Journal of Biological Chemistry

Laminin-3B11, a novel vascular type laminin capable of inducing prominent lamellipodial protrusions in microvascular endothelial cells.

Publication Date: 2010 Aug 30 PMID: 20805229
Authors: Mori, T. - Ono, K. - Kariya, Y. - Ogawa, T. - Higashi, S. - Miyazaki, K.
Journal: J Biol Chem

The basement membrane (BM) proteins laminins play critical roles in the maintenance of tissue structures and cellular functions. One of laminin alpha chains, alpha3 has two isoforms, the truncated alpha3A and the full-sized alpha3B. Although the alpha3A-containing laminin laminin-3A32 (previously, laminin-5) has been extensively studied, there is little information about alpha3B-containing laminins. To date, only alpha4- and alpha5-types of laminins, most typically laminin-411 and laminin-511, have been identified in the BMs of blood vessels. Here we for the first time identified and characterized a novel vascular laminin isoform, laminin-3B11 (Lm3B11), which consists of laminin alpha3B, beta1 and gamma1 chains. Firstly, we found that cultured human umbilical vein endothelial cells expressed Lm3B11 but at a very low level. Next, we overexpressed recombinant Lm3B11 by introducing the cDNAs of the three chains into HEK-293 cells and purified it to homogeneity. Purified Lm3B11 exhibited relatively weak cell adhesion activity through both alpha3beta1 and alpha6beta1 integrins. Most characteristically, Lm3B11 strongly stimulated microvascular endothelial (MVE) cells to extend many lamellipodial protrusions. This morphological change was dependent on Src- and PI3K-mdiated signal transduction. The unique activity of Lm3B11 appears to be favorable to the branching or network formation of capillaries and venules.

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The antioxidant transcription factor Nrf2 negatively regulates autophagy and growth arrest induced by the anticancer redox agent mitoquinine.

Publication Date: 2010 Aug 30 PMID: 20805228
Authors: Rao, V. A. - Klein, S. R. - Bonar, S. J. - Zielonka, J. - Mizuno, N. - Dickey, J. S. - Keller, P. W. - Joseph, J. - Kalyanaraman, B. - Shacter, E.
Journal: J Biol Chem

Mitoquinone (MitoQ) is a synthetically-modified, redox-active ubiquinone compound that accumulates predominantly in mitochondria. We found that MitoQ is 30-fold more cytotoxic to breast cancer cells than to healthy mammary cells. MitoQ treatment led to irreversible inhibition of clonogenic growth of breast cancer cells through a combination of autophagy and apoptotic cell death mechanisms. Relatively limited cytotoxicity was seen with the parent ubiquinone coenzyme Q10. Inhibition of cancer cell growth by MitoQ was associated with G1/S cell cycle arrest and phosphorylation of the checkpoint kinases Chk1 and Chk2. The possible role of oxidative stress in MitoQ activity was investigated by measuring the products of hydroethidine oxidation. Increases in ethidium and dihydroethidium levels, markers of one electron oxidation of hydroethidine, were observed at cytotoxic concentrations of MitoQ. Keap1, an oxidative stress sensor protein that regulates the antioxidant transcription factor Nrf2, underwent oxidation, degradation, and dissociation from Nrf2 in MitoQ-treated cells. Nrf2 protein levels, nuclear localization, and transcriptional activity also increased following MitoQ treatment. Knockdown of Nrf2 caused a 2-fold increase in autophagy and an increase in G1 cell cycle arrest in response to MitoQ, but had no apparent effect on apoptosis. The Nrf2-regulated enzyme NQO1 is partly responsible for controlling the level of autophagy. Keap1 and Nrf2 act as redox sensors for oxidative perturbations that lead to autophagy. MitoQ and similar compounds should be further evaluated for novel anticancer activity.

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Conformational dynamics of the bovine mitochondrial ADP/ATP carrier isoform 1 revealed by hydrogen/deuterium exchange coupled to mass spectrometry.

Publication Date: 2010 Aug 30 PMID: 20805227
Authors: Rey, M. - Man, P. - Clemencon, B. - Trezeguet, V. - Brandolin, G. - Forest, E. - Pelosi, L.
Journal: J Biol Chem

The mitochondrial adenine nucleotide carrier (Ancp) catalyzes the transport of ADP and ATP across the mitochondrial inner membrane, thus playing an essential role in cellular energy metabolism. During the transport mechanism, the carrier switches between two different conformations that can be blocked by two toxins: carboxyatractyloside (CATR) and bongkrekic acid (BA). Therefore, our understanding of the nucleotide transport mechanism can be improved by analysing structural differences of the individual inhibited states. We have solved the three dimensional structure of bovine carrier isoform 1 (bAnc1p) in a complex with CATR, but the structure of the carrier-BA complex and thus the detailed mechanism of transport remains unknown. Improvements in sample processing in the hydrogen/deuterium exchange technique coupled to mass spectrometry (HDX-MS) have allowed us to gain novel insights into the conformational changes undergone by bAnc1p. This paper describes the first study of bAnc1p using HDX-MS. Results obtained with the CATR-bAnc1p complex were fully in agreement with published results, thus validating our approach. On the other hand, the HDX kinetics of the two complexes displays marked differences. The BA-bAnc1p complex exhibits greater accessibility to the solvent on the matrix side whereas the CATR-bAnc1p complex is more accessible on the intermembrane side. These results are discussed with respect to the structural and biochemical data available on Ancp.

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Palmitate-induced down-regulation of sortilin and impaired GLUT4 trafficking in C2C12 myotubes.

Publication Date: 2010 Aug 30 PMID: 20805226
Authors: Tsuchiya, Y. - Hatakeyama, H. - Emoto, N. - Wagatsuma, F. - Matsushita, S. - Kanzaki, M.
Journal: J Biol Chem

Elevated saturated free fatty acids (FFAs) including palmitate (C16:0) are a primary trigger for peripheral insulin resistance characterized by impaired glucose uptake/disposal in skeletal muscle, resulting from impaired GLUT4 translocation in response to insulin. We herein demonstrate that palmitate induces down-regulation of sortilin, a sorting receptor implicated in the formation of insulin-responsive GLUT4 vesicles, via mechanisms involving PKC and TNF-alpha-converting enzyme (TACE), but not p38, JNK or mitochondrial ROS generation, leading to impaired GLUT4 trafficking in C2C12 myotubes. Intriguingly, unsaturated FFAs such as palmitoleate (C16:1) and oleate (C18:1) had no such detrimental effects, appearing instead to effectively reverse palmitate-induced impairment of insulin-responsive GLUT4 recycling along with restoration of sortilin abundance by preventing aberrant PKC activation. On the other hand, shRNA-mediated reduction of sortilin in intact C2C12 myotubes inhibited insulin-induced GLUT4 recycling without dampening Akt phosphorylation. We found that the PPARgamma agonist troglitazone prevented the palmitate-induced sortilin reduction and also ameliorated insulin-responsive GLUT4 recycling without altering the palmitate-evoked insults on signaling cascades; neither highly phosphorylated PKC states nor impaired insulin-responsive Akt phosphorylation was affected. Taken together, our data provide novel insights into the pathogenesis of PKC-dependent insulin resistance with respect to insulin-responsive GLUT4 translocation, which could occur not only through defects of insulin signaling but also via a reduction of sortilin, which directly controls trafficking/sorting of GLUT4 in skeletal muscle cells. In addition, our data suggest the insulin-sensitizing action of PPARgamma agonists to be at least partially mediated through the restoration of proper GLUT4 trafficking/sorting events governed by sortilin.

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Activation of human VPS4A by ESCRT-III proteins reveals ability of substrates to relieve enzyme autoinhibition.

Publication Date: 2010 Aug 30 PMID: 20805225
Authors: Merrill, S. A. - Hanson, P. I.
Journal: J Biol Chem

VPS4 is an AAA+ ATPase required to form multivesicular bodies, release viral particles, and complete cytokinesis. It acts by disassembling ESCRT-III heteropolymers during or after their proposed function in membrane scission. Here we show that purified human VPS4A is essentially inactive but can be stimulated to hydrolyze ATP by ESCRT-III proteins in a reaction that requires both their previously defined MIT interacting motifs (MIMs) and ~50 amino acids of adjacent sequence. Importantly, C-terminal fragments of all ESCRT-III proteins tested, including CHMP2A, CHMP1B, CHMP3, CHMP4A, CHMP6, and CHMP5, activated VPS4A suggesting that it disassembles ESCRT-III heteropolymers by affecting each component protein. VPS4A is thought to act as a ring-shaped cylindrical oligomer like other AAA+ ATPases, but this has been difficult to directly demonstrate. We found that concentrating His6-VPS4A on liposomes containing Ni2+-NTA-tagged lipid increased ATP hydrolysis, confirming the importance of inter-subunit interactions for activity. We also found that mutating pore loops expected to line the center of a cylindrical oligomer changed the response of VPS4A to ESCRT-III proteins. Based on these data, we propose that ESCRT-III proteins facilitate assembly of functional but transient VPS4A oligomers and interact with sequences inside the pore of the assembled enzyme. Deleting the N-terminal MIT domain and adjacent linker from VPS4A increased both basal and liposome-enhanced ATPase activity, indicating that these elements play a role in autoinhibiting VPS4A until it encounters ESCRT-III proteins. These findings reveal new ways in which VPS4 activity is regulated and specifically directed to ESCRT-III polymers.

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Seeded aggregation and toxicity of {alpha}-synuclein and tau: cellular models of neurodegenerative diseases.

Publication Date: 2010 Aug 30 PMID: 20805224
Authors: Nonaka, T. - Watanabe, S. T. - Iwatsubo, T. - Hasegawa, M.
Journal: J Biol Chem

The deposition of amyloid-like filaments in brain is the central event in the pathogenesis of neurodegenerative diseases. Here we report cellular models of intracytoplasmic inclusions of alpha-synuclein, generated by introducing nucleation seeds into SH-SY5Y cells with a transfection reagent. Upon introduction of preformed seeds into cells over-expressing alpha-synuclein, abundant, highly filamentous alpha-synuclein-positive inclusions, which are extensively phosphorylated and ubiquitinated and partially thioflavin-positive, were formed within the cells. SH-SY5Y cells that formed such inclusions underwent cell death, which was blocked by small-molecular compounds that inhibit beta-sheet formation. Similar seed-dependent aggregation was observed in cells expressing 4-repeat tau by introducing 4-repeat tau fibrils, but not 3-repeat tau fibrils or alpha-synuclein fibrils. No aggregate formation was observed in cells overexpressing 3-repeat tau upon treatment with 4-repeat tau fibrils. Our cellular models thus provide evidence of nucleation-dependent and protein-specific polymerization of intracellular amyloid-like proteins in cultured cells.

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O-GlcNAc transferase regulates mitotic chromatin dynamics.

Publication Date: 2010 Aug 30 PMID: 20805223
Authors: Sakabe, K. - Hart, G. W.
Journal: J Biol Chem

Mitosis must faithfully divide the genome such that each progeny inherits the same genetic material. DNA condensation is crucial in ensuring that chromosomes are correctly attached to the mitotic spindle for segregation, preventing DNA breaks or constrictions from the contractile ring. Histones form an octomeric complex of basic proteins important in regulating DNA organization and accessibility. Histone post-translational modifications (PTMs) are altered during mitosis, although the roles of these PTMs remain poorly characterized. Here, we report that O-GlcNAc Transferase (OGT), the enzyme catalyzing the addition of N-acetylglucosamine (O-GlcNAc) moieties to nuclear and cytoplasmic proteins at serine and threonine residues, regulates some aspects of mitotic chromatin dynamics. OGT protein amounts decrease during M phase. Modest over-expression of OGT alters mitotic histone post-translational modifications at Lys 9, Ser 10, Arg 17, and Lys 27 of histone H3. Over-expression of OGT also prevents mitotic phosphorylation of Coactivator Associated Arginine Methyltransferase 1 (CARM1) and prevents its correct cellular localization during mitosis. Moreover, OGT over-expression results in an increase in abnormal chromosomal bridge formation. Together, these results show that regulating the amount of OGT during mitosis is important in ensuring correct chromosomal segregation during mitosis.

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SEQUENCES FROM THE FIRST FIBRONECTIN TYPE III REPEAT OF THE NEURAL CELL ADHESION MOLECULE ALLOW O-GLYCAN POLYSIALYLATION OF AN ADHESION MOLECULE CHIMERA.

Publication Date: 2010 Aug 30 PMID: 20805222
Authors: Foley, D. A. - Swartzentruber, K. G. - Thompson, M. G. - Mendiratta, S. S. - Colley, K. J.
Journal: J Biol Chem

Polysialic acid is a developmentally regulated, anti-adhesive polymer that is added to N-glycans on the fifth immunoglobulin domain (Ig5) of the neural cell adhesion molecule, NCAM. We found that the first fibronectin type III repeat (FN1) of NCAM is required for the polysialylation of N-glycans on the adjacent Ig5 domain, and proposed that the polysialyltransferases recognize specific sequences in FN1 to position themselves for Ig5 N-glycan polysialylation. Other studies identified a novel FN1 acidic surface patch and alpha helix that play roles in NCAM polysialylation. Here we characterize the contribution of two additional FN1 sequences, Pro510Tyr511Ser512 (PYS) and Gln516Val517Gln518 (QVQ). Replacing PYS or the acidic patch dramatically decreases the O-glycan polysialylation of a truncated NCAM protein, while replacing the alpha helix or QVQ shifts polysialic acid to FN1 O-glycans in full length NCAM. We also found that the FN1 domain of the olfactory cell adhesion molecule, OCAM, a homologous, but unpolysialylated protein, could partially replace NCAM FN1. Inserting Pro510Tyr511 eliminated N-glycan polysialylation and enhanced O-glycosylation of an NCAM-OCAM chimera, while inserting other FN1 sequences unique to NCAM, predominantly the acidic patch, created a new polysialyltransferase recognition site. Taken together, our results highlight the role of the FN1 alpha helix and QVQ sequences in N-glycan polysialylation and demonstrate that the acidic patch primarily functions in O-glycan polysialylation.

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Cysteine-scanning analysis of helices TM8, TM9a, TM9b and intervening loops in the YgfO xanthine permease: a carboxyl group is essential at position Asp-276.

Publication Date: 2010 Aug 29 PMID: 20802252
Authors: Mermelekas, G. - Georgopoulou, E. - Kallis, A. - Botou, M. - Vlantos, V. - Frillingos, S.
Journal: J Biol Chem

Bacterial and fungal members of the ubiquitous nucleobase-ascorbate transporter (NAT/NCS2) family use the NAT signature motif, a conserved 11-amino acid sequence between amphipathic helices TM9a and TM9b, to define function and selectivity of the purine binding site. To examine the role of flanking helices TM9a, TM9b and TM8, we employed Cys-scanning analysis of the xanthine-specific homolog YgfO from Escherichia coli. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequences 259FLVVGTIYLLSVLEAVGDITATAMVSRRPIQGEEYQSRLKGGVLADGLVSVIASAV314 and 342TIAVMLVILGLFP354 including these TMs (underlined) was replaced individually with Cys, except the irreplaceable Glu-272 and Asp-304 which had been studied previously. Of 67 single-Cys mutants, 55 accumulate xanthine to 35-140% of the steady state observed with C-less, five (I265C, D276C, I277C, G299C, L350C) accumulate to low levels (10-20%) and seven (T278C, A279C, T280C, A281C, G305C, G351C, P354C) show negligible expression in the membrane. Extensive mutagenesis reveals that a carboxyl group is needed at position Asp-276 for high activity and that D276E differs from wild type as it recognizes 8-methylxanthine (Ki 79 muMu) but fails to recognize 2-thioxanthine, 3-methylxanthine or 6-thioxanthine; bulky replacements of Ala-279 or Thr-280 and replacements of Gly-305, Gly-351 or Pro-354 impair activity or expression. Single-Cys mutants V261C, A273C, G275C and S284C are sensitive to inactivation by N-ethylmaleimide and sensitivity of G275C (IC50 15 muMu) is enhanced in the presence of substrate. The data suggest that residues crucial for the transport mechanism cluster in two conserved motifs, at the cytoplasmic end of TM8 (ExxGDxxAT) and in TM9a (GxxxDG).

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Heat shock transcription factor 1 localizes to the sex chromatin during meiotic repression.

Publication Date: 2010 Aug 27 PMID: 20802198
Authors: Akerfelt, M. - Vihervaara, A. - Laiho, A. - Conter, A. - Christians, E. S. - Sistonen, L. - Henriksson, E.
Journal: J Biol Chem

Heat shock factor 1 (HSF1) is an important transcription factor in cellular stress responses, cancer, aging, and developmental processes including gametogenesis. Disruption of Hsf1 together with another HSF family member Hsf2, causes male sterility and complete lack of mature sperm in mice, but the specific role of HSF1 in spermatogenesis has remained unclear. Here, we show that HSF1 is transiently expressed in meiotic spermatocytes and haploid round spermatids in mouse testis. The Hsf1-/- male mice displayed regions of seminiferous tubules containing only spermatogonia, and increased morphological abnormalities in the sperm heads. In search for HSF1 target genes, we identified 742 putative promoters in mouse testis. Among them, the sex-chromosomal multicopy genes that are expressed in post-meiotic cells, were occupied by HSF1. Given that the sex chromatin mostly is repressed during and after meiosis, it is remarkable that HSF1 directly regulates the transcription of sex-linked multicopy genes during post-meiotic repression. In addition, our results show that HSF1 localizes to the sex body prior to the meiotic divisions, and to the sex chromocenter after completed meiosis. To the best of our knowledge, HSF1 is the first known transcription factor found at the repressed sex chromatin during meiosis.

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Mechanism of activation of PSI-7851 and its diastereoisomer PSI-7977.

Publication Date: 2010 Aug 26 PMID: 20801890
Authors: Murakami, E. - Tolstykh, T. - Bao, H. - Niu, C. - Micolochick Steuer, H. M. - Bao, D. - Chang, W. - Espiritu, C. - Bansal, S. - Lam, A. M. - Otto, M. J. - Sofia, M. J. - Furman, P. A.
Journal: J Biol Chem

A phosphoramidate prodrug of 2'-deoxy-2'-alpha-F-2'-beta-C-methyluridine-5'-monophosphate, PSI-7851 demonstrates potent anti-HCV activity both in vitro and in vivo. PSI-7851 is a mixture of two diastereoisomers, PSI-7976 and PSI-7977, with PSI-7977 being the more active inhibitor of HCV RNA replication in the HCV replicon assay. In order to inhibit the HCV NS5B RNA-dependent RNA polymerase, PSI-7851 must be metabolized to the active triphosphate form. The first step, hydrolysis of the carboxylester by human cathepsin A (CatA) and/or carboxylesterase 1 (CES1), is a stereospecific reaction. Western blot analysis showed that CatA and CES1 are both expressed in primary human hepatocytes. However, expression of CES1 is undetectable in Clone A replicon cells. Studies with inhibitors of CatA and/or CES1 indicated that CatA is primarily responsible for hydrolysis of the carboxylester in Clone A cells whereas in primary human hepatocytes, both CatA and CES1 contribute to the hydrolysis. Hydrolysis of the ester is followed by a putative nucleophilic attack on the phosphorus by the carboxyl group resulting in the spontaneous elimination of phenol and the production of an alaninyl phosphate metabolite, PSI-352707, which is common to both isomers. The removal of the amino acid moiety of PSI-352707 is catalyzed by histidine triad nucleotide binding protein 1 (Hint1) to give the 5'-monophosphate form, PSI-7411. siRNA-mediated Hint1 knockdown studies further indicate that Hint1 is, at least in part, responsible for converting PSI-352707 to PSI-7411. PSI-7411 is then consecutively phosphorylated to the diphosphate, PSI-7410, and to the active triphosphate metabolite, PSI-7409, by UMP-CMP kinase and nucleoside diphosphate kinase, respectively.

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Selective fusion of azurophilic granules with Leishmania-containing phagosomes in human neutrophils.

Publication Date: 2010 Aug 26 PMID: 20801889
Authors: Mollinedo, F. - Janssen, H. - de la Iglesia-Vicente, J. - Villa-Pulgarin, J. A. - Calafat, J.
Journal: J Biol Chem

Leishmania parasites use polymorphonuclear neutrophils as intermediate hosts before their ultimate delivery to macrophages following engulfment of parasite-infected neutrophils. This leads to a silent and unrecognized entry of Leishmania into the macrophage host cell. Neutrophil function depends on its cytoplasmic granules, but their mobilization and role in how Leishmania parasites evade intracellular killing in neutrophils remain undetermined. Here, we have found by ultrastructural approaches that neutrophils ingested L. major promastigotes, and azurophilic granules fused in a preferential way with parasite-containing phagosomes, without promoting parasite killing. Azurophilic granules, identified by the granule marker myeloperoxidase, also fused with L. donovani-engulfed vacuoles in human neutrophils. In addition, the azurophilic membrane marker CD63 was also detected in the vacuole surrounding the parasite, and in the fusion of azurophilic granules with the parasite-engulfed phagosome. Tertiary and specific granules, involved in vacuole acidification and superoxide anion generation, hardly fused with Leishmania-containing phagosomes. L. major interaction with neutrophils did not elicit production of reactive oxygen species or mobilization of tertiary and specific granules. By using immunogold electron microscopy approaches in the engulfment of L. major and L. donovani by human neutrophils, we did not find a significant contribution of endoplasmic reticulum to the formation of Leishmania-containing vacuoles. Live Leishmania parasites were required to be optimally internalized by neutrophils. Our data suggest that Leishmania promastigotes modulate their uptake by neutrophils, and regulate granule fusion processes in a rather selective way to favor parasite survival in human neutrophils.

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Proteins at work: A combined SAXS and theoretical determination of the multiple structures involved on the protein kinase functional landscape.

Publication Date: 2010 Aug 26 PMID: 20801888
Authors: Jamros, M. A. - Oliveria, L. C. - Whitford, P. C. - Onuchic, J. N. - Adams, J. A. - Blumenthal, D. K. - Jennings, P. A.
Journal: J Biol Chem

C-terminal Src kinase (Csk) phosphorylates and down-regulates the Src family tyrosine kinases (SFKs). Crystallographic studies of Csk found an unusual arrangement of the SH2 and SH3 regulatory domains about the kinase core, forming a compact structure. However, recent structural studies of mutant Csk in the presence of an inhibitor indicate that the enzyme accesses an expanded structure. To investigate whether wt-Csk may also access open conformations we applied small angle X-ray scattering (SAXS). We find wt-Csk frequently occupies an extended conformation where the regulatory domains are removed from the kinase core. In addition, all-atom structure-based simulations indicate Csk occupies two free energy basins. These basins correspond to ensembles of distinct global conformations of Csk: a compact structure and an extended structure. The transitions between these structures are entropically driven and accessible via thermal fluctuations that break local interactions. We further characterized the ensemble by generating theoretical scattering curves for mixed populations of conformations from both basins and compared the predicted scattering curves to the experimental profile. This population-combination analysis is more consistent with the experimental data than any rigid model. It suggests that Csk adopts a broad ensemble of conformations in solution, populating extended conformations not observed in the crystal structure that may play an important role in the regulation of Csk. The methodology developed here is broadly applicable to biological macromolecules and will provide useful information about what ensembles of conformations are consistent with the experimental data as well as the ubiquitous dynamic reversible assembly processes inherent in biology.

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Vesicle fusion probability is determined by the specific interactions of MUNC18.

Publication Date: 2010 Aug 26 PMID: 20801887
Authors: Smyth, A. M. - Rickman, C. - Duncan, R. R.
Journal: J Biol Chem

Mammalian regulated secretion is absolutely dependent on four evolutionarily conserved proteins: three SNARE proteins and munc18. Dissecting the functional outcomes of the spatially organized protein interactions between these factors has been difficult, because of the close inter-relationship between different binding modes, and the indirect approaches available. Here, we measured the spatial distribution of single munc18 molecules at the plasma membrane of cells, combined with targeted disruption of a specific mechanism of interaction between syntaxin and munc18. Ablation of this interaction did not alter the spatial distribution of munc18 molecules, indicating that munc18 is maintained at the plasma membrane by another mode of interaction with syntaxin. However, ablating interaction of munc18 with the N-terminal peptide motif of syntaxin had a pronounced influence on the behavior of vesicles at the plasma membrane and their likelihood to undergo fusion. We therefore conclude that munc18 can be maintained at the plasma membrane by interaction with the closed form of syntaxin, but that interaction with the syntaxin N-peptide can confer differential release probabilities to vesicles and contribute to the delineation of secretory vesicle pools.

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Nesprin-2 interacts with alpha-Catenin and regulates WNT-signalling at the nuclear envelope.

Publication Date: 2010 Aug 26 PMID: 20801886
Authors: Neumann, S. - Schneider, M. - Daugherty, R. L. - Gottardi, C. J. - Eming, S. A. - Beijer, A. - Noegel, A. A. - Karakesisoglou, I.
Journal: J Biol Chem

Nesprins and Emerin are structural nuclear envelope proteins that tether nuclei to the cytoskeleton. In the present work we identified the cytoskeleton-associated alpha-(N-/E)-Catenins as novel Nesprin-2 binding partners. The association involves the C-termini of Nesprin-2 giant and alpha-(N-/E)-Catenin. alpha-(E-/T-/N)-Catenins are primarily known for their roles in Cadherin-mediated cell-cell adhesion. Here we show that in addition alpha-Catenin forms complexes with Nesprin-2, that include beta-Catenin and Emerin. We demonstrate that the depletion of Nesprin-2 reduces both the amount of active beta-Catenin inside the nucleus and TCF/LEF-dependent transcription. Taken together these findings suggest novel Nesprin-2 functions in cellular signalling. Moreover, we propose that Nesprin-2 in contrast to Emerin is a positive regulator of the Wnt-signalling pathway.

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Association with {beta}-COP regulates the trafficking of the newly synthesized Na,K-ATPase.

Publication Date: 2010 Aug 26 PMID: 20801885
Authors: Morton, M. J. - Farr, G. A. - Hull, M. - Capendeguy, O. - Horisberger, J. D. - Caplan, M. J.
Journal: J Biol Chem

Plasma membrane expression of the Na,K-ATPase requires assembly of its alpha and beta-subunits. Using a novel labeling technique to identify Na,K-ATPase partner proteins, we detected an interaction between the Na,K-ATPase alpha-subunit and the coat protein, beta-COP, a component of the COP-I complex. When expressed in the absence of the Na,K-ATPase beta-subunit, the Na,K-ATPase alpha-subunit interacts with beta-COP, is retained in the endoplasmic reticulum and targeted for degradation. In the presence of the Na,K-ATPase beta-subunit, the alpha-subunit does not interact with beta-COP and traffics to the plasma membrane. Pulse chase experiments demonstrate that, in cells expressing both the Na,K-ATPase alpha and beta-subunits, newly synthesized alpha-subunit associates with beta-COP immediately after its synthesis but that this interaction does not constitute an obligate intermediate in the assembly of the alpha and beta-subunits to form the pump holoenzyme. The interaction with beta-COP was reduced by mutating a dibasic motif at K54 in the Na,K-ATPase alpha-subunit. This mutant alpha-subunit is not retained in the endoplasmic reticulum and reaches the plasma membrane, even in the absence of Na,K-ATPase beta-subunit expression. Although the K54 alpha-subunit reaches the cell surface without need for beta-subunit assembly, it is only functional as an ion-transporting ATPase in the presence of the beta-subunit.

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3-deoxy-D-manno-octulosonic acid (Kdo) hydrolase identified in Francisella tularensis, Helicobacter pylori, and Legionella pneumophila.

Publication Date: 2010 Aug 26 PMID: 20801884
Authors: Chalabaev, S. - Kim, T. H. - Ross, R. - Derian, A. - Kasper, D. L.
Journal: J Biol Chem

KDO (3-deoxy-D-manno-octulosonic acid) is an eight-carbon sugar ubiquitous in Gram-negative bacterial lipopolysaccharides (LPS). Although its biosynthesis is well described, no protein has yet been identified as a KDO hydrolase. However, KDO hydrolase enzymatic activity has been detected in membranes of Helicobacter pylori and Francisella tularensis, and may be responsible for the removal of side-chain KDO from the LPS core saccharides. We now report the identification of genes encoding a KDO hydrolase in F. tularensis Schu S4 and LVS strains, in H. pylori 26695 strain, and in Legionella pneumophila Philadelphia 1 strain. We have renamed the genes kdhA for keto-deoxyoctulosonate hydrolase A. Deletion of kdhA abolished KDO hydrolase activity in membranes of F. tularensis LVS. The F. tularensis kdhA mutant synthesized a core oligosaccharide containing a KDO disaccharide with one of the KDO residues being a terminal side-chain. This side-chain KDO monosaccharide was absent in the wild-type core oligosaccharide. Expression in E. coli of recombinant KdhA from F. tularensis, H. pylori and L. pneumophila resulted in a reduction of membrane associated side-chain KDO. The identification of this previously faceless enzyme will accelerate study of the biosynthetic basis and biologic impact for post-biosynthetic LPS structural modification.

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NA+/H+ exchanger regulatory factor-1 is involved in chemokine receptor homodimer CCR5 internalization and signal transduction, but does not affect CXCR4 homodimer or CXCR4-CCR5 heterodimer.

Publication Date: 2010 Aug 26 PMID: 20801883
Authors: Hammad, M. M. - Kuang, Y. Q. - Yan, R. - Allen, H. - Dupre, D. J.
Journal: J Biol Chem

Chemokine receptors are members of the G protein-coupled receptor (GPCR) family. CCR5 is also the principal co-receptor for macrophage-tropic strains of human immunodeficiency virus-1 (HIV-1) and several efforts have been made to develop ligands to inhibit HIV-1 infection by promoting CCR5 receptor endocytosis. Given the nature of GPCRs and their propensity to form oligomers, one can consider ligand-based therapies as unselective in terms of the oligomeric composition of complexes. For example, a ligand targeting a CCR5 homomer could likely induce signal transduction on a heteromeric CCR5-CXCR4. Other avenues could therefore be explored. We identified, using a Bimolecular fluorescence complementation-Bioluminescence resonance energy transfer approach, a receptor adaptor capable of interacting specifically with one receptor complex, but not others. NHERF1, an adaptor known for its role in desensitization, internalization and regulation of ERK signalling cascade for several GPCRs, can interact via its PDZ2 domain with the CCR5 homodimer, but not with the CXCR4-CCR5 heterodimer or CXCR4 homodimer. To further characterize this interaction, we also show that NHERF1 increases the capacity of CCR5 to recruit arrestin2 following RANTES stimulation. NHERF1 is also involved in CCR5 internalization, as we demonstrate that coexpression of constructs bearing the PDZ2 domain can block CCR5 internalization. Finally, we show that NHERF1 potentiates RANTES-induced ERK1/2 phosphorylation, and that this activation requires NHERF1, but not arrestin2. Taken together, our results suggest that oligomeric receptor complexes can associate specifically with partners, and that in this case, NHERF1 could represent an interesting new target for the regulation of CCR5 internalization.

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Requirement of the epithelial-specific ETS transcription factor SPDEF for mucous gland cell function in the gastric antrum.

Publication Date: 2010 Aug 26 PMID: 20801882
Authors: Horst, D. - Gu, X. - Bhasin, M. - Yang, Q. - Verzi, M. - Lin, D. - Joseph, M. - Zhang, X. - Chen, W. - Li, Y. P. - Shivdasani, R. A. - Libermann, T. A.
Journal: J Biol Chem

Mucus-secreting cells of the stomach epithelium provide a protective barrier against damage that might result from bacterial colonization or other stimuli. Impaired barrier function contributes to chronic inflammation and cancer. Knockout mice for the epithelium-specific transcription factor Spdef have defects in terminal differentiation of intestinal and bronchial secretory cells. We sought to determine the physiologic function of Spdef in the stomach, another site of significant levels of Spdef expression. We used in situ hybridization and immunohistochemistry to localize Spdef-expressing cells in the mouse stomach; targeted gene disruption to generate mice lacking Spdef; and histologic, immunologic and transcriptional profiling approaches to determine its requirements in stomach epithelial homeostasis. In wild-type mice Spdef RNA and protein are expressed predominantly in mucous gland cells of the antrum and in mucous neck cells of the glandular corpus. Within 1.5 years, nearly half of homozygous mutant mice developed profound mucosal hyperplasia of the gastric antrum. Sub-mucosal infiltration of inflammatory cells preceded antral hyperplasia by several weeks. Absence of Spdef impaired terminal maturation of antral mucous gland cells, as reflected in reduced expression of Muc6 and Tff2 and reduced numbers of secretory granules. Antral gene expression abnormalities overlapped significantly with those in Spdef-/- colon, including genes implicated in secretory granule traffic and functions. Spdef is required for terminal maturation of antral mucous gland cells to protect animals from gastric inflammation and resulting hyperplasia. These requirements parallel Spdef functions in secretory intestinal cells and suggest a common molecular mechanism for maturation of gastrointestinal secretory lineages.

