extension. Given these differences, the molecular evolution of the XTH gene products in GH16 has remained enigmatic. Here, we present the biochemical and structural analysis of a unique, mixed function endoglucanase from black cottonwood (Populus trichocarpa), which reveals a small, newly recognized subfamily of GH16 members intermediate between the bacterial licheninases and plant XTH gene products. We postulate that this clade comprises an important link in the evolution of the large plant XTH gene families from a putative microbial ancestor. As such, this analysis provides new insights into the diversification of GH16 and further unites the apparently disparate members of this important family of proteins.

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S-nitrosation of glutathione transferase p1-1 is controlled by the conformation of a dynamic active site helix.

Publication Date: 2013 May 24 PMID: 23572520
Authors: Balchin, D. - Wallace, L. - Dirr, H. W.
Journal: J Biol Chem

S-Nitrosation is a post-translational modification of protein cysteine residues, which occurs in response to cellular oxidative stress. Although it is increasingly being linked to physiologically important processes, the molecular basis for protein regulation by this modification remains poorly understood. We used transient kinetic methods to determine a minimal mechanism for spontaneous S-nitrosoglutathione (GSNO)-mediated transnitrosation of human glutathione transferase (GST) P1-1, a major detoxification enzyme and key regulator of cell proliferation. Cys(47) of GSTP1-1 is S-nitrosated in two steps, with the chemical step limited by a pre-equilibrium between the open and closed conformations of helix alpha2 at the active site. Cys(101), in contrast, is S-nitrosated in a single step but is subject to negative cooperativity due to steric hindrance at the dimer interface. Despite the presence of a GSNO binding site at the active site of GSTP1-1, isothermal titration calorimetry as well as nitrosation experiments using S-nitrosocysteine demonstrate that GSNO binding does not precede S-nitrosation of GSTP1-1. Kinetics experiments using the cellular reductant glutathione show that Cys(101)-NO is substantially more resistant to denitrosation than Cys(47)-NO, suggesting a potential role for Cys(101) in long term nitric oxide storage or transfer. These results constitute the first report of the molecular mechanism of spontaneous protein transnitrosation, providing insight into the post-translational control of GSTP1-1 as well as the process of protein transnitrosation in general.

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Sterile Inflammation in Acetaminophen-induced Liver Injury Is Mediated by Cot/tpl2.

Publication Date: 2013 May 24 PMID: 23572518
Authors: Sanz-Garcia, C. - Ferrer-Mayorga, G. - Gonzalez-Rodriguez, A. - Valverde, A. M. - Martin-Duce, A. - Velasco-Martin, J. P. - Regadera, J. - Fernandez, M. - Alemany, S.
Journal: J Biol Chem

Cot/tpl2 (MAP3K8) activates MKK1/2-Erk1/2 following stimulation of the Toll-like/IL-1 receptor superfamily. Here, we investigated the role of Cot/tpl2 in sterile inflammation and drug-induced liver toxicity. Cot/tpl2 KO mice exhibited reduced hepatic injury after acetaminophen challenge, as evidenced by decreased serum levels of both alanine and aspartate aminotransferases, decreased hepatic necrosis, and increased survival relative to Wt mice. Serum levels of both alanine and aspartate aminotransferases were also lower after intraperitoneal injection of acetaminophen in mice expressing an inactive form of Cot/tpl2 compared with Wt mice, suggesting that Cot/tpl2 activity contributes to acetaminophen-induced liver injury. Furthermore, Cot/tpl2 deficiency reduced neutrophil and macrophage infiltration in the liver of mice treated with acetaminophen, as well as their hepatic and systemic levels of IL-1alpha. Intraperitoneal injection of damage-associated molecular patterns from necrotic hepatocytes also impaired the recruitment of leukocytes and decreased the levels of several cytokines in the peritoneal cavity in Cot/tpl2 KO mice compared with Wt counterparts. Moreover, similar activation profiles of intracellular pathways were observed in Wt macrophages stimulated with Wt or Cot/tpl2 KO damage-associated molecular patterns. However, upon stimulation with damage-associated molecular patterns, the activation of Erk1/2 and JNK was deficient in Cot/tpl2 KO macrophages compared with their Wt counterparts; an effect accompanied by weaker release of several cytokines, including IL-1alpha, an important component in the development of sterile inflammation. Taken together, these findings indicate that Cot/tpl2 contributes to acetaminophen-induced liver injury, providing some insight into the underlying molecular mechanisms.

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Metabolite Profiling to Characterize Disease-related Bacteria: GLUCONATE EXCRETION BY PSEUDOMONAS AERUGINOSA MUTANTS AND CLINICAL ISOLATES FROM CYSTIC FIBROSIS PATIENTS.

Publication Date: 2013 May 24 PMID: 23572517
Authors: Behrends, V. - Bell, T. J. - Liebeke, M. - Cordes-Blauert, A. - Ashraf, S. N. - Nair, C. - Zlosnik, J. E. - Williams, H. D. - Bundy, J. G.
Journal: J Biol Chem

Metabolic footprinting of supernatants has been proposed as a tool for assigning gene function. We used NMR spectroscopy to measure the exometabolome of 86 single-gene transposon insertion mutant strains (mutants from central carbon metabolism and regulatory mutants) of the opportunistic pathogen Pseudomonas aeruginosa, grown on a medium designed to represent the nutritional content of cystic fibrosis sputum. Functionally related genes had similar metabolic profiles. E.g. for two-component system mutants, the cognate response regulator and sensor kinase genes clustered tightly together. Some strains had metabolic phenotypes (metabotypes) that could be related to the known gene function. E.g. pyruvate dehydrogenase mutants accumulated large amounts of pyruvate in the medium. In other cases, the metabolic phenotypes were not easily interpretable. The rpoN mutant, which lacks the alternative sigma factor RpoN (sigma(54)), accumulated high levels of gluconate in the medium. In addition, endometabolome profiling of intracellular metabolites identified a number of systemic metabolic changes. We linked this to indirect regulation of the catabolite repression protein Crc via the non-coding RNA crcZ and found that a crcZ (but not crc) mutant also shared the high-gluconate phenotype. We profiled an additional set of relevant metabolic enzymes and transporters, including Crc targets, and showed that the Crc-regulated edd mutant (gluconate-6-phosphate dehydratase) had similar gluconate levels as the rpoN mutant. Finally, a set of clinical isolates showed patient- and random amplification of polymorphic DNA (RAPD) type-specific differences in gluconate production, which were associated significantly with resistance across four antibiotics (tobramycin, ciprofloxacin, aztreonam, and imipenem), indicating that this has potential clinical relevance.

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Highly Divergent T-cell Receptor Binding Modes Underlie Specific Recognition of a Bulged Viral Peptide bound to a Human Leukocyte Antigen Class I Molecule.