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An exceptionally potent inducer of cytoprotective enzymes: elucidation of the structural features that determine inducer potency and reactivity with KEAP1.

Publication Date: 2010 Aug 26 PMID: 20801881
Authors: Dinkova-Kostova, A. T. - Talalay, P. - Sharkey, J. - Zhang, Y. - Holtzclaw, W. D. - Wang, X. J. - David, E. - Schiavoni, K. H. - Finlayson, S. - Mierke, D. F. - Honda, T.
Journal: J Biol Chem

The Keap1/Nrf2/ARE pathway controls a network of cytoprotective genes that defend against the damaging effects of oxidative and electrophilic stress, and inflammation. Induction of this pathway is a highly effective strategy in combating the risk of cancer and chronic degenerative diseases, including atherosclerosis and neurodegeneration. An acetylenic tricyclic bis(cyano enone) bearing two highly electrophilic Michael acceptors is an extremely potent inducer in cells and in vivo. We demonstrate spectroscopically that both cyano enone functions of the tricyclic molecule react with cysteine residues of Keap1 and activate transcription of cytoprotective genes. Novel monocyclic cyano enones, representing fragments of rings A and C of the tricyclic compound, reveal that the contribution to inducer potency of the ring C Michael acceptor is much greater than that of ring A, and that potency is further enhanced by spatial proximity of an acetylenic function. Critically, the simultaneous presence of two cyano enone functions in rings A and C within a rigid three-ring system results in exceptionally high inducer potency. Detailed understanding of the structural elements that contribute to the reactivity with the protein sensor Keap1 and determine high potency of induction is essential for the development of specific and selective lead compounds as clinically-relevant chemoprotective agents.

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Molecular Basis of Phosphatidylinositol Mannoside Biosynthesis and Regulation in Mycobacteria.

Publication Date: 2010 Aug 27 PMID: 20801880
Authors: Guerin, M. E. - Kordulakova, J. - Alzari, P. M. - Brennan, P. J. - Jackson, M.
Journal: J Biol Chem

Phosphatidyl-myo-inositol mannosides (PIMs) are unique glycolipids found in abundant quantities in the inner and outer membranes of the cell envelope of all Mycobacterium species. They are based on a phosphatidyl-myo-inositol (PI) lipid anchor carrying one to six mannose residues and up to four acyl chains. PIMs are considered not only essential structural components of the cell envelope, but also the structural basis of the lipoglycans - lipomannan and lipoarabinomannan - all important molecules implicated in host-pathogen interactions in the course of tuberculosis and leprosy. Although the chemical structure of PIMs is now well established, knowledge of the enzymes and sequential events leading to their biosynthesis and regulation is still incomplete. Recent advances in the identification of key proteins involved in PIM biogenesis, and the determination of the three-dimensional structures of the essential phosphatidyl-myo-inositol mannosyltransferase PimA and the lipoprotein LpqW, have led to important insights into the molecular basis of this pathway.

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Mitotic phosphorylation of cdc25B Ser321 disrupts 14-3-3 binding to the high affinity Ser323 site.

Publication Date: 2010 Aug 27 PMID: 20801879
Authors: Astuti, P. - Boutros, R. - Ducommun, B. - Gabrielli, B.
Journal: J Biol Chem

Cdc25B is a key regulator of entry into mitosis, and its activity and localisation are regulated by binding of 14-3-3 dimer. There are three 14-3-3 binding sites on cdc25B, with Ser323 being the highest affinity binding and is highly homologous to the Ser214 14-3-3 binding site on cdc25C. Loss of 14-3-3 binding to Ser323 increases cyclin/cdk substrate access to the catalytic site thereby increasing its activity. It also affects the localization of cdc25B. Thus phosphorylation and 14-3-3 binding to this site is essential for down regulating cdc25B activity, blocking its mitosis promoting function. The question of how this inhibitory signal is relieved to allow cdc25B activation and entry into mitosis is yet to be resolved. Here we show that Ser323 phosphorylation is maintained into mitosis, but phosphorylation of Ser321 disrupts 14-3-3 binding to Ser323, mimicking the effect of inhibiting Ser323 phosphorylation on both cdc25B activity and localisation. The unphosphorylated Ser321 appears to have a role in stabilizing 14-3-3 binding to Ser323, and loss of the Ser hydroxyl group appears to be sufficient to significantly reduce 14-3-3 binding. A consequence of loss of 14-3-3 binding is dephosphorylation of Ser323. Ser321 is phosphorylated in mitosis by cdk1. The mitotic phosphorylation of Ser321 acts to maintain full activation of cdc25B by disrupting 14-3-3 binding to Ser323 and enhancing the dephosphorylation of Ser323 to block 14-3-3 binding to this site.

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Structural basis of cyclophilin B binding by the calnexin/calreticulin P-domain.

Publication Date: 2010 Aug 27 PMID: 20801878
Authors: Kozlov, G. - Bastos-Aristizabal, S. - Maattanen, P. - Rosenauer, A. - Fenglin, Z. - Killikelly, A. - Thomas, D. Y. - Gehring, K.
Journal: J Biol Chem

Little is known about how chaperones in the endoplasmic reticulum are organized into complexes to assist in the proper folding of secreted proteins. One notable exception is the complex of ERp57 and calnexin that functions as part the calnexin cycle to direct disulfide bond formation in N-glycoproteins. Here, we report three new complexes composed of the peptidyl prolyl cis-trans isomerase cyclophilin B and any of the lectin chaperones: calnexin (CNX), calreticulin (CRT), or calmegin (CMG). The 1.7 A crystal structure of cyclophilin with the proline-rich P-domain of CMG reveals that binding is mediated by the same surface that binds ERp57. We use NMR titrations and mutagenesis to measure low micromolar binding of cyclophilin to all three lectin chaperones and identify essential interfacial residues. The immunosuppressant cyclosporin A did not affect complex formation, confirming the functional independence of the P-domain-binding and proline isomerization sites of cyclophilin. Our results reveal the P-domain functions as a unique protein-protein interaction domain and implicate a peptidyl prolyl isomerase as a new element in the calnexin cycle.

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Aberrant overexpression of the RGL2 ral small GTPase-specific guanine nucleotide exchange factor promotes pancreatic cancer growth through ral-dependent and -independent mechanisms.

Publication Date: 2010 Aug 27 PMID: 20801877
Authors: Vigil, D. - Martin, T. D. - Williams, F. - Yeh, J. J. - Campbell, S. L. - Der, C. J.
Journal: J Biol Chem

Our recent studies established essential and distinct roles for RalA and RalB small GTPase activation in K-Ras mutant pancreatic ductal adenocarcinoma (PDAC) cell line tumorigencity, invasion and metastasis. However, the mechanism of Ral GTPase activation in PDAC has not been determined. There are four highly related mammalian RalGEFs (RalGDS, Rgl1, Rgl2, and Rgl3) that can serve as Ras effectors. Whether or not they share distinct or overlapping functions in K-Ras-mediated growth transformation has not been explored. We found that plasma membrane targeting to mimic persistent Ras activation enhanced the growth transforming activities of RalGEFs. Unexpectedly, transforming activity did not correlate directly with total cell steady-state levels of Ral activation. Next, we observed elevated Rgl2 expression in PDAC tumor tissue and cell lines. Expression of dominant negative Ral, which blocks RalGEF function, as well as interfering RNA suppression of Rgl2, reduced PDAC cell line steady-state Ral activity, growth in soft agar and Matrigel invasion. Surprisingly, the effect of Rgl2 on anchorage-independent growth could not be rescued by constitutively actived RalA, suggesting a novel Ral-independent function for Rgl2 in transformation. Finally, we determined that Rgl2 and RalB both localized to the leading edge, and this localization of RalB was dependent on endogenous Rgl2 expression. In summary, our observations support nonredundant roles for RalGEFs in Ras-mediated oncogenesis and a key role for Rgl2 in Ral activation and Ral-independent PDAC growth.

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Collapsin response mediator protein-2 (Crmp2) regulates trafficking by linking endocytic regulatory proteins to dynein motors.

Publication Date: 2010 Aug 27 PMID: 20801876
Authors: Rahajeng, J. - Giridharan, S. S. - Naslavsky, N. - Caplan, S.
Journal: J Biol Chem

Endocytosis is a conserved cellular process in which nutrients, lipids, and receptors are internalized and transported to early endosomes, where they are sorted and either channeled to degradative pathways or recycled to the plasma membrane. MICAL-L1 and EHD1 are two important endocytic regulatory proteins that control key transport steps within the endocytic system. However, the precise mechanisms by which they mediate transport, and particularly the mode by which they connect to motor proteins, have remained enigmatic. In this study we have identified the collapsin response mediator protein 2 (Crmp2) as an interaction partner of MICAL-L1 in non-neuronal cells. Crmp2 interacts with tubulin dimers and kinesin, and negatively regulates dynein-based transport in neuronal cells, but its expression and function in non-neuronal cells has remained poorly characterized. Upon Crmp2 depletion, we observed dramatic relocalization of internalized transferrin from peripheral vesicles to the endocytic recycling compartment (ERC), similar to the effect of depleting either MICAL-L1 or EHD1. Moreover, transferrin relocalization to the ERC could be inhibited by interfering with microtubule polymerization, consistent with a role for uncoupled motor protein based transport upon depletion of Crmp2, MICAL-L1 or EHD1. Finally, transfection of dynamitin, a component of the dynactin complex whose over-expression inhibits dynein activity, prevented the relocalization of internalized transferrin to the ERC upon depletion of Crmp2, MICAL-L1 or EHD1. These data provide the first trafficking regulatory role for Crmp2 in non-neuronal cells, and support a model in which Crmp2 is an important endocytic regulatory protein that links MICAL-L1/EHD1-based vesicular transport to dynein motors.

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Zyxin-mediated actin assembly is required for efficient wound closure.

Publication Date: 2010 Aug 27 PMID: 20801875
Authors: Nguyen, T. N. - Uemura, A. - Shih, W. - Yamada, S.
Journal: J Biol Chem

Cytoskeletal regulation of cell adhesion is vital to the organization of multi-cellular structures. The focal adhesion protein zyxin emerged as a key regulator of actin assembly since zyxin recruits Ena/VASP proteins to promote actin assembly. Zyxin also localizes to the sites of cell-cell adhesion, and is thought to promote actin assembly with Ena/VASP. Using shRNA targeted to zyxin, we analyzed the roles of zyxin at adhesive contacts. In zyxin deficient cells, the actin assembly at both focal adhesion and cell-cell adhesion was limited, but their migration rate was unchanged. Cell spreading on E-cadherin coated surfaces and the formation of cell clusters was slower for zyxin deficient cells than wildtype cells. By ablating a single cell within a cell monolayer, we quantified the rate of wound closure driven by a contractile circumferential actin ring. Zyxin deficient cells failed to recruit VASP to cell-cell junctions at the wound edge, and had a slower wound closure rate than wildtype cells. Our results suggest that, by recruiting VASP, zyxin regulates actin assembly at the sites of force-bearing cell-cell adhesion.

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Proline isomerase PIN1 represses terminal differentiation and myocyte enhancer factor 2C function in skeletal muscle cells.

Publication Date: 2010 Aug 27 PMID: 20801874
Authors: Magli, A. - Angelelli, C. - Ganassi, M. - Baruffaldi, F. - Matafora, V. - Battini, R. - Bachi, A. - Messina, G. - Rustighi, A. - Del Sal, G. - Ferrari, S. - Molinari, S.
Journal: J Biol Chem

Reversible proline-directed phosphorylation at Ser/Thr-Pro motifs has an essential role in myogenesis, a multistep process strictly regulated by several signalling pathways that impinge on two families of myogenic effectors, the basic Helix Loop Helix (bHLH) myogenic transcription factors and the Myocyte Enhancer Factor 2 (MEF2) proteins. The question of how these signals are deciphered by the myogenic effectors remains largely undefined. In this study we show that the peptidyl-prolyl isomerase Pin1, which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds to induce conformational changes of its target proteins, acts as an inhibitor of muscle differentiation as its knock-down in myoblasts promotes myotube formation. With the aim of clarifying the mechanism of Pin1 function in skeletal myogenesis, we investigated whether MEF2C, a critical regulator of the myogenic program that is the endpoint of several signalling pathways, might serve as a/the target for the inhibitory effects of Pin1 on muscle differentiation. We show that Pin1 interacts selectively with phosphorylated MEF2C in skeletal muscle cells, both in vitro and in vivo. The interaction with Pin1 requires two novel critical pSer/Thr-Pro motifs in MEF2C, Ser98 and Ser110, which are phosphorylated in vivo. Overexpression of Pin1 decreases MEF2C stability and activity, and its ability to cooperate with MyoD to activate myogenic conversion. Collectively these findings reveal a novel role for Pin1 as a regulator of muscle terminal differentiation and suggest that Pin1 mediated repression of MEF2C function could contribute to this function.

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Prolyl Hydroxylase Domain (PHD)2 affects cell migration and F-actin formation via RhoA/ROCK-dependent cofilin phosphorylation.

Publication Date: 2010 Aug 27 PMID: 20801873
Authors: Vogel, S. - Wottawa, M. - Farhat, K. - Zieseniss, A. - Schnelle, M. - Le-Huu, S. - von Ahlen, M. - Malz, C. - Camenisch, G. - Katschinski, D. M.
Journal: J Biol Chem

Cells are responding to hypoxia via prolyl-4-hydroxylase domain (PHD) enzymes, which are responsible for oxygen-dependent hydroxylation of the hypoxia-inducible factor (HIF)-1alpha subunit. To further gain insight into PHD function, we generated knock down cell models for the PHD2 isoform, which is the main isoform regulating HIF-1alpha hydroxylation and thus stability in normoxia. Induction of a PHD2 knock down in Tetracycline-inducible HeLa PHD2 knock down cells resulted in increased F-actin formation as detected by phalloidin staining. A similar effect could be observed in the stably transfected PHD2 knock down cell clones 1B6 and 3B7. F-actin is at least in part responsible for shaping cell morphology as well as regulating cell migration. In line, cell migration was significantly impaired as a consequence of PHD2 knock down in a scratch assay. Mechanistically, PHD2 knock down resulted in activation of the Rho A/Rho associated kinase pathway with subsequent phosphorylation of cofilin. Since cofilin phosphorylation impairs it;s actin severing function, this may explain the F-actin phenotype thereby providing a functional link between PHD2-dependent signalling and cell motility.

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Heart-specific small subunit of myosin light chain phosphatase activates Rho-associated kinase and regulates phosphorylation of myosin phosphatase target subunit 1.

Publication Date: 2010 Aug 27 PMID: 20801872
Authors: Shichi, D. - Arimura, T. - Ishikawa, T. - Kimura, A.
Journal: J Biol Chem

Phosphorylation of myosin regulatory light chain (MLC) plays a regulatory role in muscle contraction and the level of MLC phosphorylation is balanced by MLC kinase and MLC phosphatase (MLCP). MLCP consists of a catalytic subunit, a large subunit (MYPT1 or MYPT2) and a small subunit. MLCP activity is regulated by phosphorylation of MYPTs, while the role of small subunit in the regulation remains unknown. We previously characterized a human heart-specific small subunit (hHS-M21) which increased the sensitivity to Ca2+ in muscle contraction. In this study, we investigated the role of hHS-M21 in the regulation of MLCP phosphorylation. Two isoforms of hHS-M21, hHS-M21A and hHS-M21B, preferentially bound C-terminal one-third regions of MYPT1 and MYPT2, respectively. Amino acid substitutions at a phosphorylation site of MYPT1, Ser852, impaired the binding of MYPT1 and hHS-M21. The hHS-M21 increased the phosphorylation level of MYPT1 at Thr696, which was attenuated by Rho-associated kinase (ROCK) inhibitors and small interfering RNAs for ROCK. In addition, hHS-M21 bound ROCK and enhanced the ROCK activity. These findings suggest that hHS-M21 is a heart-specific effecter of ROCK and plays a regulatory role in the MYPT1 phosphorylation at Thr696 by ROCK.

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Human alveolar type II cells secrete and absorb liquid in response to local nucleotide signaling.

Publication Date: 2010 Aug 27 PMID: 20801871
Authors: Bove, P. F. - Grubb, B. R. - Okada, S. F. - Ribeiro, C. M. - Rogers, T. D. - Randell, S. H. - O'Neal, W. K. - Boucher, R. C.
Journal: J Biol Chem

A balance sheet describing the integrated homeostasis of secretion, absorption, and surface movement of liquids on pulmonary surfaces has remained elusive. It remains unclear whether the alveolus exhibits an intra-alveolar ion/liquid transport physiology or whether it secretes ions/liquid that may communicate with airway surfaces. Studies employing isolated human AT2 cells were utilized to investigate this question. Human AT2 cells exhibited both epithelial Na+ channel (ENaC)-mediated Na+ absorption and cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- secretion, both significantly regulated by extracellular nucleotides. In addition, we observed in normal AT2 cells an absence of CFTR regulation of ENaC activity and an absence of expression/activity of reported calcium-activated chloride channels (TMEM16A, Bestrophin-1, ClC2, and SLC26A9), both features strikingly different from normal airway epithelial cells. Measurements of alveolar surface liquid (AvSL) volume revealed that normal AT2 cells: 1) achieved an extracellular nucleotide concentration-dependent steady-state alveolar surface liquid (AvSL) height of approximately 4 um in vitro; 2) absorbed liquid when the lumen was flooded; and 3) secreted liquid when treated with uridine 5 triphosphate (UTP), forskolin, or subjected to cyclic compressive stresses mimicking tidal breathing. Collectively, our studies suggest that human AT2 cells in vitro have the capacity to absorb or secrete liquid in response to local alveolar conditions.

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Mating pheromone in Cryptococcus neoformans is regulated by a transcriptional/degradative "futile" cycle.

Publication Date: 2010 Aug 27 PMID: 20801870
Authors: Park, Y. D. - Panepinto, J. - Shin, S. - Larsen, P. - Giles, S. - Williamson, P. R.
Journal: J Biol Chem

Sexual reproduction in fungi requires induction of signaling pheromones within environments that are conducive to mating. The fungus Cryptococcus neoformans is currently the fourth major cause of infectious death in regions of Africa and undergoes mating in phyto-nutrient rich environments to create spores with infectious potential. Here we show that under conditions where mating is inhibited, a ~ 17 fold excess of MFalpha pheromone transcript is synthesized and then degraded by a DEAD box protein Vad1, resulting in low steady state transcript levels. Transfer to mating media or deletion of the VAD1 gene resulted in high level accumulation of MFalpha transcripts and enhanced mating, acting in concert with the mating-related HOG1 pathway. We then investigated whether the high metabolic cost of this apparently futile transcriptional cycle could be justified by a more rapid induction of mating. Maintenance of Vad1 activity on inductive mating media by constitutive expression resulted in repressed levels of MFalpha that did not prevent, but rather prolonged the time to successful mating from 5-6 hours to 15 hours (P < 0.0001). In sum, these data suggest that VAD1 negatively regulates mating via degradation of constitutive high levels of MFalpha transcripts in a synthetic/degradative cycle, providing a mechanism of mRNA induction for time-critical cellular events such as mating induction.

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Discovery and characterization of DNA excision repair pathways: the work of Philip Courtland Hanawalt.

Publication Date: 2010 Aug 20 PMID: 20740724
Authors: Kresge, N. - Simoni, R. D. - Hill, R. L.
Journal: J Biol Chem

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Involvement of 9-O-acetyl GD3 Ganglioside in Mycobacterium Leprae Infection of Schwann Cells.

Publication Date: 2010 Aug 25 PMID: 20739294
Authors: Ribeiro-Resende, V. T. - Ribeiro-Guimaraes, M. L. - Lemes, R. M. - Nascimento, I. C. - Alves, L. - Mendez-Otero, R. - Pessolani, M. C. - Lara, F. A.
Journal: J Biol Chem

Mycobacterium leprae (ML), the etiologic agent of leprosy, mainly affects the skin and peripheral nerves, leading to demyelization and loss of axonal conductance. Schwann cells (SC) are the main cell population infected by ML in the nerves, and infection triggers changes in the SC phenotype from a myelinated to a non-myelinated state. In the present study, we show that expression of 9-O-acetyl GD3, a ganglioside involved in cellular anti-apoptotic signaling and nerve regeneration, increases in SCs following infection with ML. Observation by confocal microscopy together with co-immunoprecipitation suggested that this ganglioside participates in ML attachment and internalization by SC. Immunoblockage of 9-O-acetyl GD3 in vitro significantly reduced adhesion of ML to SC surfaces. Finally, we show that activation of the MAP Kinase (ErK1/2) pathway and SC proliferation, two known effects of ML on SCs that result in demyelization, are significantly reduced when the 9-O-acetyl GD3 ganglioside is immunoblocked. Taken together, these data suggest the involvement of 9-O-acetyl GD3 in ML infection on SCs.

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Expanding role of the Jumonji C domain as an RNA hydroxylase.

Publication Date: 2010 Aug 25 PMID: 20739293
Authors: Noma, A. - Ishitani, R. - Kato, M. - Nagao, A. - Nureki, O. - Suzuki, T.
Journal: J Biol Chem

JmjC (Jumonji C) domain-containing proteins are known to be an extensive family of Fe(II)/2-oxoglutarate -dependent oxygenases involved in epigenetic regulation of gene expression by catalyzing oxidative demethylation of methylated histones. We report here that a human JmjC protein named TYW5 unexpectedly acts in biosynthesis of a hypermodified nucleoside, hydroxywybutosine (OHyW), in tRNAPhe by catalyzing hydroxylation. The finding provides an insight into expanding role of JmjC protein as an RNA hydroxylase.

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Apolipoprotein A-I deficiency increases cerebral amyloid angiopathy and cognitive deficits in APP/PS1{Delta}E9 mice.

Publication Date: 2010 Aug 25 PMID: 20739292
Authors: Lefterov, I. - Fitz, N. F. - Cronican, A. A. - Fogg, A. - Lefterov, P. - Kodali, R. - Wetzel, R. - Koldamova, R.
Journal: J Biol Chem

A hallmark of Alzheimer's disease (AD) is the deposition of Amyloid beta (Abeta) in brain parenchyma and cerebral blood vessels accompanied by cognitive decline. Previously, we showed that human Apolipoprotein A-I (ApoA-I) decreases Abeta40 aggregation and toxicity. Here we demonstrate that ApoA-I in lipidated or non-lipidated form prevents the formation of high molecular weight aggregates of Abeta42 and decreases Abeta42 toxicity in primary brain cells. To determine the effects of ApoA-I on AD phenotype in vivo, we crossed APP/PS1DeltaE9 to ApoA-I*ko mice. Using Morris Water Maze we demonstrate that the deletion of mouse Apoa-I exacerbates memory deficits in APP/PS1DeltaE9 mice. Further characterization of APP/PS1DeltaE9/ApoA-I*ko mice showed that ApoA-I deficiency did not affect APP processing , soluble Abeta oligomer levels, Abeta plaque load, or levels of insoluble Abeta in brain parenchyma. To examine the effect of Apoa-I deletion on cerebral amyloid angiopathy (CAA) we measured insoluble Abeta isolated from cerebral blood vessels. Our data show that in APP/PS1DeltaE9/ApoA-I*ko mice insoluble Abeta40 is increased more than 10-fold and Abeta42 - 1.5-fold. The increased levels of deposited amyloid in the vessels of cortices and hippocampi of APP/PS1DeltaE9/ApoA-I*ko mice, measured by X-34 staining, confirmed the results. Finally, we demonstrate that lipidated and non-lipidated ApoA-I significantly decreased Abeta toxicity against brain vascular smooth muscle cells. We conclude that lack of ApoA-I aggravates the memory deficits in APP/PS1DeltaE9 mice in parallel to significantly increased CAA.

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ATP-binding cassette transporter A1 mediates the beneficial effects of the liver-X-receptor agonist GW3965 on object recognition memory and amyloid burden in APP/PS1 mice.

Publication Date: 2010 Aug 30 PMID: 20739291
Authors: Donkin, J. J. - Stukas, S. - Hirsch-Reinshagen, V. - Namjoshi, D. - Wilkinson, A. - May, S. - Chan, J. - Fan, J. - Collins, J. - Wellington, C. L.
Journal: J Biol Chem

The cholesterol transpoter ATP-binding cassette transporter A1 (ABCA1) moves lipids onto apolipoproteins including apolipoprotein E (apoE), which is the major cholesterol carrier in brain and an established genetic risk factor for late-onset Alzheimer&'s Disease (AD). In amyloid mouse models of AD, ABCA1 deficiency exacerbates amyloidogenesis, whereas ABCA1 overexpression ameliorates amyloid load, suggesting a role for ABCA1 in Abeta metabolism. Agonists of Liver X Receptors (LXR), including GW3965, induce transcription of several genes including ABCA1 and apoE, reduce Abeta levels and improve cognition in AD mice. However, the specific role of ABCA1 in mediating beneficial responses to LXR agonists in AD mice is unknown. We evaluated behavioral and neuropathogical outcomes in GW3965-treated female APP/PS1 mice with and without ABCA1. Treatment of APP/PS1 mice with GW3965 increased ABCA1 and apoE protein levels. ABCA1 was required to observe significantly elevated apoE levels in brain tissue and CSF upon therapeutic (33mg/kg/d) GW3965 treatment. At 33 mg/kg/d, GW3965 was also associated with a trend toward redistribution of Abeta to the carbonate-soluble pool independent of ABCA1. APP/PS1 mice treated with either 2.5 mg/kg/d or 33 mg/kg/d of GW3965 showed a clear trend toward reduced amyloid burden in hippocampus and whole brain, whereas treated APP/PS1 mice lacking ABCA1 failed to display reduced amyloid load in whole brain and showed trends toward increased hippocampal amyloid. Treatment of APP/PS1 mice with either dose of GW3965 completely restored novel object recognition (NOR) memory to wild-type levels, which required ABCA1. These results suggest that ABCA1 contributes to several beneficial effects of the LXR agonist GW3965 in APP/PS1 mice.

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Kv7-type channel currents in spiral ganglia neurons: involvement in sensorineural hearing loss.

Publication Date: 2010 Aug 25 PMID: 20739290
Authors: Lv, P. - Wei, D. - Yamoah, E. N.
Journal: J Biol Chem

Alterations in Kv7-mediated currents in excitable cells result in several diseased conditions. A case in DFNA2, an autosomal dominant version of progressive hearing loss, involves degeneration of hair cells and spiral ganglia neurons (SGNs) from basal to apical cochlea, manifesting as high-to-low frequency hearing loss and has been ascribed to mutations in Kv7.4 channels. Analyses of the cellular mechanisms of Kv7.4-mutations and progressive degeneration of SGNs have been hampered by paucity of functional data on the role Kv7 channels play in young and adult neurons. To understand the cellular mechanisms of the disease in SGNs, we examined temporal (young, 0.5 months old and senescent, 17 months old) and spatial (apical and basal) roles of Kv7-mediated currents. We report that differential contribution of Kv7 currents in mice SGNs results in distinct and profound variations of the membrane properties of basal versus apical neurons. The current produces a major impact on the resting membrane potential of basal neurons. Inhibition of the current promotes membrane depolarization, resulting in activation of Ca2+ currents and a sustained rise in intracellular Ca2+. Using TUNEL assay, we demonstrate that a sustained increase in intracellular Ca2+ mediated by inhibition of Kv7 current results in significant SGN apoptotic death. Thus, this report provides evidence of the cellular etiology and mechanisms of SGN degeneration in DFNA2.

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Pseudomonas aeruginosa homoserine lactone activates store-operated cAMP and CFTR-dependent Cl secretion by human airway epithelia.

Publication Date: 2010 Aug 26 PMID: 20739289
Authors: Schwarzer, C. - Wong, S. - Shi, J. - Matthes, E. - Illek, B. - Ianowski, J. P. - Arant, R. J. - Isacoff, E. - Vais, H. - Foskett, J. K. - Maiellaro, I. - Hofer, A. M. - Machen, T.
Journal: J Biol Chem

The ubiquitous bacterium Pseudomonas aeruginosa frequently causes hospital-acquired infections. P. aeruginosa also infect lungs of cystic fibrosis (CF) patients and secrete N-(3-oxo-dodecanoyl)-S-homoserine lactone (3O-C12) to regulate bacterial gene expression critical for P. aeruginosa persistence. In addition to its effects as a quorum-sensing gene regulator in P. aeruginosa, 3O-C12 elicits cross-kingdom effects on host cell signaling leading to both pro- or anti-inflammatory effects. We find that in addition to these slow effects mediated through changes in gene expression, 3O-C12 also rapidly increases Cl(-) and fluid secretion in CFTR-expressing airway epithelia. 3O-C12 does not stimulate Cl(-) secretion in CF cells, suggesting that the lactone activates the CFTR. 3O-C12 also appears to directly activate the inositol trisphosphate receptor 1 and release Ca(2+) from the endoplasmic reticulum (ER), lowering [Ca(2+)] in the ER and thereby activating the Ca(2+)-sensitive ER signaling protein STIM1. 3O-C12 increases cytosolic [Ca(2+)] and, strikingly, also cytosolic [cAMP], the known activator of CFTR. Activation of Cl(-) current by 3O-C12 was inhibited by a cAMP antagonist and increased by a phosphodiesterase inhibitor. Finally, a Ca(2+) buffer that lowers [Ca(2+)] in the ER similar to the effect of 3O-C12 also increased cAMP and I(Cl). The results suggest that 3O-C12 stimulates CFTR-dependent Cl(-) and fluid secretion in airway epithelial cells by activating the IP(3)R, thus, lowering [Ca(2+)] in the ER and activating STIM1 and store-operated cAMP production. In CF airways, where CFTR is absent, the adaptive ability to rapidly flush the bacteria away is compromised because the lactone cannot affect Cl(-) and fluid secretion.

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Flap endonuclease 1 mechanism analysis indicates flap base binding prior to threading.

Publication Date: 2010 Aug 25 PMID: 20739288
Authors: Gloor, J. W. - Balakrishnan, L. - Bambara, R. A.
Journal: J Biol Chem

FEN1 cleaves 5 flaps at their base to create a nicked product for ligation. FEN1 has been reported to enter the flap from the 5 end and track to the base. Current binding analyses support a very different mechanism of interaction with the flap substrate. Measurements of FEN1 binding to a flap substrate show that the nuclease binds with similar high affinity to the base of a long flap even when the 5 end is blocked with biotin/streptavidin. However, FEN1 bound to a blocked flap is more sensitive to sequestration by a competing substrate. These results are consistent with a substrate interaction mechanism in which FEN1 first binds the flap base and then threads the flap through an opening in the protein from the 5 end to the base for cleavage. Significantly, when the unblocked flap length is reduced from five to two nucleotides, FEN1 can be sequestered from the substrate to a similar extent as a blocked, long flap substrate. Apparently, interactions related to threading occur only when the flap is greater than two to four nucleotides long, implying that short flaps are cleaved without a threading requirement.

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Integrin beta3 phosphorylation dictates its complex with Shc PTB domain.

Publication Date: 2010 Aug 25 PMID: 20739287
Authors: Deshmukh, L. - Gorbatyuk, V. - Vinogradova, O.
Journal: J Biol Chem

Adaptor protein Shc plays a key role in mitogen-activated protein kinase (MAPK) signaling pathway, which can be mediated through a number of different receptors including integrins. By specifically recognizing the tyrosine phosphorylated integrin beta3, Shc has been shown to trigger integrin outside-in signaling, although the structural basis of this interaction remains nebulous. Here we present the detailed structural analysis of Shc Phosphotyrosine Binding (PTB) domain in complex with the bi-phosphorylated beta3 integrin cytoplasmic tail (CT). We show that this complex is primarily defined by the phosphorylation state of the integrin C-terminal 759Y, which fits neatly into the classical PTB pocket of Shc. In addition, we have identified a novel binding interface which concurrently accommodates phosphorylated 747Y of the highly conserved NPxY motif of beta3. The structure represents the first snapshot of an integrin cytoplasmic tail bound to a target for mediating the outside-in signaling. Detailed comparison with the known Shc PTB structure bound to a target TrkA peptide revealed some significant differences, which shed new light upon the PTB domains' specificity.

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ALLOSTERY IS AN INTRISIC PROPERTY OF THE PROTEASE DOMAIN OF DegS: IMPLICATIONS FOR ENZYME FUNCTION AND EVOLUTION.

Publication Date: 2010 Aug 24 PMID: 20739286
Authors: Sohn, J. - Grant, R. A. - Sauer, R. T.
Journal: J Biol Chem

DegS is a periplasmic E. coli protease, which functions as a trimer to catalyze the initial rate-limiting step in a proteolytic cascade that ultimately activates transcription of stress-response genes in the cytoplasm. Each DegS subunit consists of a protease domain and a PDZ domain. During protein-folding stress, DegS is allosterically activated by peptides exposed in misfolded outer-membrane porins (OMPs), which bind to the PDZ domain and stabilize the active protease. It is not known if allostery is conferred by the PDZ domains or is an intrinsic feature of the trimeric protease domain. Here, we demonstrate that free DegSPDZ equilibrates between active and inactive trimers, with the latter species predominating. Substrate binding stabilizes active DegSPDZ in a positively cooperative fashion. Mutations can also stabilize active DegSPDZ and produce an enzyme that displays hyperbolic kinetics and degrades substrate with a maximal velocity within error of that for fully activated, intact DegS. Crystal structures of multiple DegSPDZ variants, in functional and non-functional conformations, support a two-state model in which allosteric switching is mediated by changes in specific elements of tertiary structure in the context of an invariant trimeric base. Overall, our results indicate that protein substrates must bind sufficiently tightly and specifically to the functional conformation of DegSPDZ to assist their own degradation. Thus, substrate binding alone may have regulated the activities of ancestral DegS trimers, with subsequent fusion of the protease domain to a PDZ domain resulting in ligand-mediated regulation.

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The structure of RPN10 and its interactions with polyubiquitin chains and the proteasome subunit RPN12.

Publication Date: 2010 Aug 24 PMID: 20739285
Authors: Riedinger, C. - Boehringer, J. - Trempe, J. F. - Lowe, E. D. - Brown, N. R. - Gehring, K. - Noble, M. E. - Gordon, C. - Endicott, J. A.
Journal: J Biol Chem

Schizosaccharomyces pombe Rpn10 (SpRpn10) is a proteasomal ubiquitin (Ub) receptor located within the 19S regulatory particle where it binds to subunits of both the base and lid sub-particles. We have solved the structure of full-length SpRpn10 by determining the crystal structure of the VWA domain and characterising the full-length protein by NMR. We demonstrate that the single Ub interacting motif (UIM) of SpRpn10 forms a 1:1 complex with K48-linked diUb, which it binds selectively over monoUb and K63-linked diUb. We further show that the SpRpn10 UIM binds to SpRpn12, a subunit of the lid sub-particle, with an affinity comparable to Lys48-linked diUb. This is the first observation of a UIM binding other than a Ub fold and suggests that SpRpn12 could modulate the activity of SpRpn10 as a proteasomal Ub receptor.

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Crystal Structures of phosphoketolase: thiamine diphosphate-dependent dehydration mechanism.

Publication Date: 2010 Aug 24 PMID: 20739284
Authors: Suzuki, R. - Katayama, T. - Kim, B. J. - Wakagi, T. - Shoun, H. - Ashida, H. - Yamamoto, K. - Fushinobu, S.
Journal: J Biol Chem

Thiamine diphosphate (ThDP)-dependent enzymes are ubiquitously present in all organisms and catalyze essential reactions in various metabolic pathways. ThDP-dependent phosphoketolase plays key roles in the central metabolism of heterofermentative bacteria and in the pentose catabolism of various microbes. In particular, bifidobacteria, representatives of beneficial commensal bacteria, have an effective glycolytic pathway called bifid shunt, in which 2.5 moles of ATP are produced per glucose. Phosphoketolase catalyzes two steps in the bifid shunt because of its dual-substrate specificity: phosphorolytic cleavage of fructose-6-phosphate or xylulose-5-phosphate to produce aldose phosphate, acetyl phosphate, and H(2)O. The phosphoketolase reaction is different from other well-studied ThDP-dependent enzymes because it involves a dehydration step. Although phosphoketolase was discovered more than fifty years ago, its three-dimensional structure remains unclear. In this study, we report the crystal structures of xylulose-5-phosphate/fructose-6-phosphate phosphoketolase from Bifidobacterium breve. The structures of the two intermediates before and after dehydration (alpha,beta-dihydroxyethyl ThDP and 2-acetyl-ThDP) and complex with inorganic phosphate give an insight into the mechanism of each step of the enzymatic reaction.

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Implications for collagen binding from the crystallographic structure of fibronectin 6FnI1-2FnII7FnI.

Publication Date: 2010 Aug 24 PMID: 20739283
Authors: Erat, M. C. - Schwarz-Linek, U. - Pickford, A. R. - Farndale, R. W. - Campbell, I. D. - Vakonakis, I.
Journal: J Biol Chem

Collagen and fibronectin (FN) are two abundant and essential components of the vertebrate extracellular matrix; they interact directly with cellular receptors and affect cell adhesion and migration. Past studies identified a FN fragment comprising six modules, (6)FnI(1-2)FnII(7-9)FnI, and termed the gelatin binding domain (GBD) as responsible for collagen interaction. Recently, we showed that GBD binds tightly to a specific site within type I collagen and determined the structure of domains (8-9)FnI in complex with a peptide from that site. Here, we present the crystallographic structure of domains (6)FnI(1-2)FnII(7)FnI, which form a compact, globular unit through interdomain interactions. Analysis of NMR titrations with single-stranded collagen peptides reveals a dominant collagen interaction surface on domains (2)FnII and (7)FnI; a similar surface appears involved in interactions with triple-helical peptides. Models of the complete GBD, based on the new structure and the (8-9)FnI-collagen complex show a continuous putative collagen binding surface. We explore the implications of this model using long collagen peptides and discuss our findings in the context of FN interactions with collagen fibrils.

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Identification of protein-protein and protein-ribosome interacting regions of the C-terminal tail of human mitochondrial inner membrane protein Oxa1L.

Publication Date: 2010 Aug 25 PMID: 20739282
Authors: Haque, M. E. - Spremulli, L. L. - Fecko, C. J.
Journal: J Biol Chem

The mammalian mitochondrial inner membrane protein Oxa1L is involved in the insertion of a number of mitochondrial translation products into the inner membrane. During this process, the C-terminal tail of Oxa1L (Oxa1L-CTT) binds mitochondrial ribosomes and is believed to coordinate the synthesis and membrane insertion of the nascent chains into the membrane. The C-terminal tail of Oxa1L does not contain any Cys residues. Four variants of this protein have been prepared having a specifically placed Cys residue at position 4, 39, 67 or 94 of the Oxa1L-CTT. These Cys residues have been derivatized with a fluorescent probe, rhodamine maleimide for biophysical studies. Oxa1L-CTT forms oligomers cooperatively with a binding constant in the sub-muM range. Fluorescence anisotropy and fluorescence lifetime measurements indicate that contacts near a long helix close to position 39 of Oxa1L-CTT occur during oligomer formation. Fluorescence correlation spectroscopy measurements demonstrate that all the Oxa1L-CTT derivatives bind to mammalian mitochondrial ribosomes. Steady state fluorescence quenching and fluorescent lifetime data indicate that there are extensive contacts between Oxa1L-CTT and the ribosome encompassing regions around positions 39, 67 and 94. The present study suggests that Oxa1L-CTT undergoes conformational changes and induced oligomer formation when it binds to the ribosome.

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In vitro and in vivo analysis of the binding of the C-terminus of the HDL Receptor SR-BI to the PDZ1 domain of its adaptor protein PDZK1.

Publication Date: 2010 Aug 25 PMID: 20739281
Authors: Kocher, O. - Birrane, G. - Tsukamoto, K. - Fenske, S. - Yesilaltay, A. - Pal, R. - Daniels, K. - Ladias, J. A. - Krieger, M.
Journal: J Biol Chem

The PDZ1 domain of the four PDZ domain-containing protein PDZK1 has been reported to bind the C-terminus of the HDL receptor SR-BI and to control hepatic SR-BI expression and function. We generated wild-type (WT) and mutant murine PDZ1 domains, the mutants bearing single amino acid substitutions in their carboxylate binding loop (14-KQEGQNYGFFL-24) and measured their binding affinity for a 7-residue peptide corresponding to the C-terminus of SR-BI (503-VLQEAKL-509). The 20Tyr-->Ala and 21Gly-->Tyr substitutions abrogated all binding activity. Surprisingly, binding affinities (Kd) of the 14Lys-->Ala and 22Phe-->Ala mutants were 3.2 and 4.0 muM, respectively, similar to 2.6 muM measured for the WT PDZ1. To understand these findings, we determined the high resolution structure of WT PDZ1 bound to a 5-residue sequence from SR-BI C-terminus (505-QEAKL-509) using X-ray crystallography. In addition, we incorporated the 14Lys-->Ala and 20Tyr-->Ala substitutions into full-length PDZK1 liver-specific transgenes, and expressed them in WT and PDZK1 knockout (KO) mice. In WT mice, the transgenes did not alter endogenous hepatic SR-BI protein expression (intracellular distribution or amount) or lipoprotein metabolism (total plasma cholesterol, lipoprotein size distribution). In PDZK1 KO mice, as expected, the 14Lys-->Ala mutant behaved like wild-type PDZK1 and completely corrected their hepatic SR-BI and plasma lipoprotein abnormalities. Unexpectedly, the 10 to 20-fold overexpressed 20Tyr-->Ala mutant also substantially, but not completely, corrected these abnormalities. The results suggest that there may be an additional site(s) within PDZK1 that bind(s) SR-BI and mediate(s) productive SR-BI-PDZK1 interaction previously attributed exclusively to the canonical binding of SR-BI C-terminus to PDZ1.

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MUTATIONAL INSIGHTS INTO THE ROLES OF AMINO ACID RESIDUES IN LIGAND BINDING FOR TWO CLOSELY RELATED FAMILY 16 CARBOHYDRATE BINDING MODULES.

Publication Date: 2010 Aug 25 PMID: 20739280
Authors: Su, X. - Agarwal, V. - Dodd, D. - Bae, B. - Mackie, R. I. - Nair, S. K. - Cann, I. K.
Journal: J Biol Chem

Carbohydrate binding modules (CBMs) are specialized proteins that bind to polysaccharides and oligosaccharides. Caldanaerobius polysaccharolyticus Man5ACBM16-1/CBM16-2 bind to glucose-, mannose-, and glucose/mannose-configured substrates. The crystal structures of the two proteins represent the only examples in CBM family 16, and studies that evaluate the roles of amino acid residues in ligand binding in this family are lacking. In this study, we probed the roles of amino acids (selected based on CBM16-1/ligand co-crystal structures) on substrate binding. Two tryptophan (W20 and W125) and two glutamine (Q81 and Q93) residues are shown to be critical in ligand binding. Additionally, several polar residues that flank the critical residues also contribute to ligand binding. The CBM16-1 Q121E mutation increased affinity for all substrates tested, while the Q21G and N97R mutants exhibited decreased substrate affinity. We solved CBM/substrate co-crystal structures to elucidate the molecular basis of the increased substrate binding by CBM16-1 Q121E. The Q121, Q21, and N97 residues can be manipulated to fine tune ligand binding by the Man5A CBMs. Surprisingly, none of the eight residues investigated was absolutely conserved in CBM family 16. Thus, the critical residues in the Man5A CBMs are either not essential for substrate binding in the other members of this family, or the two CBMs are evolutionarily distinct from the members available in the current protein database. Man5A is dependent on its CBMs for robust activity, and insights from this study should serve to enhance our understanding of the interdependence of its catalytic and substrate binding modules.

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N-glycosylation at the SynCAM immunoglobulin interface modulates synaptic adhesion.

Publication Date: 2010 Aug 25 PMID: 20739279
Authors: Fogel, A. I. - Li, Y. - Giza, J. - Wang, Q. - Lam, T. T. - Modis, Y. - Biederer, T.
Journal: J Biol Chem

Select adhesion molecules connect pre- and postsynaptic membranes and organize developing synapses. The regulation of these trans-synaptic interactions is an important neurobiological question. We have previously shown that the synaptic cell adhesion molecules SynCAM 1 and 2 engage in homo- and heterophilic interactions and bridge the synaptic cleft to induce presynaptic terminals. Here, we demonstrate that site-specific N-glycosylation impacts the structure and function of adhesive SynCAM interactions. Through crystallographic analysis of SynCAM 2, we identified within the adhesive interface of its Ig1 domain an N-glycan on residue N60. Structural modeling of the corresponding SynCAM 1 Ig1 domain indicates that its glycosylation sites N70/N104 flank the binding interface of this domain. Mass-spectrometric and mutational studies confirm and characterize the modification of these three sites. These site-specific N-glycans affect SynCAM adhesion, yet act in a differential manner. While glycosylation of SynCAM 2 at N60 reduces adhesion, N-glycans at N70/N104 of SynCAM 1 increase its interactions. The modification of SynCAM 1 with sialic acids contributes to the glycan-dependent strengthening of its binding. Functionally, N-glycosylation promotes the trans-synaptic interactions of SynCAM 1 and is required for synapse induction. These results demonstrate that N-glycosylation of SynCAM proteins differentially affects their binding interface and implicate post-translational modification as a mechanism to regulate trans-synaptic adhesion.

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Crystal structure of an exo-1,5-{alpha}-L-arabinofuranosidase from Streptomyces avermitilis provides insights into the mechanism of substrate discrimination between exo- and endo-type enzymes in glycoside hydrolase family 43.

Publication Date: 2010 Aug 25 PMID: 20739278
Authors: Fujimoto, Z. - Ichinose, H. - Maehara, T. - Honda, M. - Kitaoka, M. - Kaneko, S.
Journal: J Biol Chem

Exo-1,5-alpha-L-arabinofuranosidases belonging to glycoside hydrolase family 43 have strict substrate specificity. These enzymes hydrolyze only the alpha-1,5-linkages of linear arabinan and arabino-oligosaccharides in an exo-acting manner. The enzyme from Streptomyces avermitilis contains a core catalytic domain belonging to glycoside hydrolase family 43 and a C-terminal arabinan-binding module belonging to carbohydrate-binding module family 42. We determined the crystal structure of intact exo-1,5-alpha-L-arabinofuranosidase. The catalytic module is composed of a 5-bladed beta-propeller topologically identical to the other family 43 enzymes. The arabinan-binding module had 3 similar subdomains assembled against one another around a pseudo-3-fold axis, forming a beta-trefoil fold. A sugar complex structure with alpha-1,5-L-arabinofuranotriose revealed 3 subsites in the catalytic domain, and a sugar complex structure with alpha-L-arabinofuranosyl azide revealed 3 arabinose binding sites in the carbohydrate-binding module. Mutagenesis study revealed that substrate specificity was regulated by residues Asn159, Tyr192, and Leu289 located at the aglycon side of the substrate-binding pocket. The exo-acting manner of the enzyme was attributed to the strict pocket structure of subsite -1, formed by the flexible loop region Tyr281-Arg294 and the side chain of Tyr40, which occupied the positions corresponding to the catalytic glycon cleft of GH43 endo-acting enzymes.

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Novel mechanisms in the regulation of G protein-coupled receptor trafficking to the plasma membrane.

Publication Date: 2010 Aug 25 PMID: 20739277
Authors: Tholanikunnel, B. G. - Joseph, K. - Kandasamy, K. - Baldys, A. - Raymond, J. R. - Luttrell, L. M. - McDermott, P. J. - Fernandes, D. J.
Journal: J Biol Chem

Beta(2) adrenergic receptors (beta(2)-AR) are low-abundance, integral membrane proteins that mediate the effects of catecholamines at the cell surface. While the processes governing desensitization of activated beta(2)-ARs and their subsequent removal from the cell surface have been characterized in considerable detail little is known about the mechanisms controlling trafficking of neo-synthesized receptors to the cell surface. Since the discovery of the signal peptide, the targeting of the integral membrane proteins to plasma membrane has been thought to be determined by structural features of the amino acid sequence alone. Here we report that localization of translationally silenced beta(2)-AR mRNA to the peripheral cytoplasmic regions is critical for receptor localization to the plasma membrane. beta(2)-AR mRNA is recognized by the nucleocytoplasmic shuttling RNA-binding protein HuR, which silences translational initiation while chaperoning the mRNA-protein complex to the cell periphery. When HuR expression is down regulated, beta(2)-AR mRNA translation is initiated prematurely in perinuclear polyribosomes, leading to overproduction of receptors but defective trafficking to the plasma membrane. Our results underscore the importance of the spatiotemporal relationship between beta(2)-AR mRNA localization, translation and trafficking to the plasma membrane, and establish a novel mechanism whereby G protein-coupled receptor (GPCR) responsiveness is regulated by RNA-based signals.

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Nucleosome interactions and stability in an ordered nucleosome array model system.

Publication Date: 2010 Aug 25 PMID: 20739276
Authors: Blacketer, M. J. - Feely, S. J. - Shogren-Knaak, M. A.
Journal: J Biol Chem

While it is well established that the majority of eukaryotic DNA is sequestered as nucleosomes, the higher-order structure resulting from nucleosome interactions, as well as the dynamics of nucleosome stability, are not as well understood. To characterize the structural and functional contribution of individual nucleosomal sites, we have developed a chromatin model system containing up to four nucleosomes, where the array composition, saturation, and length can be varied via the ordered ligation of distinct mononucleosomes. Using this system we find that the ligated tetranucleosomal arrays undergo intra-array compaction. However, this compaction is less extensive than for longer arrays and is histone H4 tail independent, suggesting that well-ordered stretches of four or fewer nucleosomes do not fully compact to the 30 nm fiber. Like longer arrays, the tetranucleosomal arrays exhibit cooperative self-association to form species composed of many copies of the array. This propensity for self-association decreases when the fraction of nucleosomes lacking H4 tails is systematically increased. However, even tetranucleosomal arrays with only two octamers possessing H4 tails recapitulate most of the interarray self-association. Varying array length shows that systems as short as dinucleosomes demonstrate significant self-association, confirming that relatively few determinants are required for interarray interactions, and suggesting that in vivo, multiple interactions of short runs of nucleosomes might contribute to complex fiber-fiber interactions. Additionally, we find that the stability of nucleosomes toward octamer loss increases with array length and saturation, suggesting that in vivo stretches of ordered, saturated nucleosomes could serve to protect these regions from histone ejection.

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Regulation of human cytidine triphosphate synthetase 2 by phosphorylation.

Publication Date: 2010 Aug 25 PMID: 20739275
Authors: Kassel, K. M. - Au, D. R. - Higgins, M. J. - Hines, M. - Graves, L. M.
Journal: J Biol Chem

Cytidine triphosphate synthetase (CTPS) is the rate-limiting enzyme in de novo CTP synthesis and is required for the formation of RNA, DNA, and phospholipids. This study determined the kinetic properties of the individual human CTPS isozymes (hCTPS1 and hCTPS2) and regulation through substrate concentration, oligomerization, and phosphorylation. Kinetic analysis demonstrated that both hCTPS1 and hCTPS2 were maximally active at physiological concentrations of ATP, GTP, and glutamine, whereas the Km and IC50 values for the substrate UTP and the product CTP, respectively, were close to their physiological concentrations, indicating that the intracellular concentrations of UTP and CTP may precisely regulate hCTPS activity. Low serum treatment increased hCTPS2 phosphorylation and five probable phosphorylation sites were identified in the hCTPS2 carboxyl-terminal domain. Metabolic labeling of hCTPS2 with [32P]-H3PO4 demonstrated that S568 and S571 were 2 major phosphorylation sites and additional studies demonstrated S568 was phosphorylated by CK1 both in vitro and in vivo. Interestingly, mutation of S568 (S568A) but not S571 significantly increased hCTPS2 activity, demonstrating that S568 is a major inhibitory phosphorylation site. The S568A mutation had a greater effect on the glutamine, rather than ammonia-dependent activity, indicating that phosphorylation of this site may influence the glutaminase domain of hCTPS2. Deletion of the carboxyl-terminal regulatory domain of hCTPS1 also greatly increased the Vmax of this enzyme. In summary, this is the first study to characterize the kinetic properties of hCTPS1 and hCTPS2 and to identify S568 as a major site of CTPS2 regulation by phosphorylation.

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Epigenetic silencing of {beta}-spectrin a TGF-{beta} signaling/scaffolding protein in a human cancer stem cell disorder: Beckwith-Wiedemann syndrome.

Publication Date: 2010 Aug 25 PMID: 20739274
Authors: Yao, Z. X. - Jogunoori, W. S. - Choufani, S. - Rashid, A. - Blake, T. - Yao, W. - Kreishman, P. - Amin, R. - Sidawy, A. A. - Evans, S. R. - Finegold, M. - Reddy, E. P. - Mishra, B. - Weksberg, R. - Kumar, R. - Mishra, L.
Journal: J Biol Chem

Hereditary cancer syndromes provide powerful insights into dysfunctional signaling pathways that lead to sporadic cancers. Beckwith-Wiedemann syndrome (BWS) is a hereditary human cancer stem cell syndrome currently linked to deregulated imprinting at chromosome 11p15 and uniparental disomy. However, causal molecular defects and genetic models have remained elusive to date in the majority of cases. The non-pleckstrin homology (PH) domain beta-spectrin (beta2SP)[Official name for human: Spectrin, beta, non-erythrocytic 1 (SPTBN1), isoform 2; Official name for mouse: Spectrin beta 2 (Spnb2), isoform 2], a scaffolding protein, functions as a potent TGF-beta signaling member adaptor in tumor suppression and development. Yet, the role of the beta2SP in human tumor syndromes remains unclear. Here, we report beta2SP(+/-) mice are born with many phenotypic characteristics observed in BWS patients suggesting that beta2SP mutant mice phenocopy BWS and beta2SP loss could be one of the mechanisms associated with BWS. Our results also suggest that epigenetic silencing of beta2SP is a new potential causal factor in human BWS patients. Furthermore, beta2SP(+/-) mice provide an important animal model for BWS, as well as sporadic cancers associated with it, including lethal gastrointestinal and pancreatic cancer. Thus, these studies could lead to further insight into defects generated by dysfunctional stem cells and identification of new treatment strategies and functional markers for the early detection of these lethal cancers that otherwise cannot be detected at an early stage.

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p15RS attenuates Wnt/{beta}-catenin signaling by disrupting {beta}-catenin/TCF4 interaction.

Publication Date: 2010 Aug 25 PMID: 20739273
Authors: Wu, Y. - Zhang, Y. - Zhang, H. - Yang, X. - Wang, Y. - Ren, F. - Liu, H. - Zhai, Y. - Jia, B. - Yu, J. - Chang, Z.
Journal: J Biol Chem

The formation of a beta-catenin/TCF4 complex in the nucleus of cells is well known as a prerequisite for the transcription of Wnt target genes. Although many co-factors have been identified to regulate the activity of the beta-catenin/TCF4 complex, it remains unclear how the complex association is negatively regulated. In this study, we report that p15RS, a negative regulator of the cell cycle, blocks beta-catenin/TCF4 complex formation and inhibits Wnt signaling. We observed that p15RS interact with beta-catenin and TCF4. Interestingly, while the interaction of p15RS with beta-catenin is increased, its interaction with TCF4 is decreased upon Wnt1 stimulation. Moreover, over-expression of p15RS reduces the interaction of beta-catenin with TCF4, while the depletion of p15RS enhances their interaction. We further demonstrate that over-expression of p15RS suppresses canonical Wnt signaling and results in retarded cell growth, while the depletion of p15RS shows an enhanced effect on Wnt signaling. We analyzed that inhibition of Wnt signaling by p15RS leads to decreased expression of Cyclin D1 and c-Myc, two Wnt targeted genes critical for cell growth. Our data suggest that p15RS inhibits Wnt signaling by interrupting beta-catenin/TCF4 complex formation and that Wnt signaling initiates downstream gene expression by removing p15RS from promoters.

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Double-strand break unwinding and resection by the mycobacterial helicase-nuclease AdnAB in the presence of mycobacterial SSB.

Publication Date: 2010 Aug 23 PMID: 20736178
Authors: Unciuleac, M. C. - Shuman, S.
Journal: J Biol Chem

Mycobacterial AdnAB is a heterodimeric DNA helicase-nuclease and 3'-to-5' DNA translocase implicated in the repair of double-strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Inclusion of mycobacterial SSB (single strand DNA binding protein) in reactions containing linear plasmid dsDNA allowed us to study the AdnAB helicase under conditions in which the unwound single strands are coated by SSB and thereby prevented from reannealing or promoting ongoing ATP hydrolysis. We found that the AdnAB motor catalyzed processive unwinding of 2.7 to 11.2-kbp linear duplex DNAs at a rate of ~250 bp s-1, while hydrolyzing ~5 ATPs per bp unwound. Crippling the AdnA phosphohydrolase active site did not affect the rate of unwinding, but lowered energy consumption slightly, to ~4.2 ATPs bp-1. Mutation of the AdnB phosphohydrolase abolished duplex unwinding, consistent with a model in which the leading AdnB motor propagates a Y-fork by translocation along the 3' DNA strand, ahead of the lagging AdnA motor domain. By tracking the resection of the 5' and 3' strands at the DSB ends, we illuminated a division of labor among the AdnA and AdnB nuclease modules during dsDNA unwinding, whereby the AdnA nuclease processes the unwound 5' strand to liberate a short oligonucleotide product while the AdnB nuclease incises the 3' strand on which the motor translocates. These results extend our understanding of presynaptic DSB processing by AdnAB and engender instructive comparisons to the RecBCD and AddAB clades.

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Inter-subunit Hydrophobic Interactions in Pf1 Filamentous Phage.

Publication Date: 2010 Aug 23 PMID: 20736177
Authors: Goldbourt, A. - Day, L. A. - McDermott, A. E.
Journal: J Biol Chem

Magic angle spinning solid state NMR has been used to study the structural changes in the Pf1 filamentous bacteriophage, which occur near 10 degrees C. Comparisons of NMR spectra recorded above and below 10 degrees C reveal reversible perturbations in many NMR chemical shifts, most of which are assigned to atoms of hydrophobic side-chains of the 46-residue subunit. The changes mainly involve groups located in patches on the interfaces between neighboring capsid subunits. The observations show that the transition adjusts the hydrophobic interfaces between fairly rigid subunits. The low temperature form has been generally more amenable to structure determination; spin diffusion experiments on this form revealed unambiguous contacts between side-chains of neighboring subunits. These contacts are important constraints for structure modeling.

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COT/TPL2 (MAP3K8) mediates myeloperoxidase activity and hypernociception following peripheral inflammation.

Publication Date: 2010 Aug 24 PMID: 20736176
Authors: Soria Castro, I. - Krzyzanowska, A. - Lopez Pelaez, M. - Regadera, J. - Ferrer, G. - Montoliu, L. - Rodriguez-Ramos, R. - Fernandez, M. - Alemany, S.
Journal: J Biol Chem

Cot/tpl2 (also known as MAP3K8) has emerged as a new and potentially interesting therapeutic anti-inflammatory target. Here we report the first study of Cot/tpl2 involvement in acute peripheral inflammation in vivo. Six hours following an intraplantar injection of zymosan, Cot/tpl2-/- mice showed a 47% reduction in myeloperoxidase activity, concomitant with a 46% lower neutrophil recruitment and a 40% decreased luminol-mediated bioluminescence imaging in vivo. Accordingly, Cot/tpl2 deficiency provoked a 25-30% reduction in luminol-mediated bioluminescence and neutrophil recruitment together with a 65% lower macrophage recruitment 4 hours following zymosan-induced peritonitis. Significantly impaired levels of G-CSF and GM-CSF, and of other cytokines such as TNFalpha IL-1beta and IL-6, as well as some chemokines such as MCP-1, MIP-1beta and KC were detected during the acute zymosan-induced intraplantar inflammatory response in Cot/tpl2-/- mice. Moreover, Cot/tpl2 deficiency dramatically decreased the production of the hypernociceptive ligand NGF at the inflammatory site during the course of inflammation. Most importantly, Cot/tpl2 deficiency significantly reduced zymosan-induced inflammatory hypernociception in mice, with a most pronounced effect of a 50% decrease in comparison to Wt, at 24 hours following intraplantar injection of zymosan. At this time Cot/tpl2-/- mice showed significantly reduced NGF, TNFalpha and PGE2 levels in comparison to Wt littermates. In conclusion, our study demonstrates an important role of Cot/tpl2 in the NGF, G-CSF and GM-CSF production and myeloperoxidase activity in the acute inflammatory response process and its implication in inflammatory hypernociception.

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Recombinant membrane targeted form of CD59 inhibits the growth of choroidal neovascular complex in mice.

Publication Date: 2010 Aug 24 PMID: 20736175
Authors: Bora, N. S. - Jha, P. - Lyzogubov, V. V. - Kaliappan, S. - Liu, J. - Tytarenko, R. G. - Fraser, D. A. - Morgan, B. P. - Bora, P. S.
Journal: J Biol Chem

This study was designed to explore the effect of recombinant, membrane-targeted CD59 (rCD59- APT542) on the growth and size of fully developed neo-vascular complex using the murine model of laser induced choroidal neovascularization (CNV). CNV was induced by laser photocoagulation in C57BL/6 mice using an Argon laser and the animals received rCD59-APT542 via intravitreal (ivt) route. Western blot analysis, immunohistochemistry and total complement hemolytic assay demonstrated that exogenously administered rCD59-APT542 was incorporated as well as retained in RPE and choroid and was functionally active in vivo. Single ivt injection during the growth of CNV (i.e. at day 3 post laser) resulted in approximately 79% inhibition of the further growth of neovascular complex. The size of the CNV complex was significantly (p<0.05) reduced by the administration of rCD59-APT542 after the CNV complex has fully developed (i.e. at day 7 post-laser). Treatment with rCD59 APT542 blocked the formation of membrane attack complex (MAC), increased apoptosis and decreased cell proliferation in the neovascular complex. On the basis of results presented here we conclude that recombinant membrane targeted CD59 inhibited the growth of the CNV complex and reduced the size of fully developed CNV in the laser-induced mouse model. We propose that a combination of two mechanisms increased apoptosis and decreased cell proliferation both resulting from local inhibition of MAC may be responsible for inhibition of CNV by rCD59-APT542.

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Cathepsin X deficient gastric epithelial cells in coculture with macrophages-characterization of cytokine response and migration capability after H. pylori infection.

Publication Date: 2010 Aug 24 PMID: 20736174
Authors: Bernhardt, A. - Kuester, D. - Roessner, A. - Reinheckel, T. - Krueger, S.
Journal: J Biol Chem

Our previous studies have shown an association between Helicobacter pylori infection, the strong up-regulation of cathepsin X (CTSX, also called cathepsin Z/P), and the development of gastric cancer. In the present study, we analyzed primary and conventional gastric epithelial cell lines to establish an optimal in-vitro mouse model system for the examination of H. pylori-induced over-expression of Ctsx in a functional way. Gastric epithelial cells were isolated from stomachs of wild-type C57BL/6 and Ctsx-/- mice and compared with the gastric cancer cell line CLS103. Indirect cocultures of epithelial cells and macrophages were infected with H. pylori strain SS1 and analyzed for the expression of cathepsins, cytokines, and adhesion factors. Cellular interactions, migration capability, and adherence of H. pylori were assessed using time-lapse video microscopy and colony-forming assays. Isolated primary cells from wild-type and transgenic mice revealed qualities and expression profiles similar to those of corresponding tissue samples. Adherence of H. pylori was significantly higher in primary compared to commercially cells. Thus, induction of cathepsins, cytokines, and adhesion proteins was detected solely in primary cells and cocultured macrophages. Microarray and migration experiments indicated that Ctsx is involved in B-/T-cell proliferation/migration and adhesion of macrophages. Primary epithelial cells from stomach of Ctsx-/- mice represent an excellent model of H. pylori gastritis to elaborate the special functions of Ctsx in regulating the immune response to H. pylori.

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Divergent Intracellular Sorting of Fc{gamma}RIIA and Fc{gamma}RIIB2.

Publication Date: 2010 Aug 24 PMID: 20736173
Authors: Zhang, C. Y. - Booth, J. W.
Journal: J Biol Chem

The human low affinity FcgammaRII family includes both the activating receptor FcgammaRIIA and the inhibitory receptor FcgammaRIIB2. These receptors have opposing signalling functions but are both capable of internalizing IgG-containing immune complexes through clathrin-mediated endocytosis. We demonstrate that upon engagement by multivalent aggregated human IgG, FcgammaRIIA expressed in ts20 Chinese hamster fibroblasts is delivered along with its ligand to lysosomal compartments for degradation, while FcgammaRIIB2 dissociates from the ligand and is routed separately into the recycling pathway. FcgammaRIIA sorting to lysosomes requires receptor multimerization, but does not require either Src family kinase activity or ubiquitylation of receptor lysine residues. The sorting of FcgammaRIIB2 away from a degradative fate is not due to its lower affinity for IgG and occurs even upon persistent receptor aggregation. Upon co-engagement of FcgammaRIIA and FcgammaRIIB2, the receptors are sorted independently to distinct final fates after dissociation of co-clustering ligand. These results reveal fundamental differences in the trafficking behaviour of different Fcgamma receptors.