Publication Date: 2013 May 31 PMID: 23569211
Authors: Liu, Y. C. - Miles, J. J. - Neller, M. A. - Gostick, E. - Price, D. A. - Purcell, A. W. - McCluskey, J. - Burrows, S. R. - Rossjohn, J. - Gras, S.
Journal: J Biol Chem

Human leukocyte antigen (HLA)-I molecules can present long peptides, yet the mechanisms by which T-cell receptors (TCRs) recognize featured pHLA-I landscapes are unclear. We compared the binding modes of three distinct human TCRs, CA5, SB27, and SB47, complexed with a "super-bulged" viral peptide (LPEPLPQGQLTAY) restricted by HLA-B*35:08. The CA5 and SB27 TCRs engaged HLA-B*35:08(LPEP) similarly, straddling the central region of the peptide but making limited contacts with HLA-B*35:08. Remarkably, the CA5 TCR did not contact the alpha1-helix of HLA-B*35:08. Differences in the CDR3beta loop between the CA5 and SB27 TCRs caused altered fine specificities. Surprisingly, the SB47 TCR engaged HLA-B*35:08(LPEP) using a completely distinct binding mechanism, namely "bypassing" the bulged peptide and making extensive contacts with the extreme N-terminal end of HLA-B*35:08. This docking footprint included HLA-I residues not observed previously as TCR contact sites. The three TCRs exhibited differing patterns of alloreactivity toward closely related or distinct HLA-I allotypes. Thus, the human T-cell repertoire comprises a range of TCRs that can interact with "bulged" pHLA-I epitopes using unpredictable strategies, including the adoption of atypical footprints on the MHC-I.

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A single divergent exon inhibits ankyrin-B association with the plasma membrane.

Publication Date: 2013 May 24 PMID: 23569209
Authors: He, M. - Tseng, W. C. - Bennett, V.
Journal: J Biol Chem

Vertebrate ankyrin-B and ankyrin-G exhibit divergent subcellular localization and function despite their high sequence and structural similarity and common origin from a single ancestral gene at the onset of chordate evolution. Previous studies of ankyrin family diversity have focused on the C-terminal regulatory domain. Here, we identify an ankyrin-B-specific linker peptide connecting the ankyrin repeat domain to the ZU52-UPA module that inhibits binding of ankyrin-B to membrane protein partners E-cadherin and neurofascin 186 and prevents association of ankyrin-B with epithelial lateral membranes as well as neuronal plasma membranes. The residues of the ankyrin-B linker required for autoinhibition are encoded by a small exon that is highly divergent between ankyrin family members but conserved in the ankyrin-B lineage. We show that the ankyrin-B linker suppresses activity of the ANK repeat domain through an intramolecular interaction, likely with a groove on the surface of the ANK repeat solenoid, thereby regulating the affinities between ankyrin-B and its binding partners. These results provide a simple evolutionary explanation for how ankyrin-B and ankyrin-G have acquired striking differences in their plasma membrane association while maintaining overall high levels of sequence similarity.

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Cdc37 (Cell Division Cycle 37) Restricts Hsp90 (Heat Shock Protein 90) Motility by Interaction with N-terminal and Middle Domain Binding Sites.

Publication Date: 2013 May 31 PMID: 23569206
Authors: Eckl, J. M. - Rutz, D. A. - Haslbeck, V. - Zierer, B. K. - Reinstein, J. - Richter, K.
Journal: J Biol Chem

The ATPase-driven dimeric molecular Hsp90 (heat shock protein 90) and its cofactor Cdc37 (cell division cycle 37 protein) are crucial to prevent the cellular depletion of many protein kinases. In complex with Hsp90, Cdc37 is thought to bind an important lid structure in the ATPase domain of Hsp90 and inhibit ATP turnover by Hsp90. As different interaction modes have been reported, we were interested in the interaction mechanism of Hsp90 and Cdc37. We find that Cdc37 can bind to one subunit of the Hsp90 dimer. The inhibition of the ATPase activity is caused by a reduction in the closing rate of Hsp90 without obviously bridging the two subunits or affecting nucleotide accessibility to the binding site. Although human Cdc37 binds to the N-terminal domain of Hsp90, nematodal Cdc37 preferentially interacts with the middle domain of CeHsp90 and hHsp90, exposing two Cdc37 interaction sites. A previously unreported site in CeCdc37 is utilized for the middle domain interaction. Dephosphorylation of CeCdc37 by the Hsp90-associated phosphatase PPH-5, a step required during the kinase activation process, proceeds normally, even if only the new interaction site is used. This shows that the second interaction site is also functionally relevant and highlights that Cdc37, similar to the Hsp90 cofactors Sti1 and Aha1, may utilize two different attachment sites to restrict the conformational freedom and the ATP turnover of Hsp90.

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Structural and Functional Characterization of a Phosphatase Domain within Yeast General Transcription Factor IIIC.

Publication Date: 2013 May 24 PMID: 23569204
Authors: Taylor, N. M. - Glatt, S. - Hennrich, M. L. - von Scheven, G. - Grotsch, H. - Fernandez-Tornero, C. - Rybin, V. - Gavin, A. C. - Kolb, P. - Muller, C. W.
Journal: J Biol Chem

Saccharomyces cerevisiae tau55, a subunit of the RNA polymerase III-specific general transcription factor TFIIIC, comprises an N-terminal histidine phosphatase domain (tau55-HPD) whose catalytic activity and cellular function is poorly understood. We solved the crystal structures of tau55-HPD and its closely related paralogue Huf and used in silico docking methods to identify phosphoserine- and phosphotyrosine-containing peptides as possible substrates that were subsequently validated using in vitro phosphatase assays. A comparative phosphoproteomic study identified additional phosphopeptides as possible targets that show the involvement of these two phosphatases in the regulation of a variety of cellular functions. Our results identify tau55-HPD and Huf as bona fide protein phosphatases, characterize their substrate specificities, and provide a small set of regulated phosphosite targets in vivo.

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Phosphorylation of Serine 779 in Fibroblast Growth Factor Receptor 1 and 2 by Protein Kinase C{epsilon} Regulates Ras/Mitogen-activated Protein Kinase Signaling and Neuronal Differentiation.

Publication Date: 2013 May 24 PMID: 23564461
Authors: Lonic, A. - Powell, J. A. - Kong, Y. - Thomas, D. - Holien, J. K. - Truong, N. - Parker, M. W. - Guthridge, M. A.
Journal: J Biol Chem

The FGF receptors (FGFRs) control a multitude of cellular processes both during development and in the adult through the initiation of signaling cascades that regulate proliferation, survival, and differentiation. Although FGFR tyrosine phosphorylation and the recruitment of Src homology 2 domain proteins have been widely described, we have previously shown that FGFR is also phosphorylated on Ser(779) in response to ligand and binds the 14-3-3 family of phosphoserine/threonine-binding adaptor/scaffold proteins. However, whether this receptor phosphoserine mode of signaling is able to regulate specific signaling pathways and biological responses is unclear. Using PC12 pheochromocytoma cells and primary mouse bone marrow stromal cells as models for growth factor-regulated neuronal differentiation, we show that Ser(779) in the cytoplasmic domains of FGFR1 and FGFR2 is required for the sustained activation of Ras and ERK but not for other FGFR phosphotyrosine pathways. The regulation of Ras and ERK signaling by Ser(779) was critical not only for neuronal differentiation but also for cell survival under limiting growth factor concentrations. PKCepsilon can phosphorylate Ser(779) in vitro, whereas overexpression of PKCepsilon results in constitutive Ser(779) phosphorylation and enhanced PC12 cell differentiation. Furthermore, siRNA knockdown of PKCepsilon reduces both growth factor-induced Ser(779) phosphorylation and neuronal differentiation. Our findings show that in addition to FGFR tyrosine phosphorylation, the phosphorylation of a conserved serine residue, Ser(779), can quantitatively control Ras/MAPK signaling to promote specific cellular responses.

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Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-mediated Calcium Signaling and Arrhythmias in the Heart Evoked by beta-Adrenergic Stimulation.

Publication Date: 2013 May 31 PMID: 23564460
Authors: Nebel, M. - Schwoerer, A. P. - Warszta, D. - Siebrands, C. C. - Limbrock, A. C. - Swarbrick, J. M. - Fliegert, R. - Weber, K. - Bruhn, S. - Hohenegger, M. - Geisler, A. - Herich, L. - Schlegel, S. - Carrier, L. - Eschenhagen, T. - Potter, B. V. - Ehmke, H. - Guse, A. H.
Journal: J Biol Chem

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca(2+)-releasing second messenger known to date. Here, we report a new role for NAADP in arrhythmogenic Ca(2+) release in cardiac myocytes evoked by beta-adrenergic stimulation. Infusion of NAADP into intact cardiac myocytes induced global Ca(2+) signals sensitive to inhibitors of both acidic Ca(2+) stores and ryanodine receptors and to NAADP antagonist BZ194. Furthermore, in electrically paced cardiac myocytes BZ194 blocked spontaneous diastolic Ca(2+) transients caused by high concentrations of the beta-adrenergic agonist isoproterenol. Ca(2+) transients were recorded both as increases of the free cytosolic Ca(2+) concentration and as decreases of the sarcoplasmic luminal Ca(2+) concentration. Importantly, NAADP antagonist BZ194 largely ameliorated isoproterenol-induced arrhythmias in awake mice. We provide strong evidence that NAADP-mediated modulation of couplon activity plays a role for triggering spontaneous diastolic Ca(2+) transients in isolated cardiac myocytes and arrhythmias in the intact animal. Thus, NAADP signaling appears an attractive novel target for antiarrhythmic therapy.

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Energetics and location of phosphoinositide binding in human kir2.1 channels.

Publication Date: 2013 Jun 7 PMID: 23564459
Authors: D'Avanzo, N. - Lee, S. J. - Cheng, W. W. - Nichols, C. G.
Journal: J Biol Chem

Kir2.1 channels are uniquely activated by phosphoinositide 4,5-bisphosphate (PI(4,5)P2) and can be inhibited by other phosphoinositides (PIPs). Using biochemical and computational approaches, we assess PIP-channel interactions and distinguish residues that are energetically critical for binding from those that alter PIP sensitivity by shifting the open-closed equilibrium. Intriguingly, binding of each PIP is disrupted by a different subset of mutations. In silico ligand docking indicates that PIPs bind to two sites. The second minor site may correspond to the secondary anionic phospholipid site required for channel activation. However, 96-99% of PIP binding localizes to the first cluster, which corresponds to the general PI(4,5)P2 binding location in recent Kir crystal structures. PIPs can encompass multiple orientations; each di- and triphosphorylated species binds with comparable energies and is favored over monophosphorylated PIPs. The data suggest that selective activation by PI(4,5)P2 involves orientational specificity and that other PIPs inhibit this activation through direct competition.

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Amot130 Adapts Atrophin-1 Interacting Protein 4 to Inhibit Yes-associated Protein Signaling and Cell Growth.

Publication Date: 2013 May 24 PMID: 23564455
Authors: Adler, J. J. - Heller, B. L. - Bringman, L. R. - Ranahan, W. P. - Cocklin, R. R. - Goebl, M. G. - Oh, M. - Lim, H. S. - Ingham, R. J. - Wells, C. D.
Journal: J Biol Chem

The adaptor protein Amot130 scaffolds components of the Hippo pathway to promote the inhibition of cell growth. This study describes how Amot130 through binding and activating the ubiquitin ligase AIP4/Itch achieves these effects. AIP4 is found to bind and ubiquitinate Amot130 at residue Lys-481. This both stabilizes Amot130 and promotes its residence at the plasma membrane. Furthermore, Amot130 is shown to scaffold a complex containing overexpressed AIP4 and the transcriptional co-activator Yes-associated protein (YAP). Consequently, Amot130 promotes the ubiquitination of YAP by AIP4 and prevents AIP4 from binding to large tumor suppressor 1. Amot130 is found to reduce YAP stability. Importantly, Amot130 inhibition of YAP dependent transcription is reversed by AIP4 silencing, whereas Amot130 and AIP4 expression interdependently suppress cell growth. Thus, Amot130 repurposes AIP4 from its previously described role in degrading large tumor suppressor 1 to the inhibition of YAP and cell growth.

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Minimal interleukin 6 (IL-6) receptor stalk composition for IL-6 receptor shedding and IL-6 classic signaling.

Publication Date: 2013 May 24 PMID: 23564454
Authors: Baran, P. - Nitz, R. - Grotzinger, J. - Scheller, J. - Garbers, C.
Journal: J Biol Chem

Signaling of the pleiotropic cytokine Interleukin-6 (IL-6) is coordinated by membrane-bound and soluble forms of the IL-6 receptor (IL-6R) in processes called classic and trans-signaling, respectively. The soluble IL-6R is mainly generated by ADAM10- and ADAM17-mediated ectodomain shedding. Little is known about the role of the 52-amino acid-residue-long IL-6R stalk region in shedding and signal transduction. Therefore, we generated and analyzed IL-6R stalk region deletion variants for cleavability and biological activity. Deletion of 10 amino acids of the stalk region surrounding the ADAM17 cleavage site substantially blocked IL-6R proteolysis by ADAM17 but only slightly affected proteolysis by ADAM10. Interestingly, additional deletion of the remaining five juxtamembrane-located amino acids also abrogated ADAM10-mediated IL-6R shedding. Larger deletions within the stalk region, that do not necessarily include the ADAM17 cleavage site, also reduced ADAM10 and ADAM17-mediated IL-6R shedding, questioning the importance of cleavage site recognition. Furthermore, we show that a 22-amino acid-long stalk region is minimally required for IL-6 classic signaling. The gp130 cytokine binding sites are separated from the plasma membrane by approximately 96 A. 22 amino acid residues, however, span maximally 83.6 A (3.8 A/amino acid), indicating that the three juxtamembrane fibronectin domains of gp130 are not necessarily elongated but somehow flexed to allow IL-6 classic signaling. Our findings underline a dual role of the IL-6R stalk region in IL-6 signaling. In IL-6 trans-signaling, it regulates proper proteolysis by ADAM10 and ADAM17. In IL-6 classic-signaling, it acts as a spacer to ensure IL-6.IL-6R.gp130 signal complex formation.

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Pituitary Adenylate Cyclase-activating Polypeptide (PACAP) Promotes Both Survival and Neuritogenesis in PC12 Cells through Activation of Nuclear Factor kappaB (NF-kappaB) Pathway: INVOLVEMENT OF EXTRACELLULAR SIGNAL-REGULATED KINASE (ERK), CALCIUM, AND c-REL.