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Identification of a lipid peroxidation product as the source of oxidation-specific epitopes recognized by anti-DNA autoantibodies.

Publication Date: 2010 Aug 24 PMID: 20736172
Authors: Otaki, N. - Chikazawa, M. - Nagae, R. - Shimozu, Y. - Shibata, T. - Ito, S. - Takasaki, Y. - Fujii, J. - Uchida, K.
Journal: J Biol Chem

Lipid peroxidation in tissue and in tissue fractions represents a degradative process, which is the consequence of the production and the propagation of free radical reactions primarily involving membrane polyunsaturated fatty acids, and has been implicated in the pathogenesis of numerous diseases, including systemic lupus erythematosus (SLE). We have found that bovine serum albumin incubated with peroxidized polyunsaturated fatty acids significantly cross-reacted with the sera from MRL-lpr mice, a representative murine model of SLE. To identify the active substances responsible for the generation of autoantigenic epitopes recognized by the SLE sera, we performed the activity-guiding separation of a principal source from 13-hydroperoxy-9Z,11E-octadecadienoic acid and identified 4-oxo-2-nonenal (ONE), a highly reactive aldehyde originating from the peroxidation of omega 6 polyunsaturated fatty acids, as the source of the autoantigenic epitopes. When the age-dependent change in the antibody titer against the ONE-modified protein was measured in the sera from MRL-lpr mice and control MRL-MpJ mice, all of the MRL-lpr mice developed an anti-ONE titer, which was comparable to the anti-DNA titer. Strikingly, a subset of the anti-DNA monoclonal antibodies generated from the SLE mice, showing recognition specificity toward DNA, cross-reacted with the ONE-specific epitopes. Furthermore, these dual-specific antibodies rapidly bound and internalized into live cells. These findings raised the possibility that the enhanced lipid peroxidation followed by the generation of ONE may be involved in the pathogenesis of autoimmune disorders.

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The role of PPX in avoidance by Neisseria meningitidis of complement-mediated killing.

Publication Date: 2010 Aug 24 PMID: 20736171
Authors: Zhang, Q. - Li, Y. - Tang, C. M.
Journal: J Biol Chem

The complement system is critical for immunity against the important human pathogen, Neisseria meningitidis. We describe the isolation of a meningococcal mutant lacking PPX, an exopolyphosphatase responsible for is responsible for cleaving cellular polyphosphate (poly P), a polymer of tens to hundreds of orthophosphate residues found in virtually all living cells. Bacteria lacking PPX exhibit increased resistance to complement-mediated killing. By site directed mutagenesis, we define amino acids necessary for the biochemical activity of meningococcal PPX, including a conserved glutamate (E117) and residues in the Walker B box predicted to be involved in binding to phosphate. We show that the biochemical activity of PPX is necessary for interactions with the complement. The relative resistance of the ppx mutant does not result from changes in structures (such as capsule, lipopolysaccharide and factor H binding protein) which are known to be required for evasion of this key aspect of host immunity. Instead, expression of PPX modifies the interaction of N. meningitidis with the alternative pathway of complement activation.

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Metabolism of pentose sugars in the hyperthermophilic archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius.

Publication Date: 2010 Aug 24 PMID: 20736170
Authors: Nunn, C. E. - Johnsen, U. - Schonheit, P. - Fuhrer, T. - Sauer, U. - Hough, D. W. - Danson, M. J.
Journal: J Biol Chem

We have previously shown that the hyperthermophilic archaeon, Sulfolobus solfataricus, catabolises D-glucose and D-galactose to pyruvate and glyceraldehyde via a non-phosphorylative version of the Entner-Doudoroff pathway. At each step, one enzyme is active with both C6 epimers, leading to a metabolically-promiscuous pathway. On further investigation, the catalytic promiscuity of the first enzyme in this pathway, glucose dehydrogenase, has been shown to extend to the C5 sugars, D-xylose and L-arabinose. In the current paper, we establish that this promiscuity for C6 and C5 metabolites is also exhibited by the third enzyme in the pathway, 2-keto-3-deoxygluconate aldolase, but that the second step requires a specific C5-dehydratase, the gluconate dehydratase being active only with C6 metabolites. The products of this pathway for the catabolism of D-xylose and L-arabinose are pyruvate and glycolaldehyde, pyruvate entering the citric acid cycle after oxidative decarboxylation to acetyl-coenzyme A. We have identified and characterised the enzymes, both native and recombinant, that catalyse the conversion of glycolaldehyde to glycolate and then to glyoxylate, which can enter the citric acid cycle via the action of malate synthase. Evidence is also presented that similar enzymes for this pentose sugar pathway are present in Sulfolobus acidocaldarius, and metabolic tracer studies in this archaeon demonstrate its in vivo operation, in parallel with a route involving no aldol cleavage of the 2-keto-3-deoxy-pentanoates but direct conversion to the citric acid cycle C5-metabolite, 2-oxoglutarate.

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Characterization of 5-Chloro-5-Deoxy-D-Ribose-1-Dehydrogenase in Chloroethylmalonyl-Coenzyme A Biosynthesis: Substrate and Reaction Profiling.

Publication Date: 2010 Aug 24 PMID: 20736169
Authors: Kale, A. - McGlinchey, R. - Moore, B.
Journal: J Biol Chem

SalM is a short-chain dehydrogenase/reductase enzyme from the marine actinomycete Salinispora tropica that is involved in the biosynthesis of chloroethylmalonyl-CoA, a novel halogenated polyketide synthase extender unit of the proteasome inhibitor salinosporamide A. SalM was heterologously overexpressed in Escherichia coli and characterized in vitro for its substrate specificity, kinetics, and reaction profile. A sensitive, real-time 13C NMR assay was developed to visualize the oxidation of 5-chloro-5-deoxy-D-ribose to 5-chloro-5-deoxy-D-ribono-gamma-lactone in a NAD+-dependent reaction followed by spontaneous lactone hydrolysis to 5-chloro-5-deoxy-D-ribonate. While short-chain dehydrogenase/reductase enzymes are widely regarded as metal independent, a strong divalent metal cation dependence for Mg2+ or Mn2+ was observed with SalM. Oxidative activity was also measured with the alternative substrates D-erythrose and D-ribose, making SalM the first reported stereospecific non-phosphorylated ribose-1-dehydrogenase.

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Overoxidation of 2-Cys peroxiredoxin in prokaryotes: cyanobacterial 2-Cys peroxiredoxins sensitive to oxidative stress.

Publication Date: 2010 Aug 24 PMID: 20736168
Authors: Pascual, M. B. - Mata-Cabana, A. - Florencio, F. J. - Lindahl, M. - Cejudo, F. J.
Journal: J Biol Chem

In eukaryotic organisms hydrogen peroxide has a dual effect; it is potentially toxic for the cell, but has also an important signaling activity. According to the previously proposed floodgate hypothesis, the signaling activity of hydrogen peroxide in eukaryotes requires a transient increase in its concentration, which is due to the inactivation by overoxidation of 2-Cys peroxiredoxin (2-Cys Prx). Sensitivity to overoxidation depends on the structural GGLG and YF motifs present in eukaryotic 2-Cys Prxs and is believed to be absent from prokaryotic enzymes, thus representing a paradoxical gain-of-function exclusive to eukaryotic organisms. Here we show that 2-Cys Prxs from several prokaryotic organisms, including cyanobacteria, contain the GG(L/V/I)G and YF motifs characteristic of sensitive enzymes. In search of the existence of overoxidation-sensitive 2-Cys Prxs in prokaryotes, we have analyzed the sensitivity to overoxidation of 2-Cys Prxs from two cyanobacterial strains, Anabaena sp. PCC7120 and Synechocystis sp. PCC6803. In vitro analysis of wild type and mutant variants of the Anabaena 2-Cys Prx showed that this enzyme is overoxidized at the peroxidatic cysteine residue, thus constituting an exception among prokaryotes. Moreover, the 2-Cys Prx from Anabaena is readily and reversibly overoxidized in vivo in response to high light and hydrogen peroxide showing higher sensitivity to overoxidation than the Synechocystis enzyme. These cyanobacterial strains have different strategies to cope with hydrogen peroxide. While Synechocystis has low content of less-sensitive 2-Cys Prx and high catalase activity, Anabaena contains abundant and sensitive 2-Cys Prx, but low catalase activity, which is remarkably similar to the chloroplast system.

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The direct association of Sprouty-related protein with an EVH1 domain (SPRED) 1 or SPRED2 with DYRK1A modifies substrate/kinase interactions.

Publication Date: 2010 Aug 24 PMID: 20736167
Authors: Li, D. - Jackson, R. A. - Yusoff, P. - Guy, G. R.
Journal: J Biol Chem

The mammalian SPRED (Sprouty-related protein with an EVH1 domain) proteins comprise a family of three members: SPRED1-3. Currently, little is known about their biochemistry. The best described, SPRED1, has been shown to inhibit the Ras/ERK pathway downstream of Ras. All three SPREDs have a cysteine-rich domain (CRD) that has high homology to the CRD of the Sprouty family of proteins, several of which are also Ras/ERK inhibitors. In the belief that binding partners would clarify SPRED function, we assayed for their associated proteins. Here, we describe the direct and endogenous interaction of SPRED1 and SPRED2 with the novel kinase, DYRK1A. DYRK1A has become the subject of recent research focus as it plays a central role in C. elegans oocyte maturation and egg activation, and there is strong evidence that it could be involved in Down Syndrome in humans. Both SPRED1 and SPRED2 inhibit the ability of DYRK1A to phosphorylate its substrates: Tau and STAT3. This inhibition occurs via an interaction of the CRD of the SPREDs with the kinase domain of DYRK1A. DYRK1A substrates must bind to the kinase to enable phosphorylation, and SPRED proteins compete for the same binding site to modify this process. Our accumulated evidence indicates that the SPRED proteins are likely physiological modifiers of DYRK1A.

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Blocking interferon-beta stimulates vascular smooth muscle cell proliferation and arteriogenesis.

Publication Date: 2010 Aug 24 PMID: 20736166
Authors: Schirmer, S. H. - Bot, P. T. - Fledderus, J. O. - van der Laan, A. M. - Volger, O. L. - Laufs, U. - Boehm, M. - de Vries, C. J. - Horrevoets, A. J. - Piek, J. J. - Hoefer, I. E. - van Royen, N.
Journal: J Biol Chem

Increased interferon (IFN)-beta signaling in patients with insufficient coronary collateralization and an inhibitory effect of IFNbeta on collateral artery growth in mice have been reported. The mechanisms of IFNbeta-induced inhibition of arteriogenesis are unknown. In stimulated monocytes from patients with chronic total coronary artery occlusion and decreased arteriogenic response, whole genome expression analysis showed increased expression of IFNbeta-regulated genes. Immunohistochemically, the IFNbeta receptor was localized in the vascular media of murine collateral arteries. Treatment of vascular smooth muscle cells (VSMC) with IFNbeta resulted in an attenuated proliferation, cell-cycle arrest and increased expression of cyclin-dependent kinase inhibitor-1A (p21). Growth-inhibitory effect of IFNbeta was attenuated by inhibition of p21 by RNA-interference. IFNbeta-treated THP1 monocytes showed enhanced apoptosis. Subsequently, we tested if collateral artery growth can be stimulated by inhibition of IFNbeta-signaling. RNA-interference of the IFNbeta receptor-1 (IFNAR1) increased VSMC proliferation, cell cycle progression, and reduced p21 gene expression. IFNbeta-signaling and FAS and TRAIL expression were attenuated in monocytes from IFNAR1-/- mice, indicating reduced monocyte apoptosis. Hindlimb perfusion restoration one week after femoral artery ligation was improved in IFNAR1-/- mice compared to wildtype mice as assessed by infusion of fluorescent microspheres. These results demonstrate that IFNbeta inhibits collateral artery growth and VSMC proliferation through p21-dependent cell cycle arrest and induction of monocyte apoptosis. Inhibition of IFNbeta stimulates VSMC proliferation and collateral artery growth.

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NHE3 activity is dependent on direct phosphoinositide binding at the N-terminus of its intracellular cytosolic region.

Publication Date: 2010 Aug 24 PMID: 20736165
Authors: Mohan, S. - Tse, C. M. - Gabelli, S. B. - Sarker, R. - Cha, B. - Fahie, K. - Nadella, M. - Zachos, N. C. - Tu-Sekine, B. - Raben, D. - Amzel, L. M. - Donowitz, M.
Journal: J Biol Chem

The small intestinal BB Na+/H+ antiporter NHE3 accounts for the majority of intestinal Na and water absorption. It is highly regulated, with both postprandial inhibition and stimulation sequentially occurring. Phosphoinositide PI(4,5)P2 and PI(3,4,5)P3 binding is involved with regulation of multiple transporters. We tested the hypothesis that phosphoinositides bind NHE3 under basal conditions and are necessary for its acute regulation. His6-proteins were made from the NHE3 C-terminal region divided into four parts: F1 (amino acids 475 to 589), F2 (590 to 667), F3 (668 to 747) and F4 (748 to 832) and purified by Ni column. Mutations were made in F1 region of NHE3 and cloned in pet30a and pcDNA3.1 vectors. PI(4,5)P2 and PI(3,4,5)P3 bound only to the NHE3 F1 fusion protein (aminoacids 475-589) on liposomal pull down assays. Mutations were made in the putative lipid binding region of the F1 domain and studied for alterations in lipid binding and Na+/H+ exchange: Y501A/R503A/ K505A; F509A/R511A/R512A; R511L/R512L; R520/FR527F; R551L/R552L. Our results indicate that (1) The F1 domain of NHE3 C-terminus has phosphoinositide binding regions. Mutations of these regions alter PI(4,5)P2 and PI(3,4,5)P3 binding and basal NHE3 activity. (2) Magnitude of Serum stimulation of NHE3 correlates with PI(4,5)P2 and PI(3,4,5)P3 binding of NHE3. (3) Wortmannin inhibition of PI 3-Kinase did not correlate with PI(4,5)P2 or PI(3,4,5)P3 binding of NHE3. (4) Two functionally distinct phosphoinositides binding regions (Y501-R512 and R520-R552) are present in NHE3 F1 domain; both regions are important for serum stimulation but they display differences in phosphoinositides binding, and the latter but not the former alters NHE3 surface expression.

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Regulation of the epidermal growth factor receptor ubiquitination and trafficking by the USP8/STAM complex.

Publication Date: 2010 Aug 24 PMID: 20736164
Authors: Berlin, I. - Schwartz, H. - Nash, P. D.
Journal: J Biol Chem

Reversible ubiquitination of activated receptor complexes signals their sorting between recycling and degradation and thereby dictates receptor fate. The deubiquitinating enzyme Ubiquitin-Specific Protease 8 (USP8/UBPy) has been previously implicated in the regulation of the Epidermal Growth Factor Receptor (EGFR), however, the molecular mechanisms governing its recruitment and activity in this context remain unclear. Herein, we investigate the role of USP8 in countering ligand-induced ubiquitination and down-regulation of EGFR, and characterize a subset of protein-protein interaction determinants critical for this function. USP8 depletion accelerates receptor turnover, while loss of Hrs rescues this phenotype, indicating that USP8 protects EGFR from degradation via an Hrs-dependent pathway. Catalytic inactivation of USP8 incurs EGFR hyperubiquitination and promotes receptor localization to endosomes marked by high ubiquitin content. These phenotypes require the central region of USP8, containing three extended Arg-X-X-Lys (RXXK) motifs that specify direct low-affinity interactions with the SH3 domain(s) of ESCRT-0 proteins, STAM1/2. The USP8/STAM complex critically impinges on receptor ubiquitination status and modulates ubiquitin dynamics on EGFR-positive endosomes. Consequently, USP8-mediated deubiquitination slows progression of EGFR past the early-to-recycling endosome circuit in a manner dependent upon the RXXK motifs. Collectively, these findings demonstrate a role for the USP8/STAM complex as a protective mechanism regulating early endosomal sorting of EGFR between pathways destined for lysosomal degradation and recycling.

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A novel cytoplasmic adaptor for RAR and TR functions as a derepressor of RAR in the absence of retinoic acid.

Publication Date: 2010 Aug 24 PMID: 20736163
Authors: Park, U. H. - Kim, E. J. - Um, S. J.
Journal: J Biol Chem

In most mammalian cells, the retinoic acid receptor (RAR) is nuclear rather than cytoplasmic, regardless of its cognate ligand, retinoic acid (RA). In testis Sertoli cells, however, RAR is retained in the cytoplasm and moves to the nucleus only when RA is supplied. This led us to identify a protein that regulates the translocation of RAR. From yeast two-hybrid screening, we identified a novel RAR-interacting protein called the cytoplasmic adaptor for RAR and TR (CART1). Systematic interaction assays using deletion mutants showed that the C-terminal CoRNR box of CART1 was responsible for the interaction with the NCoR-binding region of RAR and TR. Such interaction was impaired in the presence of ligand RA, as further determined by GST pull-down in vitro and immunoprecipitation assays in vivo. Fluorescence microscopy showed that unliganded RAR was captured by CART1 in the cytoplasm, whereas liganded RAR was liberated and moved to the nucleus. Over expression of CART1 blocked the transcriptional repressing activity of unliganded apo-RAR, mediated by corepressor NCoR in the nucleus. CART1 siRNA treatment in a mouse Sertoli cell line, TM4, allowed RAR to move to the nucleus and blocked the derepressing function of CART1, suggesting that CART1 might be a cytoplasmic, testis-specific derepressor of RAR.

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Transamination Is Required for {alpha}-Ketoisocaproate but not Leucine to Stimulate Insulin Secretion.

Publication Date: 2010 Aug 24 PMID: 20736162
Authors: Zhou, Y. - Jetton, T. L. - Goshorn, S. - Lynch, C. J. - She, P.
Journal: J Biol Chem

It remains unclear how alpha-ketoisocaproate (KIC) and leucine are metabolized to stimulate insulin secretion. Mitochondrial branched-chain aminotransferase (BCATm) catalyzes reversible transamination of leucine and alpha-ketoglutarate to KIC and glutamate, the first step of leucine catabolism. We investigated the biochemical mechanisms of KIC and leucine-stimulated insulin secretion (KICSIS and LSIS, respectively) using BCATm-/- mice. In static incubation, BCATm disruption abolished insulin secretion by KIC, DL-alpha-keto-beta-methylvalerate and alpha-ketocaproate without altering stimulation by glucose, leucine, or alpha-ketoglutarate. Similarly, during pancreas perfusions in BCATm-/- mice, glucose and arginine stimulated insulin release, whereas KICSIS was largely abolished. During islet perifusions, KIC plus 2mM glutamine caused robust dose-dependent insulin secretion in BCATm+/+ not BCATm-/- islets, whereas LSIS was unaffected. Consistently, in contrast to BCATm+/+ islets, the increases of the ATP concentration and NADPH/NADP+ ratio in response to KIC were largely blunted in BCATm-/- islets. Compared to non-treated islets, the combination of KIC/glutamine (10/2mM) did not influence alpha-ketoglutarate concentrations, but caused 120% and 33% increases in malate in BCATm+/+ and BCATm-/- islets, respectively. While leucine oxidation and KIC transamination were blocked in BCATm-/- islets, KIC oxidation was unaltered. These data indicate that KICSIS requires transamination of KIC and glutamate to leucine and alpha-ketoglutarate, respectively. LSIS does not require leucine catabolism and may be through leucine activation of glutamate dehydrogenase. Thus, KICSIS and LSIS occur by enhancing the metabolism of glutamine/glutamate to alpha-ketoglutarate, which in turn is metabolized to produce the intracellular signals such as ATP and NADPH for insulin secretion.

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FUNCTIONAL DIFFERENCES OF INVARIANT AND HIGHLY CONSERVED RESIDUES IN THE EXTRACELLULAR DOMAIN OF THE GLYCOPROTEIN HORMONE RECEPTORS.

Publication Date: 2010 Aug 24 PMID: 20736161
Authors: Angelova, K. - de Jonge, H. - Granneman, J. C. - Puett, D. - Bogerd, J.
Journal: J Biol Chem

Multiple interactions exist between human follicle-stimulating hormone (FSH) and the N-terminal hormone-binding fragment of the human FSH receptor (FSHR) extracellular domain (ECD). Binding of the other human glycoprotein hormones to their cognate human receptors (luteinizing hormone receptor [LHR] and thyroid-stimulating hormone receptor [TSHR]) is expected to be similar. This study focuses on amino acid residues in beta-strands 2 (K(74)), 4 (Y(124), N(129), T(130)) and 5 (D(150), D(153)) of the FSHR ECD identified in the human FSH/FSHR ECD crystal structure as contact sites with the common glycoprotein hormone alpha-subunit, and non-contact residues in beta-strands 2 (S(78)) and 8 (D(224), S(226)) as controls. These nine residues are either invariant or highly conserved in LHR and TSHR. Mutagenesis and functional characterization of these residues in all three human receptors allowed an assessment of their contribution to binding and receptor activation. Surprisingly, the six reported alpha-subunit contact residues of the FSHR ECD could be replaced without a significant loss of FSH binding, while cAMP signaling potency was diminished significantly with several replacements. Comparative studies of the homologous residues in LHR and TSHR revealed both similarities and differences. The results for FSH/FSHR were analyzed on the basis of the crystal structure of the FSH/FSHR ECD complex, while comparative modeling was used to generate structures for domains, proteins and complexes for which no structures were available. While structural information of hormone-receptor interaction allows the identification of hormone-receptor contact sites, functional analysis of each contact site is necessary to assess its contribution to hormone binding and receptor activation.

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A new hierarchical phosphorylation pathway of the translational repressor eIF4E-binding protein 1 (4E-BP1) in ischemia-reperfusion stress.

Publication Date: 2010 Aug 24 PMID: 20736160
Authors: Ayuso, M. I. - Hernandez-Jimenez, M. - Martin, M. E. - Salinas, M. - Alcazar, A.
Journal: J Biol Chem

Eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) is a translational repressor that is characterized by its capacity to bind specifically to eIF4E and inhibit its interaction with eIF4G. Phosphorylation of 4E-BP1 regulates eIF4E availability, and therefore, cap-dependent translation, in cell stress. This paper reports a physiological study of 4E-BP1 regulation by phosphorylation using control conditions and a stress-induced translational repression condition, ischemia-reperfusion (IR) stress, in brain tissue. In control conditions, 4E-BP1 was found in four phosphorylation states that were detected by 2-D gel electrophoresis and Western blotting, which corresponded to Thr69-phosphorylated alone, Thr69- and Thr36/Thr45-phosphorylated, all these plus Ser64 phosphorylation, and dephosphorylation of the sites analyzed. In control or IR conditions, no Thr36/Thr45 phosphorylation alone was detected without Thr69 phosphorylation, and neither was Ser64 phosphorylation without Thr36/Thr45/Thr69 phosphorylation detected. Ischemic stress induced 4E-BP1 dephosphorylation at Thr69, Thr36/Thr45 and Ser64 residues, with 4E-BP1 remaining phosphorylated at Thr69 alone or dephosphorylated. In the subsequent reperfusion, 4E-BP1 phosphorylation was induced at Thr36/Thr45 and Ser64, in addition to Thr69. Changes in 4E-BP1 phosphorylation after IR were according to those found for Akt and mTOR kinases. These results demonstrate a new hierarchical phosphorylation for 4E-BP1 regulation, in which Thr69 is phosphorylated first followed by Thr36/Thr45 phosphorylation, and Ser64 is phosphorylated last. Thr69 phosphorylation alone allows binding to eIF4E, and subsequent Thr36/Thr45 phosphorylation was sufficient to dissociate 4E-BP1 from eIF4E which led to eIF4E-4G interaction. These data help to elucidate the physiological role of 4E-BP1 phosphorylation in controlling protein synthesis.

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Expression of miR-33 from an SREBP2 intron inhibits cholesterol export and fatty acid oxidation.

Publication Date: 2010 Aug 22 PMID: 20732877
Authors: Gerin, I. - Clerbaux, L. A. - Haumont, O. - Lanthier, N. - Das, A. K. - Burant, C. F. - Leclercq, I. A. - Macdougald, O. A. - Bommer, G. T.
Journal: J Biol Chem

The regulation of synthesis, degradation, and distribution of lipids is crucial for homeostasis of organisms and cells. The sterol regulatory element binding protein (SREBP) transcription factor family is post-translationally activated in situations of reduced lipid abundance and activates numerous genes involved in cholesterol, fatty acid and phospholipid synthesis. In this study, we provide evidence that the primary transcript of SREBP2 contains an intronic miRNA (miR-33) that reduces cellular cholesterol export via inhibition of translation of the cholesterol export pump ABCA1. Notably, miR-33 also inhibits translation of several transcripts encoding proteins involved in fatty acid beta-oxidation including CPT1A, HADHB and CROT, thereby reducing fatty acid degradation. The genetic locus encoding SREBP2 and miR-33 therefore contains a protein that increases lipid synthesis and a miRNA that prevents export and degradation of newly synthesized lipids. These results add an additional layer of complexity to our understanding of lipid homeostasis and might open possibilities for future therapeutic intervention.

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ARACHIDONIC ACID-METABOLIZING CYTOCHROME P450 ENZYMES ARE TARGETS OF OMEGA-3 FATTY ACIDS.

Publication Date: 2010 Aug 23 PMID: 20732876
Authors: Arnold, C. - Markovic, M. - Blossey, K. - Wallukat, G. - Fischer, R. - Dechend, R. - Konkel, A. - von Schacky, C. - Luft, F. C. - Muller, D. N. - Rothe, M. - Schunck, W. H.
Journal: J Biol Chem

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) protect against cardiovascular disease by largely unknown mechanisms. We tested the hypothesis that EPA and DHA may compete with arachidonic acid (AA) for the conversion by cytochrome P450 (CYP) enzymes resulting in the formation of alternative, physiologically active, metabolites. Renal and hepatic microsomes, as well as various CYP-isoforms, displayed equal or elevated activities when metabolizing EPA or DHA instead of AA. CYP2C/2J-isoforms converting AA to epoxyeicosatrienoic acids (EETs) preferentially epoxidized the omega-3 double bond and thereby produced 17,18-epoxyeicosatetraenoic (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP) from EPA and DHA. We found that these omega-3 epoxides are highly active as antiarrhythmic agents, suppressing Ca2+-induced increased rate of spontaneous beating of neonatal rat cardiomyocytes, at low nanomolar concentrations. CYP4A/4F-isoforms omega-hydroxylating AA were less regioselective towards EPA and DHA, catalyzing predominantly omega- and omega minus-1 hydroxylation. Rats given dietary EPA/DHA supplementation exhibited substantial replacement of AA by EPA and DHA in membrane phospholipids in plasma, heart, kidney, liver, lung, and pancreas, with less pronounced changes in brain. The changes in fatty acids were accompanied by concomitant changes in endogenous CYP metabolite profiles (e.g. altering the EET:EEQ:EDP ratio from 87:0:13 to 27:18:55 in the heart). These results demonstrate that CYP-enzymes efficiently convert EPA and DHA to novel epoxy- and hydroxy-metabolites that could mediate some of the beneficial cardiovascular effects of dietary omega-3 fatty acids.

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Testicular expression of Adora3i2 in Adora3-knockout mice reveals a Role of mouse A3Ri2 and human A3Ri3 adenosine receptors in sperm.

Publication Date: 2010 Aug 23 PMID: 20732875
Authors: Burnett, L. A. - Blais, E. M. - Unadkat, J. D. - Hille, B. - Tilley, S. L. - Babcock, D. F.
Journal: J Biol Chem

Adenosine is a candidate modulator of sperm motility in the female reproductive tract that increases sperm flagellar beat frequency in vitro. Past work suggested that this acceleration may involve equilibrative (ENT) and concentrative (CNT) nucleoside transporters. Here we show that Slc29a1 (ENT-1) is the predominant nucleoside transporter expressed in the mouse testis. Unexpectedly, the beat of Slc29a1-null sperm still accelerates in response to 2-chloro-2'-deoxyadenosine (Cl-dAdo). Moreover, in wild-type sperm neither blockade of CNTs by removal of external Na+, nor inhibition of ENTs with nitrobenzylthioionosine, prevents acceleration of the sperm beat by Cl-dAdo. In contrast, pertussis toxin produces strong blockade, indicating involvement of a Galphai/o-coupled adenosine receptor. Although agonists selective for adenosine receptors A1R, A2aR, and A2bR are ineffective, A3R-selective agonists Cl-IB-MECA and IB-MECA do accelerate the beat. Consistent with this pharmacological profile, the predominant Adora transcripts in the testis are products of the nested Adora3i1 and Adora3i2 genes. Surprisingly, Cl-IB-MECA and Cl-dAdo still accelerate the beat of Adora3i1-null sperm indicating that the remaining Adora3i2 transcript produces an A3R that functions in sperm. When cloned Adora3i2 is heterologously expressed in tsA-201 cells, Cl-dAdo decreases forskolin-evoked accumulation of cAMP, indicating that Adora3i2 specifies a functional A3Ri2 adenosine receptor that couples through Galphai. Database mining reveals that mouse Adora3i2 is expressed primarily in testis, almost exclusively in spermatids. Expression of the orthologous ADORA3i3 transcript also is most prominent in human testis; presumably producing an A3Ri3 receptor that is functional in sperm and that may be a target for development of male-directed contraceptives.

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Activated PKC{delta} and PKC{epsilon} inhibit epithelial chloride secretion response to cAMP via inducing internalization of the Na+-K+-2Cl- cotransporter NKCC1.

Publication Date: 2010 Aug 23 PMID: 20732874
Authors: Tang, J. - Bouyer, P. - Mykoniatis, A. - Buschmann, M. - Matlin, K. S. - Matthews, J. B.
Journal: J Biol Chem

The basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) is a key determinant of transepithelial chloride secretion and dysregulation of chloride secretion is a common feature of many diseases including secretory diarrhea. We have previously shown that activation of protein kinase C (PKC) markedly reduces transepithelial chloride secretion in human colonic T84 cells, which correlates with both functional inhibition and loss of the NKCC1 surface expression. In the present study, we defined the specific roles of PKC isoforms in regulating epithelial NKCC1 and chloride secretion utilizing adenoviral vectors that express shRNAs targeting human PKC isoforms (alpha,delta,epsilon)(shPKCs) or LacZ (shLacZ, non-targeting control). After 72h of adenoviral transduction, protein levels of the PKC isoforms in shPKCs-T84 cells were decreased by ~90% compared to the shLacZ-control. Activation of PKCs by phorbol 12-myristate 13-acetate (PMA) caused a redistribution of NKCC1 immunostaining from the basolateral membrane to intracellular vesicles in both shLacZ- and shPKCalpha- T84 cells, whereas the effect of PMA was not observed in shPKCdelta- and shPKCepsilon- cells. These results were further confirmed by basolateral surface biotinylation. Furthermore, activation of PKCs by PMA inhibited cAMP-stimulated chloride secretion in the uninfected, shLacZ- and shPKCalpha-T84 monolayers, but the inhibitory effect was significantly attenuated in shPKCdelta- and shPKCepsilon-T84 monolayers. In conclusion, the activated novel isoform PKCdelta or PKCepsilon, but not the conventional isoform PKCalpha, inhibits the transepithelial chloride secretion through inducing internalization of the basolateral surface NKCC1. Our study reveals that the novel PKC isoforms-regulated NKCC1 surface expression plays an important role in the regulation of chloride secretion.

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Effects of ER stress on group via PLA2 (iPLA2{beta}) in beta cells include tyrosine phosphorylation and increased association with calnexin.

Publication Date: 2010 Aug 23 PMID: 20732873
Authors: Song, H. - Rohrs, H. - Tan, M. - Wohltmann, M. - Ladenson, J. H. - Turk, J.
Journal: J Biol Chem

The Group VIA PLA(2) (iPLA(2)beta) hydrolyzes glycerophospholipids at the sn-2 position to yield a free fatty acid and a 2-lysophospholipid, and iPLA(2)beta has been reported to participate in apoptosis, phospholipid remodeling, insulin secretion, transcriptional regulation, and other processes. Induction of Endoplasmic Reticulum (ER) stress in beta-cells and vascular myocytes with SERCA inhibitors activates iPLA(2)beta, resulting in hydrolysis of arachidonic acid from membrane phospholipids, by a mechanism that is not well understood. Regulatory proteins interact with iPLA(2)beta, including the Ca(2+)/Calmodulin-Dependent Protein Kinase IIbeta, and we have characterized the iPLA(2)beta interactome further using affinity capture and LC/ESI/MS/MS. An iPLA(2)beta-FLAG fusion protein was expressed in an INS-1 insulinoma cell line and then adsorbed to an anti-FLAG matrix after cell lysis. iPLA(2)beta and any associated proteins were then displaced with FLAG peptide and analyzed by SDS-PAGE. Gel sections were digested with trypsin, and the resultant peptide mixtures were analyzed by LC/MS/MS with database searching. This identified 37 proteins that associate with iPLA(2)beta, and nearly half of them reside in ER or mitochondria. They include the ER chaperone Calnexin, whose association with iPLA(2)beta increases upon induction of ER stress. Phosphorylation of iPLA(2)beta at Y616 also occurs upon induction of ER stress, and the phosphoprotein associates with Calnexin. The activity of iPLA(2)beta in vitro increases upon co-incubation with Calnexin, and overexpression of Calnexin in INS-1 cells results in augmentation of ER stress-induced, iPLA(2)beta-catalyzed hydrolysis of arachidonic acid from membrane phospholipids, reflecting the functional significance of the interaction. Similar results were obtained with mouse pancreatic islets.