Publication Date: 2013 May 24 PMID: 23564451
Authors: Manecka, D. L. - Mahmood, S. F. - Grumolato, L. - Lihrmann, I. - Anouar, Y.
Journal: J Biol Chem

The pituitary adenylate cyclase-activating polypeptide (PACAP) is a trophic factor that promotes neuronal survival and neurite outgrowth. However, the signaling pathways and the transcriptional mechanisms involved are not completely elucidated. Our previous studies aimed at characterizing the transcriptome of PACAP-differentiated PC12 cells revealed an increase in the expression of nuclear factor kappaB2 (NF-kappaB2) gene coding for p100/p52 subunit of NF-kappaB transcription factor. Here, we examined the role of the NF-kappaB pathway in neuronal differentiation promoted by PACAP. We first showed that PACAP-driven survival and neuritic extension in PC12 cells are inhibited following NF-kappaB pathway blockade. PACAP stimulated both c-Rel and p52 NF-kappaB subunit gene expression and nuclear translocation, whereas c-Rel down-regulation inhibited cell survival and neuritogenesis elicited by the neuropeptide. PACAP-induced c-Rel nuclear translocation was inhibited by ERK1/2 and Ca(2+) blockers. Furthermore, the neuropeptide stimulated NF-kappaB p100 subunit processing into p52, indicative of activation of the NF-kappaB alternative pathway. Taken together, our data show that PACAP promotes both survival and neuritogenesis in PC12 cells by activating NF-kappaB pathway, most likely via classical and alternative signaling cascades involving ERK1/2 kinases, Ca(2+), and c-Rel/p52 dimers.

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Heterogeneous Nuclear Ribonucleoprotein K (hnRNP-K) Promotes Tumor Metastasis by Induction of Genes Involved in Extracellular Matrix, Cell Movement, and Angiogenesis.

Publication Date: 2013 May 24 PMID: 23564449
Authors: Gao, R. - Yu, Y. - Inoue, A. - Widodo, N. - Kaul, S. C. - Wadhwa, R.
Journal: J Biol Chem

Cancer is a leading cause of death and still awaits effective therapies. Rapid industrialization has contributed to increase in incidence of cancer. One of the reasons why most of the cancers fail therapy is due to their metastatic property. Hence identification of factors leading to metastasis is highly important to design effective and novel anti-cancer therapeutics. In our earlier study (Inoue, A., Sawata, S. Y., Taira, K., and Wadhwa, R. (2007) Loss-of-function screening by randomized intracellular antibodies: identification of hnRNP-K as a potential target for metastasis. Proc. Natl. Acad. Sci. U.S.A. 104, 8983-8988), we had reported that the involvement of heterogeneous nuclear ribonucleoprotein K (hnRNP-K) in metastasis. Here, we established hnRNP-K-overexpressing and -underexpressing derivative cell lines and examined their proliferation and metastatic properties in vitro and in vivo. Whereas hnRNP-K compromised cells showed delayed tumor growth, its overexpression resulted in enhanced malignancy and metastasis. Molecular basis of the hnRNP-K induced malignant and metastatic phenotypes was dissected by cDNA microarray and pathway analyses. We found that the hnRNP-K regulates extracellular matrix, cell motility, and angiogenesis pathways. Involvement of the selected genes (Cck, Mmp-3, Ptgs2, and Ctgf) and pathways was validated by gene-specific expression analysis. Our results demonstrated that the hnRNP-K is a potential target for metastasis therapy.

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Disruption of Ttll5/Stamp Gene (Tubulin Tyrosine Ligase-like Protein 5/SRC-1 and TIF2-associated Modulatory Protein Gene) in Male Mice Causes Sperm Malformation and Infertility.

Publication Date: 2013 May 24 PMID: 23558686
Authors: Lee, G. S. - He, Y. - Dougherty, E. J. - Jimenez-Movilla, M. - Avella, M. - Grullon, S. - Sharlin, D. S. - Guo, C. - Blackford, J. A. Jr - Awasthi, S. - Zhang, Z. - Armstrong, S. P. - London, E. C. - Chen, W. - Dean, J. - Simons, S. S. Jr
Journal: J Biol Chem

TTLL5/STAMP (tubulin tyrosine ligase-like family member 5) has multiple activities in cells. TTLL5 is one of 13 TTLLs, has polyglutamylation activity, augments the activity of p160 coactivators (SRC-1 and TIF2) in glucocorticoid receptor-regulated gene induction and repression, and displays steroid-independent growth activity with several cell types. To examine TTLL5/STAMP functions in whole animals, mice were prepared with an internal deletion that eliminated several activities of the Stamp gene. This mutation causes both reduced levels of STAMP mRNA and C-terminal truncation of STAMP protein. Homozygous targeted mutant (Stamp(tm/tm)) mice appear normal except for marked decreases in male fertility associated with defects in progressive sperm motility. Abnormal axonemal structures with loss of tubulin doublets occur in most Stamp(tm/tm) sperm tails in conjunction with substantial reduction in alpha-tubulin polyglutamylation, which closely correlates with the reduction in mutant STAMP mRNA. The axonemes in other structures appear unaffected. There is no obvious change in the organs for sperm development of WT versus Stamp(tm/tm) males despite the levels of WT STAMP mRNA in testes being 20-fold higher than in any other organ examined. This defect in male fertility is unrelated to other Ttll genes or 24 genes previously identified as important for sperm function. Thus, STAMP appears to participate in a unique, tissue-selective TTLL-mediated pathway for alpha-tubulin polyglutamylation that is required for sperm maturation and motility and may be relevant for male fertility.

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Aberrant Assembly of RNA Recognition Motif 1 Links to Pathogenic Conversion of TAR DNA-binding Protein of 43 kDa (TDP-43).

Publication Date: 2013 May 24 PMID: 23558684
Authors: Shodai, A. - Morimura, T. - Ido, A. - Uchida, T. - Ayaki, T. - Takahashi, R. - Kitazawa, S. - Suzuki, S. - Shirouzu, M. - Kigawa, T. - Muto, Y. - Yokoyama, S. - Takahashi, R. - Kitahara, R. - Ito, H. - Fujiwara, N. - Urushitani, M.
Journal: J Biol Chem

Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is a pathological signature of amyotrophic lateral sclerosis (ALS). Although accumulating evidence suggests the involvement of RNA recognition motifs (RRMs) in TDP-43 proteinopathy, it remains unclear how native TDP-43 is converted to pathogenic forms. To elucidate the role of homeostasis of RRM1 structure in ALS pathogenesis, conformations of RRM1 under high pressure were monitored by NMR. We first found that RRM1 was prone to aggregation and had three regions showing stable chemical shifts during misfolding. Moreover, mass spectrometric analysis of aggregated RRM1 revealed that one of the regions was located on protease-resistant beta-strands containing two cysteines (Cys-173 and Cys-175), indicating that this region served as a core assembly interface in RRM1 aggregation. Although a fraction of RRM1 aggregates comprised disulfide-bonded oligomers, the substitution of cysteine(s) to serine(s) (C/S) resulted in unexpected acceleration of amyloid fibrils of RRM1 and disulfide-independent aggregate formation of full-length TDP-43. Notably, TDP-43 aggregates with RRM1-C/S required the C terminus, and replicated cytopathologies of ALS, including mislocalization, impaired RNA splicing, ubiquitination, phosphorylation, and motor neuron toxicity. Furthermore, RRM1-C/S accentuated inclusions of familial ALS-linked TDP-43 mutants in the C terminus. The relevance of RRM1-C/S-induced TDP-43 aggregates in ALS pathogenesis was verified by immunolabeling of inclusions of ALS patients and cultured cells overexpressing the RRM1-C/S TDP-43 with antibody targeting misfolding-relevant regions. Our results indicate that cysteines in RRM1 crucially govern the conformation of TDP-43, and aberrant self-assembly of RRM1 at amyloidogenic regions contributes to pathogenic conversion of TDP-43 in ALS.

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Interactions between NF-kappaB and SP3 Connect Inflammatory Signaling with Reduced FGF-10 Expression.

Publication Date: 2013 May 24 PMID: 23558680
Authors: Carver, B. J. - Plosa, E. J. - Stinnett, A. M. - Blackwell, T. S. - Prince, L. S.
Journal: J Biol Chem

Inflammation inhibits normal lung morphogenesis in preterm infants. Soluble inflammatory mediators present in the lungs of patients developing bronchopulmonary dysplasia disrupt expression of multiple genes critical for development. However, the mechanisms linking innate immune signaling and developmental programs are not clear. NF-kappaB activation inhibits expression of the critical morphogen FGF-10. Here, we show that interactions between the RELA subunit of NF-kappaB and SP3 suppress SP1-mediated FGF-10 expression. SP3 co-expression reduced SP1-mediated Fgf-10 promoter activity, suggesting antagonistic interactions between SP1 and SP3. Chromatin immunoprecipitation of LPS-treated primary mouse fetal lung mesenchymal cells detected increased interactions between SP3, RELA, and the Fgf-10 promoter. Expression of a constitutively active IkappaB kinase beta mutant not only decreased Fgf-10 promoter activity but also increased RELA-SP3 nuclear interactions. Expression of a dominant-negative IkappaB, which blocks NF-kappaB nuclear translocation, prevented inhibition of FGF-10 by SP3. The inhibitory functions of SP3 required sequences located in the N-terminal region of the protein. These data suggested that inhibition of FGF-10 by inflammatory signaling involves the NF-kappaB-dependent interactions between RELA, SP3, and the Fgf-10 promoter. NF-kappaB activation may therefore lead to reduced gene expression by recruiting inhibitory factors to specific gene promoters following exposure to inflammatory stimuli.

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Annexin A2 Regulates beta1 Integrin Internalization and Intestinal Epithelial Cell Migration.

Publication Date: 2013 May 24 PMID: 23558678
Authors: Rankin, C. R. - Hilgarth, R. S. - Leoni, G. - Kwon, M. - Den Beste, K. A. - Parkos, C. A. - Nusrat, A.
Journal: J Biol Chem

The gastrointestinal epithelium functions as an important barrier that separates luminal contents from the underlying tissue compartment and is vital in maintaining mucosal homeostasis. Mucosal wounds in inflammatory disorders compromise the critical epithelial barrier. In response to injury, intestinal epithelial cells (IECs) rapidly migrate to reseal wounds. We have previously observed that a membrane-associated, actin binding protein, annexin A2 (AnxA2), is up-regulated in migrating IECs and plays an important role in promoting wound closure. To identify the mechanisms by which AnxA2 promotes IEC movement and wound closure, we used a loss of function approach. AnxA2-specific shRNA was utilized to generate IECs with stable down-regulation of AnxA2. Loss of AnxA2 inhibited IEC migration while promoting enhanced cell-matrix adhesion. These functional effects were associated with increased levels of beta1 integrin protein, which is reported to play an important role in mediating the cell-matrix adhesive properties of epithelial cells. Because cell migration requires dynamic turnover of integrin-based adhesions, we tested whether AnxA2 modulates internalization of cell surface beta1 integrin required for forward cell movement. Indeed, pulse-chase biotinylation experiments in IECs lacking AnxA2 demonstrated a significant increase in cell surface beta1 integrin that was accompanied by decreased beta1 integrin internalization and degradation. These findings support an important role of AnxA2 in controlling dynamics of beta1 integrin at the cell surface that in turn is required for the active turnover of cell-matrix associations, cell migration, and wound closure.

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Molecular Bases of Multimodal Regulation of a Fungal Transient Receptor Potential (TRP) Channel.

Publication Date: 2013 May 24 PMID: 23553631
Authors: Ihara, M. - Hamamoto, S. - Miyanoiri, Y. - Takeda, M. - Kainosho, M. - Yabe, I. - Uozumi, N. - Yamashita, A.
Journal: J Biol Chem

Multimodal activation by various stimuli is a fundamental characteristic of TRP channels. We identified a fungal TRP channel, TRPGz, exhibiting activation by hyperosmolarity, temperature increase, cytosolic Ca(2+) elevation, membrane potential, and H2O2 application, and thus it is expected to represent a prototypic multimodal TRP channel. TRPGz possesses a cytosolic C-terminal domain (CTD), primarily composed of intrinsically disordered regions with some regulatory modules, a putative coiled-coil region and a basic residue cluster. The CTD oligomerization mediated by the coiled-coil region is required for the hyperosmotic and temperature increase activations but not for the tetrameric channel formation or other activation modalities. In contrast, the basic cluster is responsible for general channel inhibition, by binding to phosphatidylinositol phosphates. The crystal structure of the presumed coiled-coil region revealed a tetrameric assembly in an offset spiral rather than a canonical coiled-coil. This structure underlies the observed moderate oligomerization affinity enabling the dynamic assembly and disassembly of the CTD during channel functions, which are compatible with the multimodal regulation mediated by each functional module.

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Dbf4 and Cdc7 Proteins Promote DNA Replication through Interactions with Distinct Mcm2-7 Protein Subunits.

Publication Date: 2013 May 24 PMID: 23549044
Authors: Ramer, M. D. - Suman, E. S. - Richter, H. - Stanger, K. - Spranger, M. - Bieberstein, N. - Duncker, B. P.
Journal: J Biol Chem

The essential cell cycle target of the Dbf4/Cdc7 kinase (DDK) is the Mcm2-7 helicase complex. Although Mcm4 has been identified as the critical DDK phosphorylation target for DNA replication, it is not well understood which of the six Mcm2-7 subunits actually mediate(s) docking of this kinase complex. We systematically examined the interaction between each Mcm2-7 subunit with Dbf4 and Cdc7 through two-hybrid and co-immunoprecipitation analyses. Strikingly different binding patterns were observed, as Dbf4 interacted most strongly with Mcm2, whereas Cdc7 displayed association with both Mcm4 and Mcm5. We identified an N-terminal Mcm2 region required for interaction with Dbf4. Cells expressing either an Mcm2 mutant lacking this docking domain (Mcm2DeltaDDD) or an Mcm4 mutant lacking a previously identified DDK docking domain (Mcm4DeltaDDD) displayed modest DNA replication and growth defects. In contrast, combining these two mutations resulted in synthetic lethality, suggesting that Mcm2 and Mcm4 play overlapping roles in the association of DDK with MCM rings at replication origins. Consistent with this model, growth inhibition could be induced in Mcm4DeltaDDD cells through Mcm2 overexpression as a means of titrating the Dbf4-MCM ring interaction. This growth inhibition was exacerbated by exposing the cells to either hydroxyurea or methyl methanesulfonate, lending support for a DDK role in stabilizing or restarting replication forks under S phase checkpoint conditions. Finally, constitutive overexpression of each individual MCM subunit was examined, and genotoxic sensitivity was found to be specific to Mcm2 or Mcm4 overexpression, further pointing to the importance of the DDK-MCM ring interaction.