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Cyclic AMP phosphodiesterase 4D (PDE4D) tethers EPAC1 in a VE-cadherin (VECAD)-based signaling complex and controls cAMP-mediated vascular permeability.

Publication Date: 2010 Aug 23 PMID: 20732872
Authors: Rampersad, S. N. - Ovens, J. D. - Houston, E. - Umana, M. B. - Wilson, L. S. - Netherton, S. J. - Lynch, M. J. - Baillie, G. S. - Houslay, M. D. - Maurice, D. H.
Journal: J Biol Chem

Vascular endothelial cell (VEC) permeability is largely dependent on the integrity of VE-cadherin (VECAD)-based intercellular adhesions. Activators of protein kinase A (PKA) or of exchange protein activated by cAMP (EPAC) reduce VEC permeability largely by stabilizing VECAD-based intercellular adhesions. Currently, little is known concerning the nature and composition of the signaling complexes which allow PKA or EPAC to regulate VECAD-based structures and through these actions control permeability. Using pharmacological, biochemical and cell biological approaches we identify and determine the composition and functionality of a signaling complex that coordinate cAMP-mediated control of VECAD-based adhesions and VEC permeability. Thus, we report that PKA, EPAC1 and cyclic nucleotide phosphodiesterase 4D (PDE4D) enzymes integrate into VECAD-based signaling complexes in human arterial endothelial cells (HAECs). Importantly, we show that protein-protein interactions between EPAC1 and PDE4D serve to foster their integration into VECAD-based complexes and allow robust local regulation of EPAC1-based stabilization of VECAD-based adhesions. Of potential translational importance, we mapped the EPAC1 peptide motif involved in binding PDE4D and show that a cell-permeable variant of this peptide antagonizes EPAC1-PDE4D binding and directly alters VEC permeability. Collectively, our data indicate that PDE4D regulates both the activity and subcellular localization of EPAC1 and identify a novel mechanism for regulated EPAC1 signaling in these cells.

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The Leo1 subunit of yeast Paf1 complex binds RNA and contributes to complex recruitment.

Publication Date: 2010 Aug 23 PMID: 20732871
Authors: Dermody, J. L. - Buratowski, S.
Journal: J Biol Chem

The Paf1 complex (Paf1C) affects RNA polymerase II transcription by coordinating co-transcriptional chromatin modifications and helping recruit mRNA 3' end processing factors. Paf1C crosslinks to transcribed genes, but not downstream of the cleavage and polyadenylation site, suggesting it may interact with the nascent mRNA. Paf1C purified from Saccharomyces cerevisiae binds RNA in vitro, as do the purified Leo1 and Rtf1 subunits of the complex. In vivo crosslinking and immunoprecipitation of RNA associated with Paf1C (RNA-IP) shows that Leo1, but not Rtf1, is necessary for the complex to bind RNA. Cells lacking Leo1 have reduced Paf1C recruitment as well as decreased levels of histone H3 and tri-methylated H3K4 within transcribed chromatin. Together, these results suggest that association of Paf1C with RNA stabilizes its localization at actively transcribed regions where it influences chromatin structure.

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Tethering of poly(A) binding protein interferes with non-translated mRNA decay from 5' end in yeast.

Publication Date: 2010 Aug 23 PMID: 20732870
Authors: Tsuboi, T. - Inada, T.
Journal: J Biol Chem

&The decay of eukaryotic mRNA is triggered mainly by deadenylation, which leads to decapping and degradation from the 5' end of an mRNA. Poly(A) binding protein has been proposed to inhibit the decapping process and to stabilize mRNA by blocking the recruitment of mRNA to the P-bodies where mRNA degradation takes place following stimulation of translation initiation. In contrast, several lines of evidence show that poly(A) binding protein (Pab1p) has distinct functions in mRNA decay and translation in yeast. To address the translation-independent function of Pab1p in inhibition of decapping, we examined the contribution of Pab1p to the stability of non-translated mRNAs, an AUG codon-less mRNA or an mRNA containing a stable stem-loop structure at the 5'-UTR. Tethering of Pab1p stabilized non-translated mRNAs, and this stabilization did not require either the eIF4G-interacting domain of Pab1p, or the Pab1p-interacting domain of eIF4G. In a ski2Delta mutant in which 3[prime] to 5' mRNA degradation activity is defective, stabilization of non-translated mRNAs by the tethering of Pab1p lacking an eIF4G-interacting domain (Pab1-34Cp) requires a cap structure but not a poly(A) tail. In wild type cells, stabilization of non-translated mRNA by tethered Pab1-34Cp results in the accumulation of deadenylated mRNA. These results strongly suggest that tethering of Pab1p may inhibit the decapping reaction after deadenylation, independent of translation. We propose that Pab1p inhibits the decapping reaction in a translation-independent manner in vivo.

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Both transcriptional regulation and translational control of ATF4 is central to the Integrated Stress Response.

Publication Date: 2010 Aug 23 PMID: 20732869
Authors: Dey, S. - Baird, T. D. - Zhou, D. - Palam, L. R. - Spandau, D. F. - Wek, R. C.
Journal: J Biol Chem

IIn response to different environmental stresses, phosphorylation of eIF2 (eIF2~P) represses global translation coincident with preferential translation of ATF4. ATF4 is a transcriptional activator of the Integrated Stress Response, a program of gene expression involved in metabolism, nutrient uptake, anti-oxidation, and the activation of additional transcription factors, such as CHOP/GADD153, that can induce apoptosis. While eIF2-P elicits translational control in response to many different stress arrangements, there are selected stresses, such as exposure to UV irradiation, that do not increase ATF4 expression despite robust eIF2~P. In this study, we addressed the underlying mechanism for variable expression of ATF4 in response to eIF2~P during different stress conditions, and the biological significance of omission of enhanced ATF4 function. We show that in addition to translational control, ATF4 expression is subject to transcriptional regulation. Stress conditions, such as endoplasmic reticulum stress, induce both transcription and translation of ATF4, which together enhance expression of ATF4 and its target genes in response to eIF2~P. By contrast, UV irradiation represses ATF4 transcription, which diminishes ATF4 mRNA available for translation during eIF2~P. eIF2~P enhances cell survival in response to UV irradiation. However, forced expression of ATF4, and its target gene CHOP, leads to increased sensitivity to UV irradiation. This combination of transcriptional regulation and translational control allows the eIF2 kinase pathway to selectively repress or activate key regulatory genes subject to preferential translation, providing the Integrated Stress Response versatility to direct the transcriptome that is essential for maintaining the balance between stress remediation and apoptosis.

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The effect of electrostatics on the marginal cooperativity of an ultrafast folding protein.

Publication Date: 2010 Aug 22 PMID: 20729560
Authors: Desai, T. M. - Cerminara, M. - Sadqi, M. - Munoz, V.
Journal: J Biol Chem

Proteins fold up by coordinating the different segments of their polypeptide chain through a network of weak cooperative interactions. Such cooperativity results in unfolding curves that are typically sigmoidal. However, we still do not know what factors modulate folding cooperativity, or what is the minimal amount that ensures folding into specific three-dimensional structures. Here we address these issues on BBL, a small helical protein that folds in microseconds via a marginally cooperative downhill process (Li,P., Oliva,F.Y., Naganathan, A.N., Munoz, V. (2009) Proc. Natl. Acad. Sci. USA. 106, 103-108). Particularly, we explore the effects of salt-induced screening of the electrostatic interactions in BBL at neutral pH and in acid-denatured BBL. Our results show that electrostatic screening stabilizes the native state of the neutral and protonated forms, inducing complete refolding of acid-denatured BBL. Furthermore, without net electrostatic interactions the unfolding process becomes much less cooperative, as judged by the broadness of the equilibrium unfolding curve and the relaxation rate. Our experiments show that the marginally cooperative unfolding of BBL can still be made twice as broad while the protein retains its ability to fold into the native 3D structure in microseconds. This result demonstrates experimentally that efficient folding does not require cooperativity, confirming predictions from theory and computer simulations, and challenging the conventional biochemical paradigm. Furthermore, we conclude that electrostatic interactions are an important factor in determining folding cooperativity. Thus, electrostatic modulation by pH-salt and/or mutagenesis of charged residues emerges as an attractive tool for tuning folding cooperativity.

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Chemoenzymatic design of heparan sulfate oligosaccharides.

Publication Date: 2010 Aug 21 PMID: 20729556
Authors: Liu, R. - Xu, Y. - Chen, M. - Weiwer, M. - Zhou, X. - Bridges, A. S. - Deangelis, P. L. - Zhang, Q. - Linhardt, R. J. - Liu, J.
Journal: J Biol Chem

Heparan sulfate is a sulfated glycan that exhibits essential physiological functions. Interrogation of the specificity of heparan sulfate-mediated activities demands a library of structurally defined oligosaccharides. Chemical synthesis of large heparan sulfate oligosaccharides remains challenging. We report the synthesis of oligosaccharides with different sulfation patterns and sizes from a disaccharide building block using glycosyltransferases, heparan sulfate C5-epimerase and sulfotransferases. This method offers a generic approach to prepare heparan sulfate oligosaccharides possessing predictable structures.

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Lipid binding requirements for oxysterol binding protein Kes1 inhibition of autophagy and endosome-trans-golgi trafficking pathways.

Publication Date: 2010 Aug 21 PMID: 20729555
Authors: Leblanc, M. A. - McMaster, C. R.
Journal: J Biol Chem

The Saccharomyces cerevisiae protein Kes1/Osh4 is a member of the enigmatic family of oxysterol binding proteins found throughout Eukarya united by a beta-barrel structure that binds sterols and oxysterols. In this study, we determine that phosphoinositides are the major determinant in membranes that faciliate Kes1 association both in vitro and in cells. Increased expression of Kes1 in yeast cells decreased the level of both phosphatidylinositol-4-phosphate (PI-4P) and phosphatidylinositol-3-phosphate (PI-3P). Phosphoinositide and sterol binding by Kes1 were necessary for Kes1 to decrease the level of PI-4P but not PI-3P. Kes1 inhibited vesicular trafficking between the trans-Golgi and plasma membrane as evidenced by accumulation of the v-SNARE Snc1 in the cytoplasmic vesicles. Sterol and phosphoinositide binding by Kes1 both contributed to its regulation of Snc1 trafficking. This study also describes a previously unknown role for Kes1 in regulation of the autophagy/cytoplasm to vacuole trafficking pathway. The Kes1 mediated regulation of the autophagy/ cytoplasm to vacuole trafficking pathway was prevented by increasing expression of the PI 3-kinase Vps34, suggesting that it is the Kes1 mediated decrease in PI-3P level that contributes to this regulation.

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Matrilin-1 A-domains: structural and functional investigations reveal insights into their role in cartilage ecm assembly.

Publication Date: 2010 Aug 21 PMID: 20729554
Authors: Fresquet, M. - Jowitt, T. A. - Stephen, L. A. - Ylostalo, J. - Briggs, M. D.
Journal: J Biol Chem

Matrilin-1 is expressed predominantly in cartilage and co-localizes with matrilin-3 with which it can form hetero-oligomers. We recently described novel structural and functional features of the matrilin-3 A-domain (M3A) and demonstrated that it bound with high affinity to type II and IX collagens. Interactions preferentially occurred in the presence of Zn2+ suggesting that matrilin-3 has acquired a requirement for specific metal-ions for activation and/or molecular associations. To understand the interdependency of matrilin-1/-3 hetero-oligomers in extracellular matrix (ECM) interactions, we have extended these studies to include the two matrilin-1 A-domains (i.e. M1A1 and M1A2 respectively). In this study we have identified new characteristics of the matrilin-1 A-domains by describing their glycosylation state and the effect of N-glycan chains on their structure, thermal stability and protein-protein interactions. Initial characterization revealed that N-glycosylation did not affect secretion of these two proteins, nor did not it alter their folding characteristics. However, removal of the glycosylation decreased their thermal stability. We then compared the effect of different cations on the binding between both M1A domains and type II and IX collagens and showed that Zn2+ also supports their interactions. Finally, we have demonstrated that both M1A1 domains and biglycan are essential for the association of the type II/VI collagen complex. We predict that a potential role of the matrilin-1/-3 hetero-oligomer might be to increase multivalency, and therefore the ability to connect various ECM components. Differing affinities could act to regulate the integrated network, thus coordinating the organization of the macromolecular structures in the cartilage ECM.

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Binding of procollagen C-proteinase enhancer-1 (PCPE-1) to heparin/heparan sulfate: Properties and role in PCPE-1 interaction with cells.

Publication Date: 2010 Aug 21 PMID: 20729553
Authors: Weiss, T. - Ricard-Blum, S. - Moschcovich, L. - Wineman, E. - Mesilaty, S. - Kessler, E.
Journal: J Biol Chem

Procollagen C-proteinase enhancer-1 (PCPE-1) is an extracellular matrix (ECM) glycoprotein that can stimulate procollagen processing by procollagen C-proteinases (PCPs) such as bone morphogenetic protein-1 (BMP-1). The PCPs can process additional extracellular protein precursors and play fundamental roles in developmental processes and assembly of the ECM. The stimulatory activity of PCPE-1 is restricted to the processing of fibrillar procollagens, suggesting PCPE-1 is a specific regulator of collagen deposition. PCPE-1 consists of two CUB domains that bind to the procollagen C-propeptides and are required for PCP enhancing activity, and one NTR domain that binds heparin. To understand the biological role of the NTR domain, we performed surface plasmon resonance (SPR) binding assays, cell attachment assays as well as immunofluorescence and activity assays, all indicating that the NTR domain can mediate PCPE-1 binding to cell surface heparan sulfate proteoglycans (HSPGs). The SPR data revealed binding affinities to heparin/HSPGs in the high nanomolar range and dependence on calcium. Both 3T3 mouse fibroblasts and human embryonic kidney cells (HEK-293) attached to PCPE-1, an interaction that was inhibited by heparin. Cell attachment was also inhibited by an NTR-specific antibody and the NTR fragment. Immunofluorescence analysis revealed that PCPE-Flag binds to mouse fibroblasts and heparin competes for this binding. Cell-associated PCPE-Flag stimulated procollagen processing by BMP-1 several fold. Our data suggest that through interaction with cell surface HSPGs, the NTR domain can anchor PCPE-1 to the cell membrane, permitting pericellular enhancement of PCP activity. This points at the cell surface as a physiological site of PCPE-1 action.

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ZEB1 coordinately regulates laminin-332 and {beta}4 integrin expression altering the invasive phenotype of prostate cancer cells.

Publication Date: 2010 Aug 21 PMID: 20729552
Authors: Drake, J. M. - Barnes, J. M. - Madsen, J. M. - Domann, F. E. - Stipp, C. S. - Henry, M. D.
Journal: J Biol Chem

Metastasis involves the invasion of cancer cells across both extracellular matrix and cellular barriers, and an evolving theme is that epithelial-to-mesenchymal transition (EMT) may mediate invasive cellular behavior. Previously, we isolated and analyzed a subpopulation of PC-3 prostate cancer cells, TEM4-18, and found that these cells both invaded an endothelial barrier more efficiently and exhibited enhanced metastatic colonization in vivo. Transendothelial migration of these cells depended on expression of ZEB1, a known regulator of EMT. Surprisingly, these cells were much less invasive than parental PC-3 cells in assays that involve matrix barriers. Here, we report that TEM4-18 cells express significantly reduced levels of two subunits of laminin-332 (beta3 and gamma2) and that exogenous laminin-332, or co-culture with laminin-332-expressing cells, rescues the in vitro invasion phenotype in these cells. Stable knockdown of ZEB1 in prostate cancer cells upregulated LAMC2 and ITGB4 mRNA and protein, and resulted in a concomitant increase in transwell migration. Using chromatin immunoprecipitation (ChIP), we show that ZEB1 directly interacts with the promoters of LAMC2 and ITGB4. These results provide a novel molecular basis for reduced laminin-332 observed in clinical prostate cancer specimens and demonstrate a context-dependent role for EMT in invasive cellular behavior.

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Nuclear Factor I-C regulates TGF-{beta}-dependent hair follicle cycling.

Publication Date: 2010 Aug 21 PMID: 20729551
Authors: Plasari, G. - Edelmann, S. - Hogger, F. - Dusserre, Y. - Mermod, N. - Calabrese, A.
Journal: J Biol Chem

Skin appendages such as teeth and hair share several common signalling pathways. The Nuclear Factor I C (NFI-C) transcription factor has been implicated in tooth development, but a potential role in hair growth had not been assessed. In this study, we found that NFI-C regulates the onset of the hair growth cycle. NFI-C(-/-) mice were delayed in the transition from the telogen to anagen phase of the hair follicle cycle, after either experimental depilation or spontaneous hair loss. Lack of NFI-C resulted in delayed induction of the sonic hedgehog, Wnt5a and Lef1 gene expression, which are key regulators of the hair follicle growth initiation. NFI-C(-/-) mice also showed elevated levels of transforming growth factor beta1 (TGF-beta1), an inhibitor of keratinocyte proliferation, and of the cell cycle inhibitor p21 at telogen. Reduced expression of the Ki67, a marker of cell proliferation was noted at the onset of anagen, indicating impaired activation of the hair progenitor cells. These findings implicate NFI-C in the repression of TGF-beta1 signalling during telogen, thus explaining the delayed progenitor cell proliferation and hair follicle regeneration of NFI-C-deficient mice. Taken together with prior observations, these findings also designate NFI-C as a regulator of adult progenitor cell proliferation and of postnatal tissue growth or regeneration.

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Extracellular microfibrils modulate osteoblast-supported osteoclastogenesis by restricting TGF beta stimulation of RANKL production.

Publication Date: 2010 Aug 21 PMID: 20729550
Authors: Nistala, H. - Lee-Arteaga, S. - Smaldone, S. - Siciliano, G. - Ramirez, F.
Journal: J Biol Chem

Mutations in fibrillin-1 or fibrilin-2, the major structural components of extracellular microfibrils, cause pleiotropic manifestations in Marfan syndrome and congenital contractural arachnodactyly, respectively. We recently found that fibrillin-1 and fibrillin-2 control bone formation by regulating osteoblast differentiation through the differential modulation of endogenous TGF beta and BMP signals. Here, we describe in vivo and ex vivo experiments that implicate the fibrillins as negative regulators of bone resorption. Adult Fbn2-/- mice display a greater than normal osteolytic response to locally implanted lipopolysaccharide-coated titanium particles. Whereas isolated cultures of Fbn2-/- pre-osteoclasts exhibited normal differentiation and activity, these features were substantially augmented when mutant or wild-type pre-osteoclasts were co-cultured with Fbn2-/- but not wild-type osteoblasts. Greater osteoclastogenic potential of Fbn2-/- osteoblasts was largely accounted for by up-regulation of the Rankl gene secondary to heightened TGF beta activity. This conclusion was based on the findings that blockade of TGF beta signaling blunts Rankl up-regulation in Fbn2-/- osteoblasts and bones, and that systemic TGF beta antagonism improves locally induced osteolysis in Fbn2-/- mice. Abnormally high Rankl expression secondary to elevated TGF beta activity was also noted in cultured osteoblasts from Fbn1-/- mice. Collectively our data demonstrated that extracellular microfibrils balance local catabolic and anabolic signals during bone remodeling, in addition to implying distinct mechanisms of bone loss in Marfan syndrome and congenital contractural arachnodactyly.

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O-GlcNAcylation determines the solubility, filament organization and stability of keratins 8 and 18.

Publication Date: 2010 Aug 21 PMID: 20729549
Authors: Srikanth, B. - Vaidya, M. M. - Kalraiya, R. D.
Journal: J Biol Chem

Keratins 8 and 18 (K8/18) are intermediate filament proteins expressed specifically in simple epithelial tissues. Dynamic equilibrium of these phospho-glycoproteins in the soluble and filament pool is an important determinant of their cellular functions, and is known to be regulated by site specific phosphorylation. However, little is known about the role of dynamic O-GlcNAcylation on this keratin pair. Here, by comparing immortalized (Chang) and transformed hepatocyte (HepG2) cell lines we have demonstrated that O-GlcNAcylation of K8/18 exhibits a positive correlation with their solubility (NP-40 extractability). Heat stress which increases K8/18 solubility resulted in simultaneous increase in O-GlcNAc on them. Conversely, increasing O-GlcNAc levels was associated with concurrent increase in their solubility. The latter, however, was associated with notable decrease in total cellular levels of K8/18. Unaltered levels of transcripts and reduced half life of K8 and 18 indicated their decreased stability on increasing O-GlcNAcylation. On the contrary, K18 glycosylation mutant (GM) (K18 Ser 29,30,48 to Ala ) was notably more stable than the wild type (WT) K18, in Chang cells. K18-GM accumulated as aggregates upon stable expression which possibly altered endogenous filament architecture. These results strongly indicate the involvement of O-GlcNAc on K8/18 in regulating their solubility and stability which may have a bearing on functions of these keratins.

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Collagen VI microfibril formation is abolished by an {alpha}2(VI) von Willebrand factor A-domain mutation in a patient with Ullrich congenital muscular dystrophy.

Publication Date: 2010 Aug 21 PMID: 20729548
Authors: Tooley, L. D. - Zamurs, L. K. - Beecher, N. - Baker, N. L. - Peat, R. A. - Adams, N. E. - Bateman, J. F. - North, K. N. - Baldock, C. - Lamande, S. R.
Journal: J Biol Chem

Collagen VI is an extracellular protein that most often contains the three genetically distinct polypeptide chains, alpha1(VI), alpha2(VI) and alpha3(VI), although three recently identified chains, alpha4(VI), alpha5(VI) and alpha6(VI), may replace alpha3(VI) in some situations. Each chain has a triple helix flanked by N- and C- terminal globular domains that share homology with the von Willebrand Factor type-A (VWA) domains. During biosynthesis, the three chains come together to form triple helical monomers, which then assemble into dimers and tetramers. Tetramers are secreted from the cell and align end-to-end to form microfibrils. The precise molecular mechanisms responsible for assembly are unclear. Mutations in the three collagen VI genes can disrupt collagen VI biosynthesis and matrix organisation, and are the cause of the inherited disorders Bethlem myopathy and Ullrich Congenital Muscular Dystrophy (UCMD). We have identified a UCMD patient with compound heterozygous mutations in alpha2(VI). The first mutation causes skipping of exon 24, and the mRNA is degraded by nonsense-mediated decay. The second mutation is a two amino acid deletion in the C1 VWA domain. Recombinant C1 domains containing the deletion are insoluble and retained intracellularly, indicating the mutation has detrimental effects on domain folding and structure. Despite this, mutant alpha2(VI) chains retain the ability to associate into monomers, dimers and tetramers. However, we show that secreted mutant tetramers containing structurally abnormal C1 VWA domains are unable to associate further into microfibrils, directly demonstrating the critical importance of a correctly folded alpha2(VI) C1 domain in microfibril formation.

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Chondroitin sulfate synthase-2/chondroitin polymerizing factor has two variants with distinct function.

Publication Date: 2010 Aug 21 PMID: 20729547
Authors: Ogawa, H. - Shionyu, M. - Sugiura, N. - Hatano, S. - Nagai, N. - Kubota, Y. - Nishiwaki, K. - Sato, T. - Gotoh, M. - Narimatsu, H. - Shimizu, K. - Kimata, K. - Watanabe, H.
Journal: J Biol Chem

Chondroitin sulfate (CS) is a polysaccharide consisting of repeating disaccharide units of N-acetyl-D-galactosamine and D-glucuronic acid residues, modified with sulfated residues at various positions. To date, six glycosyltransferases for chondroitin synthesis have been identified, and the complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 (ChSy-1) and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor (ChPF) is assumed to play a major role in CS biosynthesis. We found an alternative splice variant of mouse CSS2 in a database, which lacks the N-terminal transmembrane domain, contrasting to the original CSS2. Here, we investigated the roles of CSS2 variants. Both the original enzyme and the splice variant, designated CSS2A and CSS2B respectively, were expressed at different levels and ratios in tissues. Western blot analysis of cultured mouse embryonic fibroblasts confirmed that both enzymes were actually synthesized as proteins, and were localized in both the endoplasmic reticulum and the Golgi apparatus. Pull-down assay revealed that either of CSS2A, CSS2B, and CSS1/ChSy-1 heterogenously and homogeneously interacts with each other, suggesting that they form a complex of multimers. In vitro glycosyltransferase assays demonstrated a reduced glucuronyltransferase activity in CSS2B and no polymerizing activity in CSS2B co-expressed with CSS1 in contrast to CSS2A co-expressed with CSS1. Radiolabeling analysis of cultured COS-7 cells overexpressing each variant revealed that, whereas CSS2A facilitated CS biosynthesis, CSS2B inhibited it. Molecular modeling of CSS2A and CSS2B provided support for their properties. These findings, implicating regulation of CS chain polymerization by CSS2 variants, provide insight in elucidating the mechanisms of CS biosynthesis.

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Angiopoietin-like 4 interacts with matrix proteins to modulate wound healing.

Publication Date: 2010 Aug 21 PMID: 20729546
Authors: Goh, Y. Y. - Pal, M. - Chong, H. C. - Zhu, P. - Tan, M. J. - Punugu, L. - Tan, C. K. - Huang, R. L. - Sze, S. K. - Tang, M. B. - Ding, J. L. - Kersten, S. - Tan, N. S.
Journal: J Biol Chem

A dynamic cell-matrix interaction is crucial for a rapid cellular response to changes in the environment. Appropriate cell behavior in response to the changing wound environment is required for efficient wound closure. However, the way in which wound keratinocytes modify the wound environment to coordinate with such cellular responses remains less studied. We demonstrated that angiopoietin-like 4 (ANGPTL4) produced by wound keratinocytes coordinates cell-matrix communication. ANGPTL4 interacts with vitronectin and fibronectin in the wound bed, delaying their proteolytic degradation by metalloproteinases. This interaction does not interfere with integrin-matrix protein recognition and directly affects cell-matrix communication by altering the availability of intact matrix proteins. These interactions stimulate integrin-FAK, 14-3-3 and PKC-mediated signaling pathways essential for effective wound healing. The deficiency of ANGPTL4 in mice delays wound re-epithelialization. Further analysis revealed that cell migration was impaired in the ANGPTL4-deficient keratinocytes. Altogether, the findings provide molecular insight into a novel control of wound healing via ANGPTL4-dependent regulation of cell-matrix communication. Given the known role of ANGPTL4 in glucose and lipid homeostasis, it is a prime therapeutic candidate for the treatment of diabetic wounds. It also underscores the importance of cell-matrix communication during angiogenesis and cancer metastasis.

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Role of DnaJ G/F-rich domain in conformational recognition and binding of protein substrates.

Publication Date: 2010 Aug 20 PMID: 20729526
Authors: Perales-Calvo, J. - Muga, A. - Moro, F.
Journal: J Biol Chem

DnaJ from Escherichia coli is a Type I Hsp40 that functions as a cochaperone of DnaK (Hsp70), stimulating its ATPase activity and delivering protein substrates. How DnaJ binds protein substrates is still poorly understood. Here we have studied the role of DnaJ G/F-rich domain in binding of several substrates with different conformational properties (folded, partially (un)folded and unfolded). Using partial proteolysis we find that RepE, a folded substrate, contacts a wide DnaJ area that involves part of the G/F-rich region and Zn-binding domain. Deletion of G/F-rich region hampers binding of native RepE and reduced the affinity for partially (un)folded substrates. However, binding of completely unfolded substrates is independent on the G/F-rich region. These data indicate that DnaJ distinguishes the substrate conformation and is able to adapt the use of the G/F-rich region to form stable substrate-complexes.

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The bell-shaped curve for peroxynitrite-mediated oxidation and nitration of NO/O2-* is alive and well.

Publication Date: 2010 Aug 27 PMID: 20729216
Authors: Jourd'heuil, D. - Lancaster, J. R. Jr - Fukuto, J. - Roberts, D. D. - Miranda, K. M. - Mayer, B. - Grisham, M. B. - Wink, D. A.
Journal: J Biol Chem

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A highly unusual thioester bond in a pilus adhesin is required for efficient host cell interaction.

Publication Date: 2010 Aug 19 PMID: 20729215
Authors: Pointon, J. A. - Smith, W. D. - Saalbach, G. - Crow, A. - Kehoe, M. A. - Banfield, M. J.
Journal: J Biol Chem

Many bacterial pathogens present adhesins at the tips of long macromolecular filaments known as pili that are often important virulence determinants. Very little is known about how pili presented by Gram positive pathogens mediate host cell binding. The crystal structure of a pilus adhesin from the important human pathogen Streptococcus pyogenes reveals an internal thioester bond formed between the side chains of a cysteine and a glutamine residue. The presence of the thioester was verified using UV-vis spectroscopy and mass spectrometry. This unusual bond has only previously been observed in thioester domains of complement and complement-like proteins, where it is used to form covalent attachment to target molecules. The structure also reveals two intramolecular isopeptide bonds, one of these formed through a Lys/Asp residue pair, that are strategically positioned to confer protein stability. Removal of the internal thioester by allele-replacement mutagenesis in S. pyogenes severely compromises bacterial adhesion to model host cells. Whilst current paradigms of bacterial/host cell interaction envisage strong non-covalent interactions, the present study suggests cell adhesion could also involve covalent bonds.

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Bacterial pathogen-associated molecular patterns stimulate biological activity of orthopaedic wear particles by activating cognate toll-like receptors.

Publication Date: 2010 Aug 19 PMID: 20729214
Authors: Greenfield, E. M. - Beidelschies, M. A. - Tatro, J. M. - Goldberg, V. M. - Hise, A. G.
Journal: J Biol Chem

Aseptic loosening of orthopaedic implants is induced by wear particles generated from the polymeric and metallic components of the implants. Substantial evidence suggests that activation of Toll-like receptors (TLRs) may contribute to the biological activity of the wear particles. Although pathogen-associated molecular patterns (PAMPs) produced by Gram-positive bacteria are likely to be more common in patients with aseptic loosening, prior studies have focused on lipopolysacharide (LPS), a TLR4 specific PAMP produced by Gram-negative bacteria. Here we show that lipoteichoic acid (LTA), a PAMP produced by Gram-positive bacteria that activates TLR2, can, like LPS, adhere to the particles and increase their biological activity. Moreover, three lines of evidence support the conclusion that TLR activation requires bacterially-derived PAMPs and that endogenously-produced danger-associated molecular patterns (DAMPs) are not sufficient. First, neither TLR2 nor TLR4 contribute to the activity of endotoxin-free particles, as would be the expected if DAMPs are sufficient to activate the TLRs. Second, non-cognate TLRs do not contribute to the activity of particles with adherent LPS or LTA, as would be expected if DAMPs are sufficient to activate the TLRs. Third, polymyxin B, which inactivates LPS blocks the activity of particles with adherent LPS. These results support the hypothesis that PAMPs produced by low levels of bacterial colonization may contribute to aseptic loosening of orthopaedic implants.

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Inhibitors of MAPK pathway ERK1/2 or p38 prevent the IL-1 beta-induced upregulation of SRP72 autoantigen in Jurkat cells.

Publication Date: 2010 Aug 19 PMID: 20729213
Authors: Arana-Argaez, V. E. - Delgado-Rizo, V. - Pizano-Martinez, O. E. - Martinez-Garcia, E. A. - Martin-Marquez, B. T. - Munoz-Gomez, A. - Petri, M. H. - Armendariz-Borunda, J. - Espinosa-Ramirez, G. - Zuniga-Tamayo, D. A. - Herrera-Esparza, R. - Vazquez-Del Mercado, M.
Journal: J Biol Chem

ABSTRACT Phosphorylation is the most important post-translational event at cellular level regulated by protein kinases. Mitogen activated protein kinase (MAPK) is a key player in the important cellular signaling pathway. It has been hypothesized that phosphorylation might have a role in the induction of break tolerance against some autoantigens such as SRP72. AIM: The aim of this study is to explore possible mechanisms related to the breakdown of tolerance against the SRP72 polypeptide using an in vitro model of Jurkat cells stimulated by rhIL-1beta in the presence of MAPK inhibitors. MATERIAL AND METHODS: We used Jurkat cells as a substrate stimulated with rhIL-1beta in the presence of MAPK inhibitors at different concentrations in a time course in vitro experiment by immunoprecipitation (IP), IP-western blotting and real time PCR. RESULTS: Our results showed that rhIL-1beta causes upregulation of protein expression and phosphorylation of SRP72 in Jurkat cells. Inhibitors of MAPK pathway ERK1/2 or p38 alpha/p38 beta; downregulate the expression of SRP72 autoantigen in Jurkat cells stimulated by rhIL-1beta. CONCLUSIONS: Our results highlight the importance of studying the pathways of activation and overexpression of autoantigens. It will be necessary to perform careful research on various kinases pathways including MAPK in Dermatomyositis and other rheumatic diseases, in order to help to explain the routes of activation and inhibition of autoantigens. The understanding of this process may help developing the design of new therapies to prevent and control the loss of the tolerance towards own molecules.

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Hyperphosphorylation by Cyclin B/CDK1 in Mitosis Resets CUX1 DNA binding Clock at each Cell Cycle.

Publication Date: 2010 Aug 19 PMID: 20729212
Authors: Sansregret, L. - Gallo, D. - Santaguida, M. - Leduy, L. - Harada, R. - Nepveu, A.
Journal: J Biol Chem

The p110 CUX1 homeodomain protein participates in the activation of DNA replication genes in part by increasing the affinity of E2F factors for the promoters of these genes. CUX1 expression is very weak in quiescent cells and increases during G1. Biochemical activities associated with transcriptional activation by CUX1 are potentiated by post-translational modifications in late G1, notably a proteolytic processing event that generates p110 CUX1. Constitutive expression of p110 CUX1, as observed in some transformed cells, leads to accelerated entry into S phase. In the present study, we investigated the post-translation regulation of CUX1 during mitosis and early G1 phases of proliferating cells. We observed a major electrophoretic mobility shift and a complete inhibition of DNA binding during mitosis. We show that cyclin B/CDK1 interacts with CUX1 and phosphorylates it at multiple sites. Serine to alanine replacement mutations at 10 SP dipeptide sites were required to restore DNA binding in mitosis. Passage into G1 was associated with the degradation of some p110 CUX1 proteins, while remaining proteins were gradually dephosphorylated. Indirect immunofluorescence and sub-fractionation assays using a phospho-specific antibody showed that most of the phosphorylated protein remained in the cytoplasm while the dephosphorylated protein was preferentially located in the nucleus. Globally our results indicate that the hyper-phosphorylation of CUX1 by cyclin B/CDK1 inhibits its DNA binding activity in mitosis and interferes with its nuclear localization following cell division and formation of the nuclear membrane, while dephosphorylation and de novo synthesis contribute to gradually restore CUX1 expression and activity in G1.

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Potentiation of polarized intestinal Caco-2 cell responsiveness to probiotics complexed with secretory IgA.

Publication Date: 2010 Aug 20 PMID: 20729211
Authors: Mathias, A. - Duc, M. - Favre, L. - Benyacoub, J. - Blum, S. - Corthesy, B.
Journal: J Biol Chem

The precise mechanisms underlying the interaction between intestinal bacteria and the host epithelium lead to multiple consequences that remain poorly understood at the molecular level. Deciphering such events can provide valuable information as to the mode of action of commensal and probiotic microorganisms in the gastrointestinal environment. Potential roles of such microorganisms along the privileged target represented by the mucosal immune system include maturation prior, during and after weaning, and the reduction of inflammatory reactions in pathogenic conditions. Using human intestinal epithelial Caco-2 cell grown as polarized monolayers, we found that association of a Lactobacillus or a Bifidobacterium with nonspecific secretory IgA (SIgA) enhanced probiotic adhesion by a factor of 3.4-fold or more. Bacteria alone or in complex with SIgA reinforced transepithelial electrical resistance, a phenomenon coupled with increased phosphorylation of tight junction proteins zonula occludens-1 and occludin. In contrast, association with SIgA resulted in both enhanced level of nuclear translocation of NF-kappaB and production of epithelial polymeric Ig receptor as compared to bacteria alone. Moreover, thymic stromal lymphopoietin production was increased upon exposure to bacteria and further enhanced with SIgA-based complexes, whereas the level of pro-inflammatory epithelial cell mediators remained unaffected. Interestingly, SIgA-mediated potentiation of the Caco-2 cell responsiveness to the two probiotics tested involved Fab-independent interaction with the bacteria. These findings add to the multiple functions of SIgA and underscore a novel role of the antibody in interaction with intestinal bacteria.

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BI-specific MHC heterodimers for characterization of cross-reactive T cells.

Publication Date: 2010 Aug 20 PMID: 20729210
Authors: Shen, Z. T. - Brehm, M. A. - Daniels, K. A. - Sigalov, A. B. - Selin, L. K. - Welsh, R. M. - Stern, L. J.
Journal: J Biol Chem

T cell cross-reactivity describes the phenomenon whereby a single T cell can recognize two or more different peptide antigens presented in complex with MHC proteins. Cross-reactive T cells previously have been characterized at the population level by cytokine secretion and MHC tetramer staining assays, but single-cell analysis is difficult or impossible using these methods. In this study, we describe development of a novel peptide-MHC heterodimer specific for cross-reactive T cells. MHC-peptide monomers were independently conjugated to hydrazide or aldehyde-containing crosslinkers using thiol-maleimide coupling at cysteine residues introduced into recombinant MHC heavy chain proteins. Hydrazone formation provided bi-specific MHC heterodimers carrying two different peptides. Using this approach we prepared heterodimers of the murine class I MHC protein H-2Kb carrying peptides from lymphocytic choriomeningitis virus (LCMV) and vaccinia virus (VV), and used these to identify cross-reactive CD8+ T cells recognizing both LCMV and vaccinia virus antigens. A similar strategy could be used to develop reagents to analyze cross-reactive T cell responses in humans.

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Suppressive Treg cell activity is potentiated by glycogen synthase kinase 3{beta} inhibition.

Publication Date: 2010 Aug 20 PMID: 20729209
Authors: Graham, J. - Fray, M. - de Haseth, S. - Lee, K. M. - Lian, M. M. - Chase, C. M. - Madsen, J. C. - Markmann, J. - Benichou, G. - Colvin, R. B. - Cosimi, A. B. - Deng, S. - Kim, J. - Alessandrini, A.
Journal: J Biol Chem

The mechanism by which regulatory T cells (Tregs) suppress the immune response is not well defined. A recent study has shown that beta-catenin prolongs Treg survival. Since beta-catenin is regulated by GSK-3beta directed phosphorylation, we focused on GSK-3beta and the role it plays in Treg function. Inhibition of GSK-3beta leads to increased suppression activity by Tregs. Inhibitor-treated Tregs exhibited prolonged FoxP3 expression and increased levels of beta-catenin and of the anti-apoptotic protein, Bcl-XL. Systemic administration of GSK-3beta inhibitor results in prolonged islet survival in an allotransplant mouse model. Our data suggest GSK-3beta could be a useful target in developing strategies designed to increase the stability and function of Tregs for inducing allotransplant tolerance or treating autoimmune conditions.

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Heme amplifies the innate immune response to microbial molecules through spleen tyrosine kinase (SYK)-dependent ROS generation.

Publication Date: 2010 Aug 20 PMID: 20729208
Authors: Fernandez, P. L. - Dutra, F. F. - Alves, L. - Figueiredo, R. T. - Mourao-Sa, D. - Fortes, G. B. - Bergstrand, S. - Lonn, D. - Cevallos, R. R. - Pereira, R. M. - Lopes, U. G. - Travassos, L. H. - Paiva, C. N. - Bozza, M. T.
Journal: J Biol Chem

Infectious diseases that cause hemolysis are among the most threatening human diseases, because of severity and/or global distribution. In these conditions hemeproteins and heme are released, but whether heme affects the inflammatory response to microorganism molecules remains to be characterized. Here, we show that heme increased the lethality and cytokine secretion induced by LPS in vivo, and enhanced the secretion of cytokines by macrophages stimulated with various agonists of innate immune receptors. Activation of nuclear factor kappaB (NF-kappaB), mitogen-activated protein kinases (MAPKs) and generation of reactive oxygen species (ROS) were essential to the increase in cytokine production induced by heme plus LPS. This synergistic effect of heme and LPS was blocked by a selective inhibitor of Spleen tyrosine kinase (Syk) and abrogated in dendritic cells deficient of Syk. Moreover, inhibition of Syk and the downstream molecules protein kinase C (PKC) and phosphoinositide 3-kinase (PI3K) reduced the ROS generation by heme. Our results highlight a mechanism by which heme amplifies the secretion of cytokines triggered by microbial molecule activation, and indicates possible pathways for therapeutic intervention during hemolytic infectious diseases.

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The binding of Toxoplasma gondii glycosylphosphatidylinositols to galectin-3 is required for their recognition by macrophages.

Publication Date: 2010 Aug 20 PMID: 20729207
Authors: Debierre-Grockiego, F. - Niehus, S. - Coddeville, B. - Elass, E. - Poirier, F. - Weingart, R. - Schmidt, R. R. - Mazurier, J. - Guerardel, Y. - Schwarz, R. T.
Journal: J Biol Chem

We showed that the production of tumor necrosis factor alpha by macrophages in response to Toxoplasma gondii glycosylphosphatidylinositols (GPIs) requires the expression of both Toll-Like Receptors TLR2 and TLR4, but not of their co-receptor CD14. Galectin-3 is a beta-galactoside-binding protein with immune-regulatory effects, which associates with TLR2. We demonstrate here by using the surface plasmon resonance method that the GPIs of T. gondii bind to human galectin-3 with strong affinity and in a dose-dependent manner. The use of a synthetic glycan and of the lipid moiety cleaved from the GPIs show that both parts are involved in the interaction with galectin-3. GPIs of T. gondii also bind to galectin-1 but with a lower affinity and only through the lipid moiety. At the cellular level, the production of tumor necrosis factor alpha induced by T. gondii GPIs in macrophages depends on the expression of galectin-3 but not of galectin-1. This study is the first identification of a galectin-3 ligand of T. gondii origin, and galectin-3 might be a co-receptor presenting the GPIs to the TLRs on macrophages.

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M-calpain activation is regulated by its membrane localization and by its binding to PIP2.

Publication Date: 2010 Aug 20 PMID: 20729206
Authors: Leloup, L. - Shao, H. - Bae, Y. H. - Deasy, B. - Stolz, D. - Roy, P. - Wells, A.
Journal: J Biol Chem

M-calpain plays a critical role in cell migration. Growth factors and chemokines regulate keratinocyte, fibroblast and endothelial cell migration by modulating m-calpain activity. Growth factor receptors activate m-calpain secondary to phosphorylation. Concurrently, activated m-calpain is localized to its inner membrane milieu by binding to PIP2. Opposing this, CXCR3 ligands inhibit m-calpain activity secondary to a PKA-mediated phosphorylation. The failure of m-calpain activation in the absence of PIP2 points to a key regulatory role, though whether this PIP2-mediated membrane localization is regulatory for m-calpain activity or merely serves as a docking site for ERK phosphorylation is uncertain. Herein, we report the effects of two CXCR3 ligands on the EGF- and VEGF-induced redistribution of m-calpain in human fibroblasts and endothelial cells. The chemokines block tail retraction and migration within minutes, preventing and reverting the growth factor-induced relocalization of m-calpain to the plasma membrane. PKA phosphorylation blocks the binding to PIP2. Unexpectedly, we found that activation was due to membrane anchorage itself and not merely serine50 phosphorylation, as farnesylation-induced anchorage of m-calpain triggers a strong activation. Moreover, the ERK and PKA phosphorylations have no effect on this membrane-anchored m-calpain. However, PIP2 is required for the activation of anchored m-calpain. In conclusion, we describe a novel mechanism of m-calpain activation by interaction with the plasma membrane and PIP2 specifically, this phosphoinositide acting as a cofactor for the enzyme. Phosphorylation of m-calpain by ERK and PKA by growth factors and chemokines, respectfully, act in cells to regulate the enzyme only indirectly by controlling its redistribution.

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Exposure to hydrogen peroxide induces oxidation and activation of AMP-activated protein kinase.

Publication Date: 2010 Aug 20 PMID: 20729205
Authors: Zmijewski, J. W. - Banerjee, S. - Bae, H. - Friggeri, A. - Lazarowski, E. R. - Abraham, E.
Journal: J Biol Chem

Although metabolic conditions associated with an increased AMP to ATP ratio are primary factors in the activation of 5'adenosine monophosphate-activated protein kinase (AMPK), a number of recent studies have shown that increased intracellular levels of reactive oxygen species (ROS) can stimulate AMPK activity, even without decrease in cellular levels of ATP. We found that exposure of recombinant AMPKalphabetagamma complex or HEK 293 cells to H(2)O(2) was associated with increased kinase activity and also resulted in oxidative modification of AMPK, including S-glutathionylation of the AMPKalpha and beta subunits. In experiments using C-terminal truncation mutants of the AMPKalpha (1-312 aa), we found that mutation of cysteine 299 to alanine diminished the ability of H(2)O(2) to induces kinase activation and mutation of cysteine 304 to alanine totally abrogated the enhancing effect of H(2)O(2) on kinase activity. Similar to the results obtained with H(2)O(2)- treated HEK 293 cells, activation and S-glutathionylation of the AMPKalpha subunit were present in the lungs of acatalasemic mice or mice treated with the catalase inhibitor aminotriazole, conditions in which intracellular steady state levels of H(2)O(2) are increased. These results demonstrate that physiologically relevant concentrations of H(2)O(2) can activate AMPK through oxidative modification of the AMPKalpha subunit. The present findings also imply that AMPK activation, in addition to being a response to alterations in intracellular metabolic pathways, is directly influenced by cellular redox status.

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Selective yolk deposition and mannose phosphorylation of lysosomal glycosidases in zebrafish.

Publication Date: 2010 Aug 20 PMID: 20729204
Authors: Fan, X. - Klein, M. - Flanagan-Steet, H. R. - Steet, R.
Journal: J Biol Chem

The regulation and function of lysosomal hydrolases during yolk consumption and embryogenesis in zebrafish are poorly understood. In an effort to better define the lysosomal biochemistry of this organism, we analyzed the developmental expression, biochemical properties and function of several glycosidases in zebrafish eggs, embryos and adult tissues. Our results demonstrated that the specific activity of most enzymes increases during embryogenesis, likely reflecting a greater need for turnover within the embryo as yolk-derived nutrients are depleted. Analysis of glycosidase activity in zebrafish and medaka eggs revealed selective deposition of enzymes required for the degradation of N-linked glycans, including an abundance of acidic mannosidases. Treatment of zebrafish embryos with the alpha-mannosidase inhibitor swainsonine resulted in the accumulation of glycosylated vitellogenin fragments, demonstrated a function for maternally deposited acid alpha-mannosidase in yolk consumption. Surprisingly, we also found that, unlike mammals, acid alpha-glucosidase from zebrafish and medaka does not appear to be modified with mannose 6-phosphate residues. We further showed these residues were not acquired on human acid alpha-glucosidase when expressed in zebrafish embryos, suggesting unique differences in the ability of the human and zebrafish N-acetylglucosamine-1-phosphotransferase to recognize and modify certain lysosomal glycosidases. Together, these results provide novel insight into the role of acidic glycosidases during yolk utilization and the evolution of the mannose 6-phosphate targeting system in vertebrates.

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MAPKKK-dependent and -independent activation of Sty1 stress MAPK in fission yeast.

Publication Date: 2010 Aug 20 PMID: 20729203
Authors: Zhou, X. - Ma, Y. - Sugiura, R. - Kobayashi, D. - Suzuki, M. - Deng, L. - Kuno, T.
Journal: J Biol Chem

In fission yeast, Sty1/Spc1/Phh1 mitogen-activated protein kinase (MAPK) pathway is known to be involved in multiple-stress responses. It is currently thought that Sty1 MAPK cascade is mediated by histidine kinases and phosphorelay proteins in response to oxidative stress signals. However, studies of the exact transduction mechanism of multiple-stress responses are lacking. Thus, in response to various stimuli, we monitored the Sty1 MAPK pathway through the downstream transcription factor Atf1 in living cells using a highly sensitive luciferase reporter gene. Surprisingly in cadmium and low-glucose (LG) medium, Atf1 activation was observed even in the absence of all of the four fission yeast MAPK kinase kinases (MAPKKKs); whereas in osmotic stress, Atf1 activation was abolished. Thus the osmotic stress likely mediates the MAPK activation via MAPKKKs, whereas cadmium or LG condition activates the MAPK in a MAPKKK-independent manner. On the other hand, knockout of tyrosine phosphatase gene pyp1(+) abolished Atf1 response to cadmium and LG, but not to osmotic stress, suggesting that Pyp1 is a sensor for cadmium and LG.

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Synergistic activation of nuclear factor kappaB (NF-kappaB)by bacterial chemoattractant and tumor necrosis factor alpha (TNFalpha) is mediated by p38MAPK-dependent RelA acetylation.

Publication Date: 2010 Aug 20 PMID: 20729202
Authors: Pan, W. W. - Li, J. D. - Huang, S. - Papadimos, T. J. - Pan, Z. K. - Chen, L. Y.
Journal: J Biol Chem

In the host immune system, leukocytes are often exposed to multiple inflammation inducers. NF-kB is of considerable importance in leukocyte function owing to its ability to activate the transcription of many proinflammatory immediate-early genes. Tremendous efforts have been made toward understanding how NF-kB is activated by various inducers. However, most research on NF-kB regulation has been focused on understanding how NF-kB is activated by a single inducer. This is unlike the situation in human immune system where multiple inflammation inducers, including both exogenous and endogenous mediators, are present concurrently. We now present evidence that fMLP, a bacterial chemoattractant, synergizes with TNFa to induce NF-kB activation and the resultant inflammatory response in vitro and in vivo. The mechanism of synergistic activation of NF-kB by bacterial fMLP and TNFa may involve in induction of RelA acetylation, which is regulated by p38 MAP kinase. Thus, the present studies provide direct evidence for the synergistic induction of NF-kB-dependent inflammatory responses by both exogenous and endogenous inducers. The ability of fMLP to synergize with TNFa and activate NF-kB represents a novel and potentially important mechanism through which bacterial fMLP can not only attracts leukocytes but also directly contribute to inflammation by synergizing with endogenous mediator TNFa.

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Rad9 is required for B cell proliferation and immunoglobulin class switch recombination.

Publication Date: 2010 Aug 20 PMID: 20729201
Authors: An, L. - Wang, Y. - Liu, Y. - Yang, X. - Liu, C. - Hu, Z. - He, W. - Song, W. - Hang, H.
Journal: J Biol Chem

B cell maturation and B cell-mediated antibody response require programmed DNA modifications such as the V(D)J recombination, the immunoglobulin (Ig) class switch recombination (CSR) and the somatic hypermutaion (SHM) to generate functional Igs. Many protein factors involved in DNA damage repair have been shown to be critical for the maturation and activation of B cells. Rad9 plays an important role in both DNA repair and cell cycle checkpoint control. However, its role in Ig generation has not been reported. In this study, we generated a conditional knockout mouse line in which Rad9 is deleted specifically in B cells and investigated the function of Rad9 in B cells. The Rad9-/- B cells isolated from the conditional knockout mice displayed impaired growth response and enhanced DNA lesions. Impaired Ig production in response to immunization in Rad9-/- mice was also detected. In addition, the Ig CSR is deficient in Rad9-/- B cells. Taken together, Rad9 plays dual roles in generating functional antibodies and in maintaining the integrity of the whole genome in B cells.

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A dynamic cpSRP43-albino3 interaction mediates translocase regulation of cpSRP targeting components.

Publication Date: 2010 Aug 20 PMID: 20729200
Authors: Lewis, N. E. - Marty, N. J. - Kathir, K. M. - Rajalingam, D. - Kight, A. D. - Daily, A. - Kumar, T. K. - Henry, R. L. - Goforth, R. L.
Journal: J Biol Chem

The chloroplast signal recognition particle (cpSRP) and its receptor, chloroplast FtsY (cpFtsY), form an essential complex with the translocase Albino3 (Alb3) during post-translational targeting of light-harvesting chlorophyll-binding proteins (LHCPs). In this manuscript, we describe a combination of studies that explore the binding interface and functional role of a previously identified cpSRP43-Alb3 interaction. Using recombinant proteins corresponding to the C-terminus of Alb3 (Alb3-Cterm) and various domains of cpSRP43, we identify the ankyrin repeat region of cpSRP43 as the domain primarily responsible for the interaction with Alb3-Cterm. Further, we show Alb3-Cterm dissociates a cpSRP-LHCP targeting complex in vitro and stimulates GTP hydrolysis by cpSRP54 and cpFtsY in a strictly cpSRP43-dependent manner. These results support a model in which interactions between the ankyrin region of cpSRP43 and the C-terminus of Alb3 promote distinct, membrane-localized events including LHCP release from cpSRP and release of targeting components from Alb3.

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Calmodulin suppresses synaptotagmin-2 transcription in cortical neurons.

Publication Date: 2010 Aug 20 PMID: 20729199
Authors: Pang, Z. P. - Xu, W. - Cao, P. - Sudhof, T. C.
Journal: J Biol Chem

Calmodulin (CaM) is a ubiquitous Ca(2+)-sensor protein that plays a pivotal role in regulating inumerable neuronal functions, including synaptic transmission. In cortical neurons, most neurotransmitter release is triggered by Ca(2+)-binding to synaptotagmin-1; however, a second delayed phase of release, referred to as asynchronous release, is triggered by Ca(2+)-binding to an unidentified secondary Ca(2+)-sensor. To test whether CaM could be the enigmatic Ca(2+)-sensor for asynchronous release, we now use in cultured neurons short-hairpin RNAs that suppress expression of ~70% of all neuronal CaM isoforms. Surprisingly, we found that in synaptotagmin-1 knockout neurons, the CaM knockdown caused a paradoxical rescue of synchronous release, instead of a block of asynchronous release. Gene and protein expression studies revealed that both in wild-type and in synaptotagmin-1 knockout neurons, the CaM knockdown altered expression of >200 genes, including that encoding synaptotagmin-2. Synaptotagmin-2 expression was increased several-fold by the CaM knockdown, which accounted for the paradoxical rescue of synchronous release in synaptotagmin-1 knockout neurons by the CaM knockdown. Interestingly, the CaM knockdown primarily activated genes that are preferentially expressed in caudal brain regions, whereas it repressed genes in rostral brain regions. Consistent with this correlation, quantifications of protein levels in adult mice uncovered an inverse relationship of CaM and synaptotagmin-2 levels in mouse forebrain, brainstem and spinal cord. Finally, we employed molecular replacement experiments using a knockdown-rescue approach to show that Ca(2+)-binding to the C- but not the N-lobe of CaM is required for suppression of synaptotagmin-2 expression in cortical neurons. Our data describe a previously unknown, Ca(2+)/CaM-dependent regulatory pathway that controls the expression of synaptic proteins in the rostral-caudal neuraxis.

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The SEF/IL-17R (SEFIR) is not enough: An extended SEFIR domain is required for IL-17RA signal transduction.

Publication Date: 2010 Aug 20 PMID: 20729198
Authors: Onishi, R. M. - Park, S. J. - Hanel, W. - Ho, A. W. - Maitra, A. - Gaffen, S. L.
Journal: J Biol Chem

IL-17, the hallmark cytokine of the Th17 population, mediates immunity to extracellular pathogens and promotes autoimmune immunopathology. The signaling mechanisms triggered by the IL-17 receptor (IL-17RA) and related receptors are strikingly different from other cytokine subclasses. Namely, IL-17Rs contain a conserved ''SEF/IL-17R'' (SEFIR) subdomain that engages Act1, leading to activation of TRAF6, NF-kappaB and other events. Although the SEFIR is critical for signaling, the molecular details of the functional subdomains within IL-17RA remain poorly characterized. Here, we provide a detailed structure-function analysis delineating the C-terminal boundary of the SEFIR-containing region of IL-17RA. We show that functionality of this domain requires a large extension to the previously-identified SEFIR motif. In contrast to the SEFIR, this extension is not conserved among IL-17R family members. Surprisingly, Act1 recruitment is not sufficient for downstream signaling activation, whereas ubiquitination of TRAF6 correlates tightly with functional receptors. We further demonstrate that IL-17RA exhibits signaling properties that are nonredundant with other IL-17R family members. Finally, we report that IL-17 signals synergistically with lymphotoxin-alpha3 (LT[alpha]3), using the same signaling motifs within IL-17RA. These studies provide new insight into the structure-function relationships of IL-17RA, and reveal distinct signaling differences among IL-17R family members.

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{alpha}2B-adrenoceptor deficiency leads to postnatal respiratory failure in mice.

Publication Date: 2010 Aug 20 PMID: 20729197
Authors: Haubold, M. - Gilsbach, R. - Hein, L.
Journal: J Biol Chem

alpha2-adrenoceptors belong to the family of adrenergic receptors, which regulate the neuronal release of norepinephrine as part of a negative feedback loop. Among the alpha2-adrenoceptors, the alpha2B-subtype may also influence developmental signalling pathways involved in angiogenesis of the placenta. Thus, the aim of the present study was to determine whether alpha2B-adrenoceptors are also involved in other developmental processes beyond placenta angiogenesis. Ablation of alpha2B-adrenoceptors led to lethality of mutant mice during the first hours after birth. Despite normal breathing and drinking behavior, mutant mice developed cyanosis which could be traced back to a defect in lung morphology with significantly reduced alveolar volume and thickened interalveolar septi. In alpha2B-deficient lungs and in isolated alveolar type II cells, expression of sonic hedgehog (Shh) was significantly increased, resulting in mesenchymal proliferation. In vitro alpha2B-adrenoceptor stimulation suppressed expression of sonic hedgehog and the cell cycle genes cyclin D1 and Ki67. In vivo inhibition of enhanced Shh signalling by the smoothened antagonist cyclopamine partially rescued perinatal lethality, lung morphology and altered gene expression in mutant mice. Thus, alpha2B-adrenoceptors in lung epithelia play an important role in suppressing sonic hedgehog-mediated proliferation of mesenchymal cells and thus prevent respiratory failure.

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The C331A mutant of neuronal nitric-oxide synthase is labilized for HSP70/chip (C-terminus of HSC70-interacting protein)-dependent ubiquitination.

Publication Date: 2010 Aug 20 PMID: 20729196
Authors: Clapp, K. M. - Peng, H. M. - Morishima, Y. - Lau, M. - Walker, V. J. - Pratt, W. B. - Osawa, Y.
Journal: J Biol Chem

It is established that suicide inactivation of neuronal nitric oxide synthase (nNOS) by drugs and other xenobiotics leads to ubiquitination and proteasomal degradation of the enzyme. The exact mechanism is not known, although it is widely thought that the covalent alteration of the active site during inactivation triggers the degradation. A mechanism that involves recognition of the altered nNOS by Hsp70 and its cochaperone CHIP, an E3-ubiquitin ligase, has been proposed. To further address how alterations of the active site trigger ubiquitination of nNOS, we examined a C331A nNOS mutant, which was reported to have impaired ability to bind L-arginine and tetrahydrobiopterin. We show here that C331A nNOS is highly susceptible to ubiquitination by a purified system containing ubiquitinating enzymes and chaperones, by the endogenous ubiquitinating system in recticulocyte lysate fraction II, and by intact HEK293 cells. The involvement of the altered heme cleft in regulating ubiquitination is confirmed by the finding that the slowly reversible inhibitor of nNOS, NG-nitro-L-arginine, but not its inactive D-isomer, protects the C331A nNOS from ubiquitination in all these experimental systems. We also show that both Hsp70 and CHIP play a major role in the ubiquitination of C331A nNOS, whereas Hsp90 protects from ubiquitination. Thus, these studies further strengthen the link between the mobility of the substrate binding cleft and chaperone-dependent ubiquitination of nNOS. These results support a general model of chaperone-mediated protein quality control and lead to a novel mechanism for substrate stabilization based on nNOS interaction with the chaperone machinery.

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The viral oncoprotein tax sequesters DNA-damage response factors by tethering MDC1 to chromatin.

Publication Date: 2010 Aug 20 PMID: 20729195
Authors: Belgnaoui, S. M. - Fryrear, K. A. - Nyalwidhe, J. O. - Guo, X. - Semmes, O. J.
Journal: J Biol Chem

Infection with Human T-cell Leukemia Virus induces cellular genomic instability mediated through the viral oncoprotein Tax. Here we present evidence that Tax undermines the cellular DNA damage response by sequestration of damage response factors. We show by confocal microscopy that Tax forms damage-independent nuclear foci that contain DNA-PK, BRCA1 and MDC1. Tax sequesters MDC1 to chromatin sites distinct from classic ionizing radiation induced foci. The recruitment of MDC1 is competitive between the two foci. The N-terminal region of Tax is sufficient for foci localization and the C-terminal half is critical for binding to MDC1 and recruitment of additional response factors. Tax expression and DDR factor recruitment repressed the formation of ionizing radiation induced Nbs1-containing foci. The Tax induced pseudo DNA damage response results in phosphorylation and monoubiquitylation of H2AX, which is ablated by siRNA suppression of MDC1. These data support a model for viral induced genomic instability in which viral oncogene-induced damage-independent foci compete with normal cellular DNA damage response.

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Degradation of the human mitotic checkpoint kinase Mps1 is cell-cycle regulated by APC/cCdc20 and APC/cCdh1 ubiquitin ligases.

Publication Date: 2010 Aug 20 PMID: 20729194
Authors: Cui, Y. - Cheng, X. - Zhang, C. - Zhang, Y. - Li, S. - Wang, C. - Guadagno, T. M.
Journal: J Biol Chem

Mps1 is a dual-specificity protein kinase with key roles in regulating the spindle assembly checkpoint and chromosome-microtubule attachments. Consistent with these mitotic functions, Mps1 protein levels fluctuate during the cell cycle, peaking at early mitosis and abruptly declining during mitotic exit and progression into G1 phase. While evidence in budding yeast indicates that Mps1 is targeted for degradation at anaphase by the APC/cCdc20 complex, little is known about the regulatory mechanisms that govern Mps1 protein levels in human cells. Here, we provide evidence for the ubiquitin ligase/proteosome pathway in regulating human Mps1 levels during late mitosis through G1 phase. First, we showed that treatment of HEK 293T cells with proteosome inhibitor MG132 resulted in an increase in both the polyubiquitination and the accumulation of Mps1 protein levels. Next, Mps1 was shown to co-precipitate with APC and its activators Cdc20 and Cdh1 in a cell-cycle dependent manner. Consistent with this, overexpression of Cdc20 or Cdh1 led to a marked reduction of endogenous Mps1 levels during anaphase or G1 phase, respectively. In contrast, depletion of Cdc20 or Cdh1 by RNAi treatment both led to the stabilization of Mps1 protein during mitosis or G1 phase, respectively. Finally, we identified a single D-box motif in human Mps1 that is required for its ubiquitination and degradation. Failure to appropriately degrade Mps1 is sufficient to trigger centrosome amplification and mitotic abnormalities in human cells. Thus, our results suggest that the sequential actions of the APC/cCdc20 and APC/cCdh1 ubiquitin ligases regulate the clearance of Mps1 levels and are critical for Mps1 functions during the cell cycle in human cells.

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The role of proline in the membrane re-entrant helix of caveolin-1.

Publication Date: 2010 Aug 20 PMID: 20729193
Authors: Aoki, S. - Thomas, A. - Decaffmeyer, M. - Brasseur, R. - Epand, R. M.
Journal: J Biol Chem

Caveolin-1 has a segment of hydrophobic amino acids comprising approximately residues 103 to 122. We have performed an in silico analysis of the conformational preference of this segment of caveolin-1 using PepLook. We find that there is one main group of stable conformations corresponding to a hydrophobic U bent model that would not traverse the membrane. Furthermore, the calculations predict that substituting the Pro(110) residue with an Ala will change the conformation to a straight hydrophobic helix that would traverse the membrane. We have expressed the P110A mutant of caveolin-1, with a FLAG tag at the amino terminus, in HEK 293 cells. We evaluate the topology of the proteins with confocal immunofluorescence microscopy in these cells. We find that FLAG tag at the amino terminus of the wild type caveolin-1 is not reactive with antibodies unless the cell membrane is permeabilized with detergent. This indicates that in these cells the hydrophobic segment of this protein is not transmembrane but takes up a bent conformation, making the protein monotopic. In contrast, the FLAG tag at the amino terminus of the P110A mutant is equally exposed to antibodies, before and after membrane permeabilization. We also find that the P110A mutation causes a large reduction of endocytosis of caveolae, cellular lipid accumulation and lipid droplet formulation. In addition, we find this mutation markedly reduces the ability of caveolin-1 to form structures with the characteristic morphology of caveolae or to partition into the detergent resistant membranes of these cells. Thus the single Pro residue in the membrane-inserting segment of caveolin-1 plays an important role in both the membrane topology and localization of the protein as well as its functions.