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Mechanism of ferrous iron binding and oxidation by ferritin from a pennate diatom.

Publication Date: 2013 May 24 PMID: 23548912
Authors: Pfaffen, S. - Abdulqadir, R. - Le Brun, N. E. - Murphy, M. E.
Journal: J Biol Chem

A novel ferritin was recently found in Pseudo-nitzschia multiseries (PmFTN), a marine pennate diatom that plays a major role in global primary production and carbon sequestration into the deep ocean. Crystals of recombinant PmFTN were soaked in iron and zinc solutions, and the structures were solved to 1.65-2.2-A resolution. Three distinct iron binding sites were identified as determined from anomalous dispersion data from aerobically grown ferrous soaked crystals. Sites A and B comprise the conserved ferroxidase active site, and site C forms a pathway leading toward the central cavity where iron storage occurs. In contrast, crystal structures derived from anaerobically grown and ferrous soaked crystals revealed only one ferrous iron in the active site occupying site A. In the presence of dioxygen, zinc is observed bound to all three sites. Iron oxidation experiments using stopped-flow absorbance spectroscopy revealed an extremely rapid phase corresponding to Fe(II) oxidation at the ferroxidase site, which is saturated after adding 48 ferrous iron to apo-PmFTN (two ferrous iron per subunit), and a much slower phase due to iron core formation. These results suggest an ordered stepwise binding of ferrous iron and dioxygen to the ferroxidase site in preparation for catalysis and a partial mobilization of iron from the site following oxidation.

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Norepinephrine Modulates the Motility of Resting and Activated Microglia via Different Adrenergic Receptors.

Publication Date: 2013 May 24 PMID: 23548902
Authors: Gyoneva, S. - Traynelis, S. F.
Journal: J Biol Chem

Microglia, the resident immune cells of the central nervous system (CNS), monitor the brain for disturbances of tissue homeostasis by constantly moving their fine processes. Microglia respond to tissue damage through activation of ATP/ADP receptors followed by directional process extension to the damaged area. A common feature of several neurodegenerative diseases is the loss of norepinephrine, which might contribute to the associated neuroinflammation. We carried out a high resolution analysis of the effects of norepinephrine (NE) on microglial process dynamics in acute brain slices from mice that exhibit microglia-specific enhanced green fluorescent protein expression. Bath application of NE to the slices resulted in significant process retraction in microglia. Analysis of adrenergic receptor expression with quantitative PCR indicated that resting microglia primarily express beta2 receptors but switch expression to alpha2A receptors under proinflammatory conditions modeled by LPS treatment. Despite the differential receptor expression, NE caused process retraction in both resting and LPS-activated microglia cultured in the gelatinous substrate Matrigel in vitro. The use of subtype-selective receptor agonists and antagonists confirmed the involvement of beta2 receptors in mediating microglial process dynamics in resting cells and alpha2A receptors in activated cells. Co-application of NE with ATP to resting microglia blocked the ATP-induced process extension and migration in isolated microglia, and beta2 receptor antagonists prolonged ATP effects in brain slice tissues, suggesting the presence of cross-talk between adrenergic and purinergic signaling in microglia. These data show that the neurotransmitter NE can modulate microglial motility, which could affect microglial functions in pathogenic situations of either elevated or reduced NE levels.

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Endomembrane H-Ras Controls Vascular Endothelial Growth Factor-induced Nitric-oxide Synthase-mediated Endothelial Cell Migration.

Publication Date: 2013 May 24 PMID: 23548900
Authors: Haeussler, D. J. - Pimentel, D. R. - Hou, X. - Burgoyne, J. R. - Cohen, R. A. - Bachschmid, M. M.
Journal: J Biol Chem

We demonstrate for the first time that endomembrane-delimited H-Ras mediates VEGF-induced activation of endothelial nitric-oxide synthase (eNOS) and migratory response of human endothelial cells. Using thiol labeling strategies and immunofluorescent cell staining, we found that only 31% of total H-Ras is S-palmitoylated, tethering the small GTPase to the plasma membrane but leaving the function of the large majority of endomembrane-localized H-Ras unexplained. Knockdown of H-Ras blocked VEGF-induced PI3K-dependent Akt (Ser-473) and eNOS (Ser-1177) phosphorylation and nitric oxide-dependent cell migration, demonstrating the essential role of H-Ras. Activation of endogenous H-Ras led to recruitment and phosphorylation of eNOS at endomembranes. The loss of migratory response in cells lacking endogenous H-Ras was fully restored by modest overexpression of an endomembrane-delimited H-Ras palmitoylation mutant. These studies define a newly recognized role for endomembrane-localized H-Ras in mediating nitric oxide-dependent proangiogenic signaling.

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Systematic Analysis of gamma-Aminobutyric Acid (GABA) Metabolism and Function in the Social Amoeba Dictyostelium discoideum.

Publication Date: 2013 May 24 PMID: 23548898
Authors: Wu, Y. - Janetopoulos, C.
Journal: J Biol Chem

While GABA has been suggested to regulate spore encapsulation in the social amoeba Dictyostelium discoideum, the metabolic profile and other potential functions of GABA during development remain unclear. In this study, we investigated the homeostasis of GABA metabolism by disrupting genes related to GABA metabolism and signaling. Extracellular levels of GABA are tightly regulated during early development, and GABA is generated by the glutamate decarboxylase, GadB, during growth and in early development. However, overexpression of the prespore-specific homologue, GadA, in the presence of GadB reduces production of extracellular GABA. Perturbation of extracellular GABA levels delays the process of aggregation. Cytosolic GABA is degraded by the GABA transaminase, GabT, in the mitochondria. Disruption of a putative vesicular GABA transporter (vGAT) homologue DdvGAT reduces secreted GABA. We identified the GABAB receptor-like family member GrlB as the major GABA receptor during early development, and either disruption or overexpression of GrlB delays aggregation. This delay is likely the result of an abolished pre-starvation response and late expression of several "early" developmental genes. Distinct genes are employed for GABA generation during sporulation. During sporulation, GadA alone is required for generating GABA and DdvGAT is likely responsible for GABA secretion. GrlE but not GrlB is the GABA receptor during late development.

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Identification of a New Interaction Mode between the Src Homology 2 Domain of C-terminal Src Kinase (Csk) and Csk-binding Protein/Phosphoprotein Associated with Glycosphingolipid Microdomains.