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BcsKC is an essential protein for the Type VI secretion system activity in Burkholderia cenocepacia that forms an outer membrane complex with BcsLB.

Publication Date: 2010 Aug 20 PMID: 20729192
Authors: Aubert, D. - Macdonald, D. K. - Valvano, M. A.
Journal: J Biol Chem

The Type VI secretion system (T6SS) contributes to the virulence of Burkholderia cenocepacia, an opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis. BcsK(C) is a highly conserved protein among the T6SSs in Gram-negative bacteria. Here, we show that BcsKC is required for Hcp secretion and cytoskeletal redistribution in macrophages upon bacterial infection. These two phenotypes are associated with a functional T6SS in B. cenocepacia. Experiments employing a bacterial two-hybrid system and pull-down assays demonstrated that BcsK(C) interacts with BcsL(B), another conserved T6SS component. Internal deletions within BcsK(C) revealed that its N-terminal domain is necessary and sufficient for interaction with BcsL(B). Fractionation experiments showed that BcsK(C) can be in the cytosol or tightly associated with the outer membrane and that BcsK(C) and BcsL(B) form a high molecular weight complex anchored to the outer membrane that requires BcsF(H) (a ClpV homolog) to be assembled. Together, our data show that BcsK(C)/BcsL(B) interaction is essential for the T6SS activity in B. cenocepacia.

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Further insights into the roles of GTP and the C-terminus of the Hepatitis C virus polymerase in the initiation of RNA synthesis.

Publication Date: 2010 Aug 20 PMID: 20729191
Authors: Harrus, D. - Ahmed-El-Sayed, N. - Simister, P. C. - Miller, S. - Triconnet, M. - Hagedorn, C. H. - Mahias, K. - Rey, F. A. - Astier-Gin, T. - Bressanelli, S.
Journal: J Biol Chem

The hepatitis C virus (HCV) NS5b protein is an RNA-dependent RNA polymerase (RdRp) essential for replication of the viral RNA genome. In vitro and presumably in vivo, NS5b initiates RNA synthesis by a de novo mechanism. Different structural elements of NS5b have been reported to participate in RNA synthesis, especially a so-called beta-flap and a C-terminal segment (designated linker) that connects the catalytic core of NS5b to a transmembrane anchor. High concentrations of GTP have also been shown to stimulate de novo RNA synthesis by HCV NS5b. Here we describe a combined structural and functional analysis of genotype 1 HCV-NS5b of strains H77 (subtype 1a), for which no structure has been previously reported, and J4 (subtype 1b). Our results highlight the linker as directly involved in lifting the first boundary to processive RNA synthesis, the formation of the first dinucleotide primer. The transition from this first dinucleotide primer state to processive RNA synthesis requires removal of the linker and of the beta-flap with which it is shown to strongly interact in crystal structures of HCV NS5b. We find that GTP specifically stimulates this transition, irrespective of its incorporation in neosynthesized RNA.

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The obligatory intestinal folate transporter PCFT (SLC46A1) is regulated by nuclear respiratory factor 1 (NRF-1).

Publication Date: 2010 Aug 19 PMID: 20724482
Authors: Gonen, N. - Assaraf, Y. G.
Journal: J Biol Chem

Folates are essential vitamins that play a key role as one-carbon donors in a spectrum of biosynthetic pathways including RNA and DNA synthesis. The proton coupled folate transporter (PCFT/SLC46A1) mediates obligatory intestinal folate absorption. Loss-of-function mutations in PCFT result in hereditary folate malabsorption, an autosomal recessive disorder characterized by very low folate levels in the blood and CSF. HFM manifests within the first months after birth with anemia, immune deficiency and neurological deficits. Here we studied the role of inducible trans-activators of PCFT gene expression. Bioinformatics identified three putative NRF-1 binding sites in the minimal promoter. The following evidence establish that PCFT is an NRF-1-responsive gene; EMSA showed NRF-1 binding to native but not mutant NRF-1 sites, whereas antibody-mediated supershift analysis and chromatin immunoprecipitation revealed NRF-1 binding to its consensus sites within the PCFT promoter. Moreover, mutational inactivation of individual or all NRF-1 binding sites resulted in 40-60% decrease in luciferase reporter activity. Consistently, overexpression of NRF-1 or a constitutively active NRF-1 VP-16 construct resulted in increased reporter activity and PCFT mRNA levels. Conversely, introduction of a dominant negative NRF-1 construct markedly repressed reporter activity and PCFT mRNA levels; likewise, introduction of NRF-1 siRNA duplexes to cells resulted in decreased PCFT transcript levels. Moreover, NRF-1 silencing down-regulated genes encoding for key folate transporters and enzymes in folate metabolism. These novel findings identify NRF-1 as a major inducible transcriptional regulator of PCFT gene expression. The implications of this linkage between folate transport and metabolism with mitochondria biogenesis and respiration are discussed.

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An ATP-dependent mechanism protects spectrin against glycation in human erythrocytes.

Publication Date: 2010 Aug 19 PMID: 20724481
Authors: Manno, S. - Mohandas, N. - Takakuwa, Y.
Journal: J Biol Chem

Human erythrocytes are continuously exposed to glucose which reacts with the amino terminus of the beta-chain of hemoglobin (Hb) to form glycated Hb, HbA1c, levels of which increase with the age of the circulating cell. In contrast to extensive insights into glycation of hemoglobin, little is known about glycation of erythrocyte membrane proteins. In the present study, we explored the conditions under which glucose and ribose can glycate spectrin, both on the intact membrane and in solution and the functional consequences of spectrin glycation. While purified spectrin could be readily glycated, membrane-associated spectrin could be glycated only after ATP depletion and consequent translocation of phosphatidylserine (PS) from the inner to the outer lipid monolayer. Glycation of membrane-associated spectrin led to a marked decrease in membrane deformability. We further observed that only PS-binding spectrin repeats are glycated. We infer that the absence of glycation in situ is the consequence of the interaction of the target lysine and arginine residues with PS and thus being inaccessible for glycation. The reduced membrane deformability following glycation in the absence of ATP is likely the result of the inability of the glycated spectrin repeats to undergo the obligatory unfolding as a consequence of inter-helix crosslinks. We thus postulate that erythrocytes through the use of an ATP-driven phospholipids translocase (flippase) have evolved a protective mechanism against spectrin glycation and thus maintain their optimal membrane function during their long circulatory life span.

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Regulation of NADPH oxidase activity in phagocytes - Relationship between FAD/NADPH binding and oxidase complex assembly.

Publication Date: 2010 Aug 19 PMID: 20724480
Authors: Debeurme, F. - Picciocchi, A. - Dagher, M. C. - Grunwald, D. - Beaumel, S. - Fieschi, F. - Stasia, M. J.
Journal: J Biol Chem

X+ linked chronic granulomatous disease variants are natural mutants characterized by defective NADPH-oxidase activity but with normal Nox2 expression. According to the 3-dimensional model of the cytosolic Nox2 domain, most of the X+CGD mutations are located in/or close to the FAD/NADPH binding regions. A structure/function study of this domain was conducted in X+CGD PLB-985 cells exactly mimicking ten human variants: T341K, C369R, G408E, G408R, P415H, P415L, 507QKT509deletion-HIWAinsert, C537R, L546P, and E568K. Diaphorase activity is defective in all these mutants. NADPH-oxidase assembly is normal for P415H/L and T341K mutants where mutation occurs in the consensus sequences of NADPH and FAD binding sites, respectively. This is in accordance with their buried position in the 3-D model of the cytosolic Nox2 domain. FAD incorporation is abolished only in the T341K mutant explaining its absence of diaphorase activity. This demonstrates that NADPH-oxidase assembly can occur without FAD incorporation. In addition a defect of NADPH binding is a plausible explanation for the diaphorase activity inhibition in the P415H, P415L and C537R mutants. In contrast C369, G408, L546 and E568 are essential for NADPH-oxidase complex assembly. However, according to their position in the 3-D model of the cytosolic domain of Nox2 only C369 could be in direct contact with cytosolic factors during oxidase assembly. In addition the defect in oxidase assembly observed in the C369R, G408E, G408R, and E568K mutants correlates with the lack of FAD incorporation. Thus the NADPH-oxidase assembly process and FAD incorporation are closely related events essential for the diaphorase activity of Nox2.

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Structure of human stabilin-1 interacting chitinase-like protein (SI-CLP) reveals a saccharide-binding cleft with lower sugar-binding selectivity.

Publication Date: 2010 Aug 19 PMID: 20724479
Authors: Geng, M. - Zhao, Y. - Bai, X. - Liu, Y. - Green, T. J. - Luo, M. - Zheng, X.
Journal: J Biol Chem

Human secreted protein stabilin-1 interacting chitinase-like protein (SI-CLP) has been identified as a novel member of Glyco_18 domain-containing proteins that is involved in host defense and inflammatory reactions. Efficient secretion of SI-CLP is mediated by its interaction with the endocytic/sorting receptor stabilin-1. SI-CLP is expressed abundantly in macrophages and neutrophils and is upregulated by Th2 cytokine IL-4 and glucocorticoid, which suggest that SI-CLP could be a marker for adverse effects of glucocorticoid therapy. To gain insight into the biological function of SI-CLP, we determined the crystal structure of SI-CLP at 2.7 Angstrom resolution by x-ray crystallography and found it featured a typical triose-phosphate isomerase-barrel fold with a putative saccharide binding cleft. Comparison with other chitinase-like proteins showed the cleft to be atypically wide and open. The saccharides binding capacity of SI-CLP was investigated and its ligand-binding specificity was found to relate to the length of the oligosaccharides, with preference for chitotetraose. Further investigations reveal that SI-CLP could bind LPS in vitro and neutralize its endotoxin effect on macrophages. Our results demonstrate the saccharide-binding property of SI-CLP by structure and in vitro biochemical analyses, and suggest the possible roles of SI-CLP in pathogen sensing and endotoxin neutralization.

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Inhibition of nicotinamide phosphoribosyl transferase: cellular bioenergetics reveals a mitochondrial insensitive NAD pool.

Publication Date: 2010 Aug 19 PMID: 20724478
Authors: Pittelli, M. - Formentini, L. - Faraco, G. - Lapucci, A. - Rapizzi, E. - Cialdai, F. - Romano, G. - Moneti, G. - Moroni, F. - Chiarugi, A.
Journal: J Biol Chem

The NAD rescue pathway consists of two enzymatic steps operated by nicotinamide phosphoribosyl transferase (Nampt) and nicotinamide mononucleotide adenylyl transferases. Recently, the potent Nampt inhibitor FK866 has been identified and evaluated in clinical trials against cancer. Yet, how Nampt inhibition affects NAD contents and bioenergetics is in part obscure. It is also unknown whether NAD rescue takes place in mitochondria, and FK866 alters NAD homeostasis within the organelle. Here, we show that FK866-dependent reduction of NAD contents is paralleled by concomitant increase of ATP in various cell types, in keeping with ATP utilization for NAD resynthesis. We also show that poly- and mono(ADP-ribosyl) transferases rather than Sirt-1 are responsible for NAD depletion in HeLa cells exposed to FK866. Mass spectrometry reveals that the drug distributes in the cytosolic and mitochondrial compartment. However, the cytoplasmic but not the mitochondrial NAD pool is reduced upon acute or chronic exposure to the drug. Accordingly, Nampt does not localize within the organelles and their bioenergetics is not affected by the drug. In the mouse, FK866-dependent reduction of NAD contents in various organs is prevented by inhibitors of poly(ADP-ribose) polymerases or the NAD precursor kynurenine. For the first time, our data indicate that mitochondria lack the canonical NAD rescue pathway, broadening current understanding of cellular bioenergetics.

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Carbon monoxide promotes VEGF expression by increasing HIF-1{alpha} protein level via two distinct mechanisms: translational activation and stabilization of HIF-1{alpha} protein.

Publication Date: 2010 Aug 19 PMID: 20724477
Authors: Choi, Y. K. - Kim, C. K. - Lee, H. - Jeoung, D. - Ha, K. S. - Kwon, Y. G. - Kim, K. W. - Kim, Y. M.
Journal: J Biol Chem

Carbon monoxide (CO) plays a significant role in vascular functions. We here examined the molecular mechanism by which CO regulates hypoxia-inducible transcription factor-1 (HIF-1)-dependent expression of vascular endothelial growth factor (VEGF), which is an important angiogenic factor. We found that astrocytes stimulated with the CO-releasing molecule (CORM-2) promoted angiogenesis by increasing VEGF expression and secretion. CORM-2 also induced hemeoxygenase-1 (HO-1) expression and increased nuclear HIF-1alpha protein level, without altering its promoter activity and mRNA level. VEGF expression was inhibited by treatment with HIF-1alpha siRNA and a HO inhibitor, indicating that CO stimulates VEGF expression via upregulation of HIF-1alpha protein level, which is partially associated with HO-1 induction. CORM-2 activated the translational regulatory proteins p70S6k and eIF-4E, as well as phosphorylated their upstream signal mediators Akt and ERK. These translational signal events and HIF-1alpha protein level were suppressed by inhibitors of phosphatidylinositol 3-kinase (PI3K), MEK, and mTOR, suggesting that the PI3K/Akt/mTOR and MEK/ERK pathways are involved in a translational increase in HIF-1alpha. In addition, CORM-2 also increased stability of the HIF-1alpha protein by suppressing its ubiquitination, without altering the proline hydroxylase-dependent HIF-1alpha degradation pathway. CORM-2 increased HIF-1alpha/HSP90alpha interaction, which is responsible for HIF-1alpha stabilization, and HSP90 specific inhibitors decreased this interaction, HIF-1alpha protein level, and VEGF expression. Furthermore, HSP90alpha knockdown suppressed CORM-2-induced increases in HIF-1alpha and VEGF protein levels. These results suggest that CO stimulates VEGF production by increasing HIF-1alpha protein level via two distinct mechanisms, translational stimulation and protein stabilization of HIF-1alpha.

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p38 MAP kinase and MAPKAP kinases MK2/3 cooperatively phosphorylate epithelial keratins.

Publication Date: 2010 Aug 19 PMID: 20724476
Authors: Menon, M. B. - Schwermann, J. - Singh, A. K. - Franz-Wachtel, M. - Pabst, O. - Seidler, U. - Omary, M. B. - Kotlyarov, A. - Gaestel, M.
Journal: J Biol Chem

The MAPKAP kinases MK2 and MK3 are directly activated via p38 MAPK phosphorylation, stabilize p38 by complex formation and contribute to the stress response. The list of substrates of MK2/3 is increasing steadily. We applied a phospho-proteomics approach to compare protein phosphorylation in MK2/3-deficient cells rescued or not by ectopic expression of MK2. Beside differences in phosphorylation of the known substrates of MK2, HSPB1 and Bag-2, we identified strong differences in phosphorylation of keratin 8 (K8). The phosphorylation of K8-S73 is catalysed directly by p38, which in turn shows MK2-dependent expression. Notably, analysis of small molecule p38-inhibitors on K8-S73 phosphorylation also demonstrated reduced phosphorylations of keratins K18-S52 and K20-S13 but not of K8-S431 or K18-S33. Interestingly, K18-S52 and K20-S13 are not directly phosphorylated by p38 in vitro, but by MK2. Furthermore, anisomycin-stimulated phosphorylations of K20-S13 and K18-S52 are inhibited by small molecule inhibitors of both, p38 and MK2. MK2 knockdown in HT29 cells leads to reduced K20-S13 phosphorylation, which further supports the notion that MK2 is responsible for K20 phosphorylation in vivo. Physiologic relevance of these findings was confirmed by differences of K20-S13 phosphorylation between the ileum of WT- and MK2/3-deficient mice and by demonstrating p38- and MK2-dependent mucin secretion of HT29 cells. Therefore, MK2 and p38 MAPK function in concert to phosphorylate K8, K18 and K20 in intestinal epithelia.

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Multiple decisive phosphorylation sites for the negative feedback regulation of SOS1 via ERK.

Publication Date: 2010 Aug 19 PMID: 20724475
Authors: Kamioka, Y. - Yasuda, S. - Fujita, Y. - Aoki, K. - Matsuda, M.
Journal: J Biol Chem

EGF-induced activation of ERK has been extensively studied by both experimental and theoretical approaches. Here, we used a simulation model based mostly on experimentally-determined parameters to study the ERK-mediated negative feedback regulation of the Ras guanine nucleotide exchange factor, son of sevenless (SOS). Because SOS1 is phosphorylated at multiple serine residues upon stimulation, we evaluated the role of the multiplicity by building two simulation models, which we termed the decisive and cooperative phosphorylation models. The two models were constrained by the duration of Ras activation and basal phosphorylation level of SOS1. Possible solutions were found only in the decisive model wherein at least three, and probably more than four, phosphorylation sites decisively suppress the SOS activity. Thus, the combination of experimental approaches and the model analysis has suggested an unexpected role of multiple phosphorylation of SOS1 in the negative regulation.

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The Sll0606 protein is required for photosystem II assembly/stability in the cyanobacterium Synechocystis sp. PCC 6803.

Publication Date: 2010 Aug 19 PMID: 20724474
Authors: Zhang, S. - Frankel, L. K. - Bricker, T. M.
Journal: J Biol Chem

An insertional transposon mutation in the sll0606 gene was found to lead to a loss of photoautotrophy, but not photoheterotrophy, in the cyanobacterium Synechocystis sp. PCC 6803. Complementation analysis of this mutant (Tsll0606) indicated that an intact sll0606 gene could fully restore photoautotrophic growth. Gene organization in the vicinity of sll0606 indicates that it is not contained in an operon. No electron transport activity was detected in Tsll0606 using water as an electron donor and 2,6 dichlorobenzoquinone as an electron acceptor, indicating that PS II was defective. Electron transport activity using dichlorophenol indolephenol plus ascorbate as an electron donor to methyl viologen, however, was the same as observed in the control strain. This indicated that electron flow through Photosystem I was normal. Fluorescence induction and decay parameters verified that Photosystem II was highly compromised. The quantum yield for energy trapping by Photosystem II (FV/FM) in the mutant was less than 10% of that observed in the control strain. The small variable fluorescence yield observed after a single saturating flash exhibited aberrant QA- reoxidation kinetics which were insensitive to dichloromethylurea. Immunological analysis indicated that while the D2 and CP47 proteins were modestly affected, the D1 and CP43 components were dramatically reduced. Analysis of two dimensional blue native/LiDS-PAGE gels indicated that no intact PS II monomer or dimers were observed in the mutant. The CP43-less PS II monomer did accumulate to detectable levels. Our results indicate that the Sll0606 protein is required for the assembly/stability of a functionally competent Photosystem II.

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2E8 binds to the high-affinity I-domain in a metal ion dependent manner: A second generation monoclonal antibody selectively targeting activated LFA-1.

Publication Date: 2010 Aug 19 PMID: 20724473
Authors: Carreno, R. - Brown, W. S. - Li, D. - Hernandez, J. A. - Wang, Y. - Kim, T. K. - Craft, J. W. - Komanduri, K. V. - Radvanyi, L. J. - Hwu, P. - Molldrem, J. J. - Legge, G. B. - McIntyre, B. W. - Ma, Q.
Journal: J Biol Chem

The activation of leukocyte function associated antigen-1 (LFA-1) plays a critical role in regulating immune responses. The metal ion dependent adhesion site (MIDAS) on the I-domain of LFA-1 alpha(L) subunit is the key recognition site for ligand binding. Upon activation, conformation changes in the I-domain can lead LFA-1 from the low-affinity (LA) state to the high-affinity (HA) state. Using the purified HA I-domain locked by disulfide-bonds for immunization, we developed a mAb, 2E8, that specifically binds to cells expressing the HA LFA-1. The surface plasmon resonance (SPR) analysis has shown that 2E8 only binds to the HA I-domain and the dissociation constant (K(D)) for HA I-domain is 197 nM. The binding of 2E8 to the HA I-domain is metal ion dependent, and the affinity decreased as Mn(2+) was replaced sequentially by Mg(2+) and Ca(2+). SPR analysis demonstrates that 2E8 inhibits the interaction of HA-I domain and ICAM-1. Furthermore, we found that 2E8 can detect activated LFA-1 on both JY and Jurkat cells using flow cytometry and parallel plate adhesion assay. In addition, 2E8 inhibits JY cell adhesion to HUVECs and homotypic aggregation. 2E8 treatment reduces the proliferation of both human CD4(+) and CD8(+) T cells upon OKT3 stimulation without the impairment of their cytolytic function. Taken together, these data demonstrate 2E8 is specific for the high affinity form of LFA-1 and that inhibits LFA-1/ICAM-1 interactions. As a novel activation-specific monoclonal antibody, 2E8 is a potentially useful reagent for blocking high-affinity LFA-1 and modulating T cell activation in research and therapeutics.

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Control of ATF4 persistence by multisite phosphorylation impacts cell cycle progression and neurogenesis.

Publication Date: 2010 Aug 19 PMID: 20724472
Authors: Frank, C. L. - Ge, X. - Xie, Z. - Zhou, Y. - Tsai, L. H.
Journal: J Biol Chem

Organogenesis is a highly integrated process with a fundamental requirement for precise cell cycle control. Mechanistically, the cell cycle is composed of transitions and thresholds that are controlled by coordinated post-translational modifications. In this study, we describe a novel mechanism controlling the persistence of the transcription factor ATF4 by multisite phosphorylation. Proline-directed phosphorylation acted additively to regulate multiple aspects of ATF4 degradation. Stabilized ATF4 mutants exhibit decreased beta-TrCP degron phosphorylation, beta-TrCP interaction, and ubiquitination, as well as elicit early G1 arrest. Expression of stabilized ATF4 also had significant consequences in the developing neocortex. Mutant ATF4 expressing cells exhibited positioning and differentiation defects that were attributed to early G1 arrest, suggesting that neurogenesis is sensitive to ATF4 dosage. We propose that precise regulation of ATF4 dosage impacts cell cycle control and impinges on neurogenesis.

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Glycan analysis and influenza a virus infection of primary swine respiratory epithelial cells: the importance of NeuAc{alpha}2-6 glycans.

Publication Date: 2010 Aug 19 PMID: 20724471
Authors: Bateman, A. C. - Karamanska, R. - Busch, M. G. - Dell, A. - Olsen, C. W. - Haslam, S. M.
Journal: J Biol Chem

To better understand influenza virus infection of pigs, we examined primary swine respiratory epithelial cells (SRECs, the primary target cells of influenza viruses in vivo), as a model system. Glycomic profiling of SRECs by mass spectrometry revealed a diverse range of glycans terminating in sialic acid or GalalphaGal. In terms of sialylation, alpha2-6 linkage was more abundant than alpha2-3 and NeuAc was more abundant than NeuGc. Virus binding and infection experiments were conducted to determine functionally important glycans for influenza virus infection, with a focus on recently emerged swine viruses. Infection of SRECs with swine and human viruses resulted in different infectivity levels. Glycan microarray analysis with a high infectivity triple reassortant virus [(A/Swine/MN/593/99 (H3N2)] that spread widely throughout the North American swine population, and a lower infectivity human virus isolated from a single pig [A/Swine/ONT/00130/97 (H3N2)] showed that both viruses bound exclusively to glycans containing NeuAcalpha2-6, with strong binding to sialylated polylactosamine and sialylated N-glycans. Treatment with mannosamine precursors of sialic acid (to alter NeuAc/NeuGc abundances) and linkage-specific sialidases prior to infection indicated that the influenza viruses tested preferentially utilize NeuAcalpha2-6 sialylated glycans to infect SRECs. Our data indicate that NeuAcalpha2-6-terminated polylactosamine and sialylated N-glycans are important determinants for influenza viruses to infect SRECs. As NeuAcalpha2-6 polylactosamine glycans play major roles in human virus infection, the importance of these receptor components in virus infection of swine cells has implications for transmission of viruses between humans and pigs, and for pigs as possible adaptation hosts of novel human influenza viruses.

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Nuclear-localized calcineurin homologous protein CHP1 interacts with upstream binding factor and inhibits ribosomal RNA synthesis.

Publication Date: 2010 Aug 18 PMID: 20720019
Authors: Jimenez-Vidal, M. - Srivastava, J. - Putney, L. K. - Barber, D. L.
Journal: J Biol Chem

Calcineurin homologous protein 1 (CHP1) is a widely expressed, 22 kDa myristoylated EF-hand Ca2+-binding protein that shares a high degree of similarity with the regulatory B subunit of calcineurin (65%) and with calmodulin (59%). CHP1 localizes to the plasma membrane, the Golgi apparatus, and the nucleus, and functions to regulate trafficking of early secretory vesicles, activation of T-cells, and expression and transport of the Na-H exchanger NHE1. Although CHP1 contains nuclear export signals, whether its nuclear and cytoplasmic localization is regulated and has distinct functions remain unknown. We show that CHP1 is predominantly in the nucleus in quiescent fibrobasts, is translocated to cytoplasmic compartments with growth medium, and that translocation is inhibited by mutations in the nuclear export motifs. In a screen for proteins co-precipitating with CHP1 in quiescent cells we identified the upstream binding factor UBF, a DNA binding protein and component of the RNA polymerase I (Pol I) complex regulating RNA synthesis. The CHP1-UBF interaction is restricted to the nucleus and inhibited by Ca2+. Nuclear retention of CHP1 attenuates the abundance of UBF in the nucleolus and inhibits RNA synthesis when quiescent cells are transferred to growth medium. These data show UBF as a newly identified CHP1 binding protein and regulation of RNA synthesis as a newly identified function for nuclear-localized CHP1, which is distinct from CHP1 functions in the cytosol.

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{gamma}-Tocotrienol but not {gamma}-tocopherol blocks STAT3 cell signaling pathway through induction of protein tyrosine phosphatase SHP-1 and sensitizes tumor cells to chemotherapeutic agents.

Publication Date: 2010 Aug 18 PMID: 20720018
Authors: Kannappan, R. - Yadav, V. R. - Aggarwal, B. B.
Journal: J Biol Chem

Although gamma-tocotrienol (T3), a vitamin E isolated primarily from palm and rice bran oil, has been linked with anticancer activities, the mechanism of this action is poorly understood. In the present report, we investigated whether gamma-T3 can modulate STAT3 cell signaling pathway, closely linked to inflammation and tumorigenesis. We found that gamma-T3 but not gamma-tocopherol, the most common saturated form of Vitamin E, inhibited constitutive activation of STAT3 in a dose- and time-dependent manner and this inhibition was not cell type specific. gamma-T3 also inhibited STAT3 DNA binding. This correlated with inhibition of Src kinase and JAK1 and JAK2 kinases. Pervanadate reversed the gamma-T3-induced downregulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase. When examined further, we found that gamma-T3 induced the expression of the tyrosine phosphatase SHP-1, and gene-silencing of the SHP-1 by small interfering RNA abolished the ability of gamma-T3 to inhibit STAT3 activation, suggesting a vital role for SHP-1 in the action of this T3. Also gamma-T3 down modulated activation of STAT3 and induced SHP-1 in in vivo. Eventually, gamma-T3 down-regulated the expression of STAT3-regulated antiapoptotic (Bcl-2, Bcl-xL, and Mcl-1), proliferative (cyclin D1), and angiogenic (VEGF) gene products; and this correlated with suppression of proliferation, the accumulation of cells in sub-G(1) phase of the cell cycle, and induction of apoptosis. This vitamin also sensitized the tumor cells to the apoptotic effects of thalidomide and bortezomib. Overall, our results suggest that gamma-T3 is a novel blocker of STAT3 activation pathway in both in vitro and in vivo and thus may have potential in prevention and treatment of cancers.

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Structure and function of the hetero-oligomeric cysteine synthase complex in plants.

Publication Date: 2010 Aug 18 PMID: 20720017
Authors: Wirtz, M. - Birke, H. - Heeg, C. - Mueller, C. - Hosp, F. - Throm, C. - Koenig, S. - Feldman-Salit, A. - Rippe, K. - Petersen, G. - Wade, R. C. - Rybin, V. - Scheffzek, K. - Hell, R.
Journal: J Biol Chem

Cysteine synthesis in bacteria and plants is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL) which form the hetero-oligomeric cysteine synthase complex (CSC). In plants, but not in bacteria, the CSC is assumed to control cellular sulfur homeostasis by reversible association of the subunits. Application of size exclusion chromatography, analytical ultracentrifugation and isothermal titration calorimetry revealed a hexameric structure of mitochondrial SAT from Arabidopsis thaliana (AtSATm) and a 2:1 ratio of the OAS-TL dimer to the SAT hexamer in the CSC. Comparable results were obtained for the composition of the cytosolic SAT from A. thaliana (AtSATc) and the cytosolic SAT from Glycine max (Glyma16g03080, GmSATc) and their corresponding CSCs. The hexameric SAT structure is also supported by the calculated binding energies between SAT trimers. The interaction sites of dimers of AtSATm trimers are identified using peptide arrays. A negative Gibbs free energy (DeltaG=-33 kcal mol-1 explains the spontaneous formation of the AtCSCs, while the measured SAT:OAS-TL affinity (KD=30 nM) is ten times weaker than that of bacterial CSCs. Free SAT from bacteria is >100-fold more sensitive to feedback inhibition by cysteine than AtSATm/c. The sensitivity of plant SATs to cysteine is further decreased by CSC formation, while the feedback inhibition of bacterial SAT by cysteine is not affected by CSC formation. The data demonstrate highly similar quaternary structures of the CSCs from bacteria and plant, but emphasize differences with respect to the affinity of CSC formation (KD) and the regulation of cysteine sensitivity of SAT within the CSC.

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Cpl-7, a lysozyme encoded by a pneumococcal bacteriophage with a novel cell wall-binding motif.

Publication Date: 2010 Aug 18 PMID: 20720016
Authors: Bustamante, N. - Campillo, N. E. - Garcia, E. - Gallego, C. - Pera, B. - Diakun, G. P. - Saiz, J. L. - Garcia, P. - Diaz, J. F. - Menendez, M.
Journal: J Biol Chem

Bacteriophage endolysins comprise a group of new antibacterials refractory to development of resistance. We present here the first structural study of the Cpl-7 endolysin, encoded by pneumococcal bacteriophage Cp-7. It contains an N-terminal catalytic module (CM) belonging to the GH25 family of glycosyl hydrolases, and a C-terminal region comprising three identical repeats of 42 amino acids (CW_7 repeats), unrelated to choline-targeting motifs present in other cell wall hydrolases produced by Streptococcus pneumoniae and its bacteriophages, that account for cell wall attachment. By combining different biophysical techniques and molecular modeling, a three-dimensional model of the overall protein structure is proposed, consistent with circular dichroism and sequence-based secondary structure prediction, small angle X-ray scattering data, and Cpl-7 hydrodynamic behavior. Cpl-7 is an approximately 115 A-long molecule with two well differentiated regions, corresponding to the CM and the cell wall-binding region (CWBR), arranged in a lateral disposition. The CM displays the (betaalpha)(5)beta(3) barrel topology characteristic of the GH25 family, and the impact of sequence differences with the CM of the Cpl-1 lysozyme in substrate binding is discussed. The CWBR is organized in three tandemly-assembled three-helical bundles whose dispositions remind a super-helical structure. Its approximate dimensions are 60 x 20 x 20 A and presents a concave face that might constitute the functional region involved in bacterial surface recognition. The distribution of CW_7 repeats in the sequences deposited in the Entrez Database has been examined, and the results drastically expanded the antimicrobial potential of the Cpl-7 endolysin.

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Structure and functional analysis of LptC, a conserved membrane protein involved in the lipopolysaccharide export pathway in Escherichia coll.

Publication Date: 2010 Aug 18 PMID: 20720015
Authors: Tran, A. X. - Dong, C. - Whitfield, C.
Journal: J Biol Chem

LptC is a conserved bitopic inner membrane protein from Escherichia coli involved in the export of lipopolysaccharide from its site of synthesis in the cytoplasmic membrane to the outer membrane. LptC forms a complex with the ATP-binding cassette transporter, LptBFG, which is thought to facilitate the extraction of lipopolysaccharide from the inner membrane and release it into a translocation pathway that includes the putative periplasmic chaperone LptA. Cysteine-modification experiments established that the catalytic domain of LptC is oriented towards the periplasm. The structure of the periplasmic domain is described at a resolution of 2.2-angstroms from X-ray crystallographic data. The periplasmic domain of LptC consists of a twisted-boat structure with two beta-sheets in apposition to each other. The beta-sheets contain seven and eight anti-parallel beta-strands, respectively. This structure bears a high degree of resemblance to the crystal structure of LptA. Like LptA, LptC binds lipopolysaccharide in vitro. In vitro, LptA can displace lipopolysaccharide from LptC (but not vice versa), consistent with their locations and their proposed placement in a unidirectional export pathway.

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Regulation of constitutive cargo transport from the trans-golgi network to plasma membrane by golgi-localized G protein {beta}{gamma} subunits.

Publication Date: 2010 Aug 18 PMID: 20720014
Authors: Irannejad, R. - Wedegaertner, P. B.
Journal: J Biol Chem

Observations of Golgi fragmentation upon introduction of G protein betagamma (Gbetagamma) subunits into cells have implicated Gbetagamma in a pathway controlling the fission at the TGN of plasma membrane (PM)-destined transport carriers. However, the subcellular location where Gbetagamma acts to provoke Golgi fragmentation is not known. Additionally, a role for Gbetagamma in regulating TGN-to-PM transport has not been demonstrated. Here we report that constitutive or inducible targeting of Gbetagamma to the Golgi, but not other subcellular locations, causes PLC- and PKD-dependent vesiculation of the Golgi in Hela cells; Golgi-targeted beta1gamma2 also activates PKD. Moreover, the novel Gbetagamma inhibitor, gallein, and the Gbetagamma sequestering protein, GRK2ct, reveal that Gbetagamma is required for the constitutive PM transport of two model cargo proteins, VSV-G and ss-HRP. Importantly, Golgi-targeted GRK2ct, but not a PM-targeted GRK2ct, also blocks protein transport to the PM. To further support a role for Golgi-localized Gbetagamma, endogenous Gbeta was detected at the Golgi in Hela cells. These results are the first to establish a role for Golgi-localized Gbetagamma in regulating protein transport from the TGN to the cell surface.

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Structure of hepatitis E virion-sized particle reveals an RNA-dependent viral assembly pathway.

Publication Date: 2010 Aug 18 PMID: 20720013
Authors: Xing, L. - Li, T. C. - Miyazaki, N. - Simon, M. N. - Wall, J. S. - Moore, M. - Wang, C. Y. - Takeda, N. - Wakita, T. - Miyamura, T. - Cheng, R. H.
Journal: J Biol Chem

Hepatitis E virus (HEV) induces acute liver failure in human with high fatality rate in pregnant women. There is a need for anti-HEV research to understand the assembly process of HEV native capsid. Here, we produced a large virion-sized and a small T=1 capsid by expressing the HEV capsid protein in insect cells with and without the N-terminal 111 residues, respectively, for comparative structural analysis. The virion-sized capsid demonstrates a T=3 icosahedral lattice and contains RNA fragment in contrast to the RNA-free T=1 capsid. However, both capsids shared common decameric organization. The in vitro assembly further demonstrated that HEV capsid protein had intrinsic ability to form decameric intermediate. Our data suggest that RNA-binding is the extrinsic factor essential for the assembly of HEV native capsids.

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A 24 residue peptide (P5), derived from P35, the CDK5 neuronal activator, specifically inhibits CDK5/P25 hyperactivity and tau hyperphosphorylation.

Publication Date: 2010 Aug 18 PMID: 20720012
Authors: Zheng, Y. L. - Amin, N. D. - Hu, Y. F. - Rudrabhatla, P. - Shukla, V. - Kanungo, J. - Keshavpany, S. - Grant, P. - Albers, W. - Pant, H. C.
Journal: J Biol Chem

The activity of Cdk5/p35 is tightly regulated in the developing and mature nervous system. Stress-induced cleavage of the activator p35 to p25 and a p10 N-terminal domain induces deregulated Cdk5 hyperactivity, perikaryal aggregations of hyperphosphorylated tau and neurofilaments (NFs), pathogenic hallmarks in neurodegenerative diseases such as Alzheimer disease (AD) and Amyotrophic lateral sclerosis (ALS), respectively. Previously, we identified a 125-residue truncated fragment of p35 called CIP that effectively and specifically inhibited Cdk5/p25 activity and tau hyperphosphorylation induced by Abeta peptides in vitro, in HEK293 cells and in neuronal cells. Although these results offer a possible therapeutic approach to those neurodegenerative diseases assumed to derive from Cdk5/p25 hyperactivity and/or Abeta induced pathology, CIP is too large for successful therapeutic regimens. To identify a smaller, more effective peptide, in this study we prepared a 24-residue peptide, p5, spanning CIP residues K245 to A277. p5 more effectively inhibited Cdk5/p25 activity than CIP in vitro. In vivo, p5 inhibited deregulated Cdk5/p25 activity in cortical neurons but had no effect on the activity of Cdk5/p35, nor on any related endogenous Cdk5/p35, nor on any related cyclin-dependent kinases in HEK 293 cells. Specificity of p5 inhibition in vivo may depend on the p10 domain in p35 which is absent in p25. Furthermore, we have demonstrated that p5 reduced Abeta1-42-induced tau hyperphosphorylation and apoptosis in cortical neurons. These results suggest that p5 peptide may be a unique and useful candidate for therapeutic studies of certain neurodegenerative diseases.

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FACTORS FROM HUMAN EMBRYONIC STEM CELL-DERIVED FIBROBLAST-LIKE CELLS PROMOTE TOPOLOGY-DEPENDENT HEPATIC DIFFERENTIATION IN PRIMATE EMBRYONIC AND INDUCED PLURIPOTENT STEM CELLS.

Publication Date: 2010 Aug 18 PMID: 20720011
Authors: Huang, H. P. - Yu, C. Y. - Chen, H. F. - Chen, P. H. - Chuang, C. Y. - Lin, S. J. - Huang, S. T. - Chan, W. H. - Ueng, T. H. - Ho, H. N. - Kuo, H. C.
Journal: J Biol Chem

The future clinical use of embryonic stem cell (ESC)-based hepatocyte replacement therapy depends on the development of an efficient procedure for differentiation of hepatocytes from ESCs. Here we report that a high density of human embryonic stem cell (ESC)-derived fibroblast-like cells (hESdFs) supported the efficient generation of hepatocyte-like cells (HLCs) with functional and mature hepatic phenotypes from primate ESCs and human induced pluripotent stem cells (iPSCs). Molecular and immunocytochemistry analyses revealed that hESdFs caused a rapid loss of pluripotency and induced a sequential endoderm-to-hepatocyte differentiation in the central area of ESC colonies. Knockdown experiments demonstrated that pluripotent stem cells were directed toward endodermal and hepatic lineages by FGF2 and Activin A secreted from hESdFs. Furthermore, we found that the central region of ESC colonies was essential for the hepatic endoderm-specific differentiation, as its removal caused a complete disruption of endodermal differentiation. In conclusion, we describe a novel in vitro differentiation model, and show that hESdF-secreted factors act in concert with regional features of ESC colonies to induce robust hepatic endoderm differentiation in primate pluripotent stem cells.

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ATBF1 inhibits ER function by selectively competing with AIB1 for binding to ER in ER-positive breast cancer cells.

Publication Date: 2010 Aug 18 PMID: 20720010
Authors: Dong, X. Y. - Sun, X. - Guo, P. - Li, Q. - Sasahara, M. - Ishii, Y. - Dong, J. T.
Journal: J Biol Chem

Loss of the q22 band of chromosome 16 is a frequent genetic event in breast cancer, and the candidate tumor suppressor gene ATBF1 has been implicated in breast cancer by genomic deletion, transcriptional downregulation, and association with better prognostic parameters. In addition, ER-positive breast cancer expresses a higher level of ATBF1, suggesting a role of ATBF1 in ER-positive breast cancer. In this study, we examined whether and how ATBF1 affects the ER function in breast cancer cells. We found that ATBF1 inhibited ER-mediated gene transcription, cell growth and proliferation in ER-positive breast cancer cells. In vitro and in vivo immunoprecipitation experiments revealed that ATBF1 physically interacted with ER, and that multiple domains in both ATBF1 and ER proteins mediated the interaction. Furthermore, we demonstrated that ATBF1 inhibited the ER function by selectively competing with the steroid receptor coactivator AIB1 but not GRIP1 or SRC1 for binding to ER. These findings not only support the concept that ATBF1 plays a tumor suppressive role in breast cancer, they also provide a mechanism for how ATBF1 functions as a tumor suppressor in breast cancer.

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Transmembrane segments prevent surface expression of sodium channel Nav1.8 and promote calnexin-dependent channel degradation.

Publication Date: 2010 Aug 18 PMID: 20720009
Authors: Li, Q. - Su, Y. Y. - Wang, H. - Li, L. - Wang, Q. - Bao, L.
Journal: J Biol Chem

The voltage-gated sodium channel (Nav) 1.8 contributes substantially to the rising phase of action potential in small dorsal root ganglion neurons. Nav1.8 is majorly localized intracellularly and its expression on the plasma membrane is regulated by exit from endoplasmic reticulum (ER). Previous work has identified an ER-retention/retrieval motif in the first intracellular loop of Nav1.8 which prevents its surface expression. Here we report that the transmembrane segments of Nav1.8 also cause this channel retained in the ER. Using transferrin receptor and CD8alpha as model molecules, immunocytochemistry showed that the first, second and third transmembrane segments in each domain of Nav1.8 reduced their surface expression. Alanine-scanning analysis revealed acidic amino acids as critical factors in the odd transmembrane segments. Furthermore, co-immunoprecipitation experiments showed that calnexin interacted with acidic amino acid-containing sequence through its transmembrane segment. Overexpression of calnexin resulted in increased degradation of those proteins through ER-associated degradation pathway, while downregulation of calnexin reversed the phenotype. Thus our results reveal a critical role and mechanism of transmembrane segments in surface expression and degradation of Nav1.8.

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Time-of-day and nutrients in feeding govern daily expression rhythms of the gene for sterol regulatory element-binding protein (SREBP)-1 in the mouse liver.

Publication Date: 2010 Aug 18 PMID: 20720008
Authors: Matsumoto, E. - Ishihara, A. - Tamai, S. - Nemoto, A. - Iwase, K. - Hiwasa, T. - Shibata, S. - Takiguchi, M.
Journal: J Biol Chem

Sterol regulatory element-binding protein-1 (SREBP-1) plays a central role in transcriptional regulation of genes for hepatic lipid synthesis that utilizes diet-derived nutrients such as carbohydrates and amino acids, and expression of SREBP-1 exhibits daily rhythms with a peak in the nocturnal feeding period under standard housing conditions of mice. Here, we report that the Srebp-1 expression rhythm shows time cue-independent and Clock mutation-sensitive circadian nature, and is synchronized with varied photoperiods apparently through entrainment of locomotor activity and food intake. Fasting caused diminution of Srebp-1 expression, while diabetic db/db and ob/ob mice showed constantly high expression with loss of rhythmicity. Time-restricted feedings during mid-light and mid-dark periods exhibited differential effects, the latter causing more severe damping of the oscillation. Therefore, ''when to eat in a day (the light/dark cycle)'', rather than ''whenever to eat in a day'', is a critical determinant to shape the daily rhythm of Srebp-1 expression. We further found that a high-carbohydrate diet and a high-protein diet, as well as a high-fat diet, cause phase shifts of the oscillation peak into the light period, underlining the importance of ''what to eat''. Daily rhythms of SREBP-1 protein levels and Akt phosphorylation levels also exhibited nutrient-responsive changes. Taken together, these findings provide a model for mechanisms by which time-of-day and nutrients in feeding shape daily rhythms of the Srebp-1 expression and possibly a number of other physiological functions with interindividual and interdaily differences in human beings and wild animals subjected to day-by-day changes in dietary timing and nutrients.

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TPC2 IS A NOVEL NAADP-SENSITIVE Ca2+-RELEASE CHANNEL, OPERATING AS A DUAL SENSOR OF LUMINAL pH AND Ca2+

Publication Date: 2010 Aug 18 PMID: 20720007
Authors: Pitt, S. J. - Funnell, T. - Sitsapesan, M. - Venturi, E. - Rietdorf, K. - Ruas, M. - Ganesan, A. - Gosain, R. - Churchill, G. C. - Zhu, M. X. - Parrington, J. - Galione, A. - Sitsapesan, R.
Journal: J Biol Chem

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a molecule capable of initiating the release of intracellular Ca(2+) required for many essential cellular processes. Recent evidence links two-pore channels (TPCs) with NAADP-induced release of Ca(2+) from lysosome-like acidic organelles, however, there has been no direct demonstration that TPCs can act as NAADP-sensitive Ca(2+)-release channels. Controversial evidence also proposes ryanodine receptors (RyR) as the primary target of NAADP. We show that TPC2, the major lysosomal targeted isoform, is a cation channel with selectivity for Ca(2+) that will enable it to act as a Ca(2+)-release channel in the cellular environment. NAADP opens TPC2 channels in a concentration-dependent manner, binding to high affinity activation and low affinity inhibition sites. At the core of this process is the luminal environment of the channel. The sensitivity of TPC2 to NAADP is steeply dependent on the luminal [Ca(2+)] allowing extremely low levels of NAADP to open the channel. In parallel, luminal pH controls NAADP affinity for TPC2 by switching from reversible activation of TPC2 at low pH to irreversible activation at neutral pH. Further evidence earmarking TPCs as the likely pathway for NAADP-induced intracellular Ca(2+)-release is obtained from the use of Ned-19, the selective blocker of cellular NAADP-induced Ca(2+)-release. Ned-19 antagonises NAADP-activation of TPC2 in a non-competitive manner at 1 muM but potentiates NAADP activation at nanomolar concentrations. This single-channel study provides a long-awaited molecular basis for the peculiar mechanistic features of NAADP signalling, and a framework for understanding how NAADP acts to mediate key physiological events.

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ALS-associated proteins TDP-43 and FUS/TLS function in a common biochemical complex to coregulate HDAC6 mRNA.

Publication Date: 2010 Aug 18 PMID: 20720006
Authors: Kim, S. H. - Shanware, N. - Bowler, M. J. - Tibbetts, R. S.
Journal: J Biol Chem

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that preferentially targets motor neurons. It was recently found that dominant mutations in two related RNA-binding proteins, TDP-43 (43 kDa TAR DNA-binding domain protein) and FUS/TLS (fused in sarcoma/translated in liposarcoma) cause a subset of ALS. The convergent ALS phenotypes associated with TDP-43 and FUS/TLS mutations are suggestive of a functional relationship; however, whether or not TDP-43 and FUS/TLS operate in common biochemical pathways is not known. Here we show that TDP-43 and FUS/TLS directly interact to form a complex at endogenous expression levels in mammalian cells. Binding was mediated by an unstructured TDP-43 C-terminal domain and occurred within the context of a 300-400 kDa complex that also contained C-terminal cleavage products of TDP-43 linked to neuropathology. TDP-43 C-terminal fragments were excluded from large molecular mass TDP-43 ribonucleoprotein complexes, but retained FUS/TLS binding activity. The functional significance of TDP-43-FUS/TLS complexes was established by showing that RNAi silencing of either TDP-43 or FUS/TLS reduced the expression of HDAC6 mRNA. TDP-43 and FUS/TLS associated with HDAC6 mRNA in intact cells and in vitro, and competition experiments suggested that the proteins occupy overlapping binding sites. The combined findings demonstrate that TDP-43 and FUS/TLS form a functional complex in intact cells and suggest that convergent ALS phenotypes associated with TDP-43 and FUS/TLS mutations may reflect their participation in common biochemical processes.

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N-type calcium channel in the pathogenesis of experimental autoimmune encephalomyelitis.

Publication Date: 2010 Aug 18 PMID: 20720005
Authors: Tokuhara, N. - Namiki, K. - Uesugi, M. - Miyamoto, C. - Ohgoh, M. - Ido, K. - Yoshinaga, T. - Yamauchi, T. - Kuromitsu, J. - Kimura, S. - Miyamoto, N. - Kasuya, Y.
Journal: J Biol Chem

One of the family of voltage-gated calcium channels (VGCC), the N-type Ca(2+) channel, is located predominantly in neurons and is associated with a variety of neuronal responses, including neurodegeneration. A precise mechanism for how the N-type Ca(2+) channel plays a role in neurodegenerative disease, however, is unknown. In this study, we immunized N-type Ca(2+) channel alpha(1B)-deficient (alpha(1B)(-/-)) mice and their wild type (WT) littermates with myelin oligodendrocyte glycoprotein 35-55 and analyzed the progression of experimental autoimmune encephalomyelitis (EAE). The neurological symptoms of EAE in the alpha(1B)(-/-) mice were less severe than in the WT mice. In conjunction with these results, sections of the spinal cord (SC) from alpha(1B)(-/-) mice revealed a reduction in both leukocytic infiltration and demyelination compared with WT mice. No differences were observed in the delayed-type hypersensitivity response, spleen cell proliferation or cytokine production from splenocytes between the two genotypes. On the other hand, western blot array analysis and RT-PCR revealed that a typical increase in the expression of MCP-1 in the SC showed a good correlation with the infiltration of leukocytes into the SC. Likewise, immunohistochemical analysis showed that the predominant source of MCP-1 was activated microglia. The cytokine-induced production of MCP-1 in primary cultured microglia from WT mice was significantly higher than that from alpha(1B)(-/-) mice and was significantly inhibited by a selective N-type Ca(2+) channel antagonist, omega-conotoxin GVIA or a withdrawal of extracellular Ca(2+). These results suggest that the N-type Ca(2+) channel is involved in the pathogenesis of EAE at least in part by regulating MCP-1 production by microglia.

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Activator-dependent p300 acetylation of chromatin in vitro: enhancement of transcription by disruption of repressive nucleosome-nucleosome interactions.

Publication Date: 2010 Aug 18 PMID: 20720004
Authors: Szerlong, H. J. - Prenni, J. E. - Nyborg, J. K. - Hansen, J. C.
Journal: J Biol Chem

Condensation of chromatin into higher order structures is mediated by intra- and inter-fiber nucleosome-nucleosome interactions. Our goals in this study were to determine the impact specific activator-dependent histone acetylation had on chromatin condensation, and to ascertain whether acetylation-induced changes in chromatin condensation were related to changes in RNA Polymerase II (RNAPII) activity. To accomplish this, an in vitro model system was constructed in which the purified transcriptional activators, Tax and phosphorylated CREB, recruited the p300 histone acetyltransferase (HAT) to nucleosomal templates containing the human T-cell leukemia virus type-1 promoter sequences. We find that activator-dependent p300 histone acetylation disrupted both inter- and intra-fiber nucleosome-nucleosome interactions, and simultaneously led to enhanced RNAPII transcription from the decondensed model chromatin. p300 HAT activity had two distinct components: non-targeted, ubiquitous activity in the absence of activators, and activator-dependent activity targeted primarily to promoter-proximal nucleosomes. Mass spectrometry identified several unique p300 acetylation sites on nucleosomal histone H3 (H3K9, H3K27, H3K36, and H3K37). Collectively, our data have important implications for understanding both the mechanism of RNAPII transcriptional regulation by chromatin and the molecular determinants of higher order chromatin structure.

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The gene encoding the hematopoietic stem cell regulator Ccn3/Nov is under direct cytokine control through the transcription factors STAT5A/B.

Publication Date: 2010 Aug 18 PMID: 20720003
Authors: Kimura, A. - Martin, C. - Robinson, G. W. - Simone, J. M. - Chen, W. - Wickre, M. C. - O'Shea, J. J. - Hennighausen, L.
Journal: J Biol Chem

Cytokines control the biology of hematopoietic stem cells (HSCs) and progenitor cells in part through the transcription factors STAT5A/B. To investigate the target genes of STAT5A/B activated by cytokines in HSCs and progenitors, we performed microarray analyses using Lineage- Sca-1+ c-Kit+ (KSL) cells in the presence and absence of STAT5A/B. Stimulation with a cocktail containing IL-3, IL-6, SCF, TPO and Flt3 ligand induced Ccn3/Nov mRNA over 100-fold in WT (control), but not Stat5a/b-null KSL cells. CCN3/NOV is a positive regulator of human HSC self-renewal and development of committed blood cells. Without stimulation, the Ccn3/Nov signal level was low in control KSL cells similar to Stat5a/b-null KSL cells. To determine which cytokine activates the Ccn3/Nov gene we analyzed Lineage- c-Kit+ (KL) and 32D cells using qPCR and ChIP assays. While stimulation with a cocktail lacking IL-3 prevented the induction of Ccn3/Nov in control KL cells, IL-3 alone could induce Ccn3/Nov mRNA in control KL and 32D cells. ChIP assays using 32D cells revealed IL-3-induced binding of STAT5A/B to a GAS site in the Ccn3/Nov gene promoter. This is the first report that Ccn3/Nov is directly induced by cytokines through STAT5A/B.

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A dual interface determines the recognition of RNA polymerase II by RNA capping enzyme.

Publication Date: 2010 Aug 18 PMID: 20720002
Authors: Suh, M. H. - Meyer, P. A. - Gu, M. - Ye, P. - Zhang, M. - Kaplan, C. D. - Lima, C. D. - Fu, J.
Journal: J Biol Chem

RNA capping enzyme (CE) is recruited specifically to RNA polymerase (Pol) II transcription sites to facilitate cotranscriptional 5(prime)-capping of pre-mRNA and other Pol II transcripts. The current model to explain this specific recruitment of CE to Pol II as opposed to Pol I and Pol III rests on the interaction between CE and the phosphorylated C-terminal domain (CTD) of Pol II largest subunit Rpb1, and more specifically between the CE nucleotidyl-transferase (NT) domain and the phosphorylated CTD (CTD-P). Through biochemical and diffraction analyses, we demonstrate the existence of a distinctive stoichiometric complex between CE and the phosphorylated Pol II (Pol IIO). Analysis of the complex revealed an additional and unexpected polymerase-CE interface (PCI) located on the multi-helical Foot domain of Rpb1. We name this interface PCI1 and the previously known NT/CTD-P interface PCI2. While PCI1 and PCI2 individually contribute to weak interactions with CE, a dramatically stabilized and stoichiometric complex is formed when PCI1 and PCI2 are combined in cis as they occur in an intact Pol IIO molecule. Disrupting either PCI1 or PCI2 by alanine-substitution or deletion diminishes CE association with Pol II and causes severe growth defects in vivo. Evidence from manipulating PCI1 indicates that the Foot domain contributes to the specificity in CE interaction with Pol II as opposed to Pol I and Pol III. Our results indicate that the dual interface based on combining PCI1 and PCI2 is required for directing CE to Pol II elongation complexes.

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Peroxisome proliferator-activated receptor {gamma} coactivator 1{alpha} (PGC-1{alpha}) promotes skeletal muscle lipid refueling in vivo by activating de-novo lipogenesis and the pentose phosphate pathway.

Publication Date: 2010 Aug 17 PMID: 20716531
Authors: Summermatter, S. - Baum, O. - Santos, G. - Hoppeler, H. - Handschin, C.
Journal: J Biol Chem

Exercise induces a pleiotropic adaptive response in skeletal muscle, largely through the peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha). PGC-1alpha enhances lipid oxidation and thereby provides energy for sustained muscle contraction. Its potential implication in promoting muscle refueling remains, however, unresolved. Here, we investigated a possible role of elevated PGC-1alpha levels in skeletal muscle lipogenesis in vivo and the molecular mechanisms that underlie PGC-1alpha-mediated de-novo lipogenesis. To this end, we studied transgenic mice with physiological overexpression of PGC-1alpha and human muscle biopsies pre- and post exercise. We demonstrate that PGC-1alpha enhances lipogenesis in skeletal muscle through liver x receptor alpha (LXRalpha) dependent activation of the fatty acid synthase (FAS) promoter and by increasing FAS activity. Using chromatin immunoprecipitation, we establish a direct interaction between PGC-1alpha and the LXR-responsive element in the FAS promoter. Moreover, we show for the first time that increased glucose uptake and activation of the pentose phosphate pathway provide substrates for RNA synthesis and cofactors for de-novo lipogenesis. Similarly, we observed increased lipogenesis and lipid levels in human muscle biopsies that were obtained post-exercise. Our findings suggest that PGC-1alpha coordinates lipogenesis, intramyocellular lipid accumulation and substrate oxidation in exercised skeletal muscle in vivo.

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Effect of Vitamin K-Dependent Protein Precursor Propeptide, Vitamin K Hydroquinone and Glutamate Substrate Binding on the {gamma}-Glutamyl Carboxylase's Structure and Function.

Publication Date: 2010 Aug 17 PMID: 20716530
Authors: Higgins-Gruber, S. L. - Mutucumarana, V. P. - Lin, P. J. - Jorgenson, J. W. - Stafford, D. W. - Straight, D. L.
Journal: J Biol Chem

The gamma-glutamyl carboxylase utilizes four substrates to catalyze carboxylation of certain glutamic acid residues in vitamin K-dependent proteins. How the enzyme brings the substrates together to promote catalysis is an important question in understanding the structure and function of this enzyme. The propeptide is the primary binding site of the vitamin K-dependent proteins to carboxylase. It is also an effector of carboxylase activity. We tested the hypothesis that binding of substrates causes changes to the carboxylase and in turn to the substrate-enzyme interactions. In addition we investigated how the sequences of the propeptides affected the substrate-enzyme interaction. To study these questions we employed fluorescently labeled propeptides to measure affinity for the carboxylase. We also measured the ability of several propeptides to increase carboxylase catalytic activity. Finally we determined the effect of substrates - vitamin K hydroquinone, FLEEL and NaHCO3 - on the propeptide-carboxylase complexes stability. We found a wide variation in the propeptide affinities for carboxylase. In contrast, the propeptides tested had similar effects on carboxylase catalytic activity. The pentapeptide carboxylase substrate, FLEEL, and vitamin K hydroquinone both stabilized the propeptide-carboxylase complex. The two together had a greater effect than either alone. We conclude that the effect of propeptide and substrates on carboxylase controls the order of substrate binding in such a way as to ensure efficient, specific carboxylation.

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Adipose tissue-specific inhibition of hypoxia inducible factor 1{alpha} induces obesity and glucose intolerance by impeding energy expenditure in mice.

Publication Date: 2010 Aug 16 PMID: 20716529
Authors: Zhang, X. - Lam, K. S. - Ye, H. - Chung, S. K. - Zhou, M. - Wang, Y. - Xu, A.
Journal: J Biol Chem

Hypoxia in adipose tissue has been postulated as a possible contributor to obesity-related chronic inflammation, insulin resistance and metabolic dysfunction. Hypoxia inducible factor 1alpha (HIF1alpha), a master signal mediator of hypoxia response, is elevated in obese adipose tissue. However, the role of HIF1alpha in obesity-related pathologies remains to be determined. Here we show that transgenic mice with adipose tissue-selective expression of a dominant negative version of HIF1alpha developed more severe obesity and were more susceptible to high fat diet-induced glucose intolerance and insulin resistance compared to their wild-type littermates. Obesity in the transgenic mice was attributed to impaired energy expenditure and reduced thermogenesis. Histological examination of interscapular brown adipose tissue (BAT) in the transgenic mice demonstrated a markedly increased size of lipid droplets and decreased mitochondrial density in adipocytes, a phenotype similar to that in white adipose tissue (WAT). These changes in BAT of the transgenic mice were accompanied by decreased mitochondrial biogenesis and reduced expression of key thermogenic genes. In the transgenic mice, angiogenesis in BAT was decreased, but was little affected in WAT. These findings support an indispensible role of HIF1alpha in maintaining the thermogenic functions of BAT, possibly through promoting angiogenesis and mitochondrial biogenesis in this tissue.

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The ATP synthase a-subunit of extreme alkaliphiles is a distinct variant. Mutations in the critical alkaliphile-specific residue Lys-180 and other residues that support alkaliphile OXPHOS.

Publication Date: 2010 Aug 17 PMID: 20716528
Authors: Fujisawa, M. - Fackelmayer, O. - Liu, J. - Krulwich, T. A. - Hicks, D. B.
Journal: J Biol Chem

A lysine residue in the putative proton uptake pathway of the ATP synthase a-subunit is found only in alkaliphilic Bacillus species and is proposed to play roles in proton capture, retention and passage to the synthase rotor. Here, Lys-180 was replaced with alanine (Ala), glycine (Gly), cysteine (Cys), arginine (Arg), or histidine (His) in the chromosome of alkaliphilic Bacillus pseudofirmus OF4. All mutants exhibited octyl glucoside (OG)-stimulated ATPase activity and beta-subunit levels at least as high as wild-type. Purified mutant F(1)F(0) ATP synthases all contained substantial a-subunit levels. The mutants exhibited diverse patterns of native (no OG) ATPase activity and a range of defects in malate growth and in vitro ATP synthesis at pH 10.5. ATP synthesis by the Ala, Gly, and His mutants was also impaired at pH 7.5 in the presence of protonophoric uncoupler. Thus Lys-180 plays a role when the protonmotive force is reduced at near neutral, not just at high pH. The Arg mutant exhibited no ATP synthesis activity in the alkaliphile setting although activity was reported for a K180R mutant of a thermoalkaliphile synthase [McMillan et al. 2007 J. Biol. Chem. 282, 17395-17404]. The hypothesis that a-subunits from extreme alkaliphiles and the thermoalkaliphile represent distinct variants was supported by demonstration of the importance of additional alkaliphile-specific a-subunit residues, not found in the thermoalkaliphile, for malate-growth of B. pseudofirmus OF4. Finally, a mutant B. pseudofirmus OF4 synthase with switched positions of Lys-180 (helix 4) and Gly-212 (helix 5) retained significant coupled synthase activity accompanied by proton leakiness.

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Tissue-specific functions in the FABP (fatty acid-binding protein) family.

Publication Date: 2010 Aug 17 PMID: 20716527
Authors: Storch, J. - Thumser, A. E.
Journal: J Biol Chem

The intracellular fatty acid-binding proteins (FABPs) are abundantly expressed in almost all tissues. They exhibit high affinity binding of a single long-chain fatty acid, with the exception of the liver FABP, which binds two fatty acids or other hydrophobic molecules. The FABPS have highly similar tertiary structures consisting of a 10-stranded anti-parallel beta-barrel and N-terminal helix-turn-helix motif. Research emerging in the last decade has suggested that the FABPs have tissue-specific functions that reflect tissue-specific aspects of lipid and fatty acid metabolism. Proposed roles for the FABPs include the assimilation of dietary lipids in the intestine; targeting of liver lipids to catabolic and anabolic pathways; regulation of lipid storage and lipid-mediated gene expression in adipose tissue and macrophages; fatty acid targeting to beta-oxidation pathways in muscle, and the maintenance of phospholipid membranes in neural tissues. The regulation of these diverse processes is accompanied by the expression of different and sometimes multiple FABPs in these tissues, and may be driven by protein-protein and protein-membrane interactions.

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Trans-membrane and trans-subunit regulation of ectodomain shedding of platelet glycoprotein Ib{alpha}

Publication Date: 2010 Aug 17 PMID: 20716526
Authors: Mo, X. - Nguyen, N. X. - Mu, F. T. - Yang, W. - Luo, S. Z. - Fan, H. - Andrews, R. K. - Berndt, M. C. - Li, R.
Journal: J Biol Chem

Ectodomain shedding of transmembrane proteins may be regulated by their cytoplasmic domains. To date, the effecting cytoplasmic domain and the shed extracellular domain have been in the same polypeptide. In this study, shedding of GPIbalpha, the ligand-binding subunit of the platelet GPIb-IX complex and a marker for platelet senescence and storage lesion, was assessed in Chinese hamster ovary cells with/without functional GPIbalpha sheddase ADAM17. Mutagenesis of the GPIb-IX complex, which contains GPIbalpha, GPIbbeta and GPIX subunits, revealed that the intracellular membrane-proximal calmodulin-binding region of GPIbbeta is critical for ADAM17-dependent shedding of GPIbalpha induced by the calmodulin inhibitor, W7. Perturbing the interaction between GPIbalpha and GPIbbeta subunits further lessened the restraint of GPIbbeta on GPIbalpha shedding. However, contrary to the widely accepted model of calmodulin regulation of ectodomain shedding, the R152E/L153E mutation in the GPIbbeta cytoplasmic domain disrupted calmodulin binding to GPIbbeta but had little effect on GPIbalpha shedding. Analysis of induction of GPIbalpha shedding by membrane-permeable GPIbbeta-derived peptides implicated the association of GPIbbeta with a to-be-identified intracellular protein in mediating regulation of GPIbalpha shedding. Overall, these results provide evidence for a novel trans-subunit mechanism for regulating ectodomain shedding.

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Characterisation of a novel WDR5 binding site that recruits RbBP5 through a conserved motif and enhances methylation of H3K4 by MLL1.

Publication Date: 2010 Aug 17 PMID: 20716525
Authors: Odho, Z. - Southall, S. M