Publication Date: 2013 May 24 PMID: 23548896
Authors: Tanaka, H. - Akagi, K. - Oneyama, C. - Tanaka, M. - Sasaki, Y. - Kanou, T. - Lee, Y. H. - Yokogawa, D. - Dobenecker, M. W. - Nakagawa, A. - Okada, M. - Ikegami, T.
Journal: J Biol Chem

Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the betaB/betaC loop of the SH2 domain.

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Nitric Oxide Modifies Global Histone Methylation by Inhibiting Jumonji C Domain-containing Demethylases.

Publication Date: 2013 May 31 PMID: 23546878
Authors: Hickok, J. R. - Vasudevan, D. - Antholine, W. E. - Thomas, D. D.
Journal: J Biol Chem

Methylation of lysine residues on histone tails is an important epigenetic modification that is dynamically regulated through the combined effects of methyltransferases and demethylases. The Jumonji C domain Fe(II) alpha-ketoglutarate family of proteins performs the majority of histone demethylation. We demonstrate that nitric oxide ((*)NO) directly inhibits the activity of the demethylase KDM3A by forming a nitrosyliron complex in the catalytic pocket. Exposing cells to either chemical or cellular sources of (*)NO resulted in a significant increase in dimethyl Lys-9 on histone 3 (H3K9me2), the preferred substrate for KDM3A. G9a, the primary methyltransferase acting on H3K9me2, was down-regulated in response to (*)NO, and changes in methylation state could not be accounted for by methylation in general. Furthermore, cellular iron sequestration via dinitrosyliron complex formation correlated with increased methylation. The mRNA of several histone demethylases and methyltransferases was also differentially regulated in response to (*)NO. Taken together, these data reveal three novel and distinct mechanisms whereby (*)NO can affect histone methylation as follows: direct inhibition of Jumonji C demethylase activity, reduction in iron cofactor availability, and regulation of expression of methyl-modifying enzymes. This model of (*)NO as an epigenetic modulator provides a novel explanation for nonclassical gene regulation by (*)NO.

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Regulatory Interactions between a Bacterial Tyrosine Kinase and Its Cognate Phosphatase.

Publication Date: 2013 May 24 PMID: 23543749
Authors: Temel, D. B. - Dutta, K. - Alphonse, S. - Nourikyan, J. - Grangeasse, C. - Ghose, R.
Journal: J Biol Chem

The cyclic process of autophosphorylation of the C-terminal tyrosine cluster (YC) of a bacterial tyrosine kinase and its subsequent dephosphorylation following interactions with a counteracting tyrosine phosphatase regulates diverse physiological processes, including the biosynthesis and export of polysaccharides responsible for the formation of biofilms or virulence-determining capsules. We provide here the first detailed insight into this hitherto uncharacterized regulatory interaction at residue-specific resolution using Escherichia coli Wzc, a canonical bacterial tyrosine kinase, and its opposing tyrosine phosphatase, Wzb. The phosphatase Wzb utilizes a surface distal to the catalytic elements of the kinase, Wzc, to dock onto its catalytic domain (WzcCD). WzcCD binds in a largely YC-independent fashion near the Wzb catalytic site, inducing allosteric changes therein. YC dephosphorylation is proximity-mediated and reliant on the elevated concentration of phosphorylated YC near the Wzb active site resulting from WzcCD docking. Wzb principally recognizes the phosphate of its phosphotyrosine substrate and further stabilizes the tyrosine moiety through ring stacking interactions with a conserved active site tyrosine.

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Regulation of primary response genes in B cells.

Publication Date: 2013 May 24 PMID: 23536186
Authors: Fowler, T. - Suh, H. - Buratowski, S. - Roy, A. L.
Journal: J Biol Chem

Deregulated gene expression in B cells often results in various lymphoid malignancies and immune deficiencies. Therefore, understanding signal-induced gene regulatory pathways involved during B cell activation is important to tackle pathologies associated with altered B cell function. Primary response genes (PRGs) are rapidly induced upon signaling in B cells and other cell types and often encode oncogenic transcription factors, which are associated with various malignancies. However, an important issue that remains unclear is whether the fundamental mechanism of activation of these genes is essentially the same under such diverse conditions. c-fos is a PRG that is induced rapidly upon activation of B cells in response to a wide variety of stimuli. Using the c-fos gene as a candidate PRG, we addressed here how it is regulated in response to tumor-promoting and antigen-mimicking signals. Our results show that although the mRNA was induced and extinguished within minutes in response to both signals, surprisingly, apparently full-length unspliced pre-mRNA persisted for several hours in both cases. However, although the mitogenic signal resulted in a more sustained mRNA response that persisted for 4 h, antigenic signaling resulted in a more robust but very transient response that lasted for <1 h. Moreover, the pre-mRNA profile exhibited significant differences between the two signals. Additionally, the splicing regulation was also observed with egr-2, but not with c-myc. Together, these results suggest a previously underappreciated regulatory step in PRG expression in B cells.

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Lewy Body-like alpha-Synuclein Aggregates Resist Degradation and Impair Macroautophagy.

Publication Date: 2013 May 24 PMID: 23532841
Authors: Tanik, S. A. - Schultheiss, C. E. - Volpicelli-Daley, L. A. - Brunden, K. R. - Lee, V. M.
Journal: J Biol Chem

Cytoplasmic alpha-synuclein (alpha-syn) aggregates, referred to as Lewy bodies, are pathological hallmarks of a number of neurodegenerative diseases, most notably Parkinson disease. Activation of macroautophagy is suggested to facilitate degradation of certain proteinaceous inclusions, but it is unclear if this pathway is capable of degrading alpha-syn aggregates. Here, we examined this issue by utilizing cellular models in which intracellular Lewy body-like alpha-syn inclusions accumulate after internalization of pre-formed alpha-syn fibrils into alpha-syn-expressing HEK293 cells or cultured primary neurons. We demonstrate that alpha-syn inclusions cannot be effectively degraded, even though they co-localize with essential components of both the autophagic and proteasomal protein degradation pathways. The alpha-syn aggregates persist even after soluble alpha-syn levels have been substantially reduced, suggesting that once formed, the alpha-syn inclusions are refractory to clearance. Importantly, we also find that alpha-syn aggregates impair overall macroautophagy by reducing autophagosome clearance, which may contribute to the increased cell death that is observed in aggregate-bearing cells.

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Structure Prediction and Analysis of DNA Transposon and LINE Retrotransposon Proteins.

Publication Date: 2013 May 31 PMID: 23530042
Authors: Abrusan, G. - Zhang, Y. - Szilagyi, A.
Journal: J Biol Chem

Despite the considerable amount of research on transposable elements, no large-scale structural analyses of the TE proteome have been performed so far. We predicted the structures of hundreds of proteins from a representative set of DNA and LINE transposable elements and used the obtained structural data to provide the first general structural characterization of TE proteins and to estimate the frequency of TE domestication and horizontal transfer events. We show that 1) ORF1 and Gag proteins of retrotransposons contain high amounts of structural disorder; thus, despite their very low conservation, the presence of disordered regions and probably their chaperone function is conserved. 2) The distribution of SCOP classes in DNA transposons and LINEs indicates that the proteins of DNA transposons are more ancient, containing folds that already existed when the first cellular organisms appeared. 3) DNA transposon proteins have lower contact order than randomly selected reference proteins, indicating rapid folding, most likely to avoid protein aggregation. 4) Structure-based searches for TE homologs indicate that the overall frequency of TE domestication events is low, whereas we found a relatively high number of cases where horizontal transfer, frequently involving parasites, is the most likely explanation for the observed homology.

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Discoidin Domain Receptor 1 is a novel modulator of megakaryocyte-collagen interactions.

Publication Date: 2013 Mar 24 PMID: 23530036
Authors: Abbonante, V. - Gruppi, C. - Rubel, D. - Gross, O. - Moratti, R. - Balduini, A.
Journal: J Biol Chem

Growing evidence demonstrates that extracellular matrices (ECMs) regulate many aspects of megakaryocyte (MK) development; however, among the different ECM receptors, integrin alpha2beta1 and GPVI are the only collagen receptors studied in platelets and MKs. In this study, we demonstrate the expression of the novel collagen receptor Discoidin Domain Receptor 1 (DDR1) by human MKs at both mRNA and protein levels and provide evidence of DDR1 involvement in the regulation of MK motility on type I collagen through a mechanism based on the activity of SHP1 phosphatase and Syk tyrosine kinase. Specifically, we demonstrated that inhibition of DDR1 binding to type I collagen, preserving the engagement of the other collagen receptors, GPVI, alpha2beta1 and LAIR-1 determines a decrease in MK migration due to the reduction in SHP1 phosphatase activity and consequent increase in the phosphorylation level of its main substrate Syk. Consistently, inhibition of Syk activity restored MK migration on type I collagen. In conclusion, we report the expression and function of a novel collagen receptor on human MKs and we point out that increasing level of complexity is necessary to better understand MK-collagen interactions in the bone marrow environment.

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Allosteric activation of trypanosomatid deoxyhypusine synthase by a catalytically dead paralog.

Publication Date: 2013 May 24 PMID: 23525104
Authors: Nguyen, S. - Jones, D. C. - Wyllie, S. - Fairlamb, A. H. - Phillips, M. A.
Journal: J Biol Chem

Polyamine biosynthesis is a key drug target in African trypanosomes. The "resurrection drug" eflornithine (difluoromethylornithine), which is used clinically to treat human African trypanosomiasis, inhibits the first step in polyamine (spermidine) biosynthesis, a highly regulated pathway in most eukaryotic cells. Previously, we showed that activity of a key trypanosomatid spermidine biosynthetic enzyme, S-adenosylmethionine decarboxylase, is regulated by heterodimer formation with a catalytically dead paralog (a prozyme). Here, we describe an expansion of this prozyme paradigm to the enzyme deoxyhypusine synthase, which is required for spermidine-dependent hypusine modification of a lysine residue in the essential translation factor eIF5A. Trypanosoma brucei encodes two deoxyhypusine synthase paralogs, one that is catalytically functional but grossly impaired, and the other is inactive. Co-expression in Escherichia coli results in heterotetramer formation with a 3000-fold increase in enzyme activity. This functional complex is also present in T. brucei, and conditional knock-out studies indicate that both DHS genes are essential for in vitro growth and infectivity in mice. The recurrent evolution of paralogous, catalytically dead enzyme-based activating mechanisms may be a consequence of the unusual gene expression in the parasites, which lack transcriptional regulation. Our results suggest that this mechanism may be more widely used by trypanosomatids to control enzyme activity and ultimately influence pathogenesis than currently appreciated.

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Identification of Novel Pretranslational Regulatory Mechanisms for NF-kappaB Activation.

Publication Date: 2013 May 31 PMID: 23515310
Authors: Huang, X. - Gong, R. - Li, X. - Virtue, A. - Yang, F. - Yang, I. H. - Tran, A. H. - Yang, X. F. - Wang, H.
Journal: J Biol Chem

NF-kappaB-controlled transcriptional regulation plays a central role in inflammatory and immune responses. Currently, understanding about NF-kappaB activation mechanism emphasizes IkappaB-tethered complex inactivation in the cytoplasm. In the case of NF-kappaB activation, IkappaB phosphorylation leads to its degradation, followed by NF-kappaB relocation to the nucleus and trans-activation of NF-kappaB-targeted genes. Pretranslational mechanism mediated NF-kappaB activation remains poorly understood. In this study, we investigated NF-kappaB pretranslational regulation by performing a series of database mining analyses and using six large national experimental databases (National Center of Biotechnology Information UniGene expressed sequence tag profile database, Gene Expression Omnibus database, Transcription Element Search System database, AceView database, and Epigenomics database) and TargetScan software. We reported the following findings: 1) NF-kappaB-signaling genes are differentially expressed in human and mouse tissues; 2) heart and vessels are the inflammation-privileged tissues and less easy to be inflamed because lacking in key NF-kappaB-signaling molecular expression; 3) NF-kappaB-signaling genes are induced by cardiovascular disease risk factors oxidized phospholipids and proinflammatory cytokines in endothelial cells; 4) transcription factors CCAAT/enhancer-binding proteins and NF-kappaB have higher binding site frequencies in the promoters of proinflammatory cytokine-induced NF-kappaB genes; 5) most NF-kappaB-signaling genes have multiple alternative promoters and alternatively spliced isoforms; 6) NF-kappaB family genes can be regulated by DNA methylation; and 7) 27 of 38 NF-kappaB-signaling genes can be regulated by microRNAs. Our findings provide important insight into the mechanism of NF-kappaB activation, which may contribute to cardiovascular disease, inflammatory diseases, and immunological disorders.

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Selective Inhibition of Human Group IIA-secreted Phospholipase A2 (hGIIA) Signaling Reveals Arachidonic Acid Metabolism Is Associated with Colocalization of hGIIA to Vimentin in Rheumatoid Synoviocytes.

Publication Date: 2013 May 24 PMID: 23482564
Authors: Lee, L. K. - Bryant, K. J. - Bouveret, R. - Lei, P. W. - Duff, A. P. - Harrop, S. J. - Huang, E. P. - Harvey, R. P. - Gelb, M. H. - Gray, P. P. - Curmi, P. M. - Cunningham, A. M. - Church, W. B. - Scott, K. F.
Journal: J Biol Chem

Human group IIA secreted phospholipase A2 (hGIIA) promotes tumor growth and inflammation and can act independently of its well described catalytic lipase activity via an alternative poorly understood signaling pathway. With six chemically diverse inhibitors we show that it is possible to selectively inhibit hGIIA signaling over catalysis, and x-ray crystal structures illustrate that signaling involves a pharmacologically distinct surface to the catalytic site. We demonstrate in rheumatoid fibroblast-like synoviocytes that non-catalytic signaling is associated with rapid internalization of the enzyme and colocalization with vimentin. Trafficking of exogenous hGIIA was monitored with immunofluorescence studies, which revealed that vimentin localization is disrupted by inhibitors of signaling that belong to a rare class of small molecule inhibitors that modulate protein-protein interactions. This study provides structural and pharmacological evidence for an association between vimentin, hGIIA, and arachidonic acid metabolism in synovial inflammation, avenues for selective interrogation of hGIIA signaling, and new strategies for therapeutic hGIIA inhibitor design.

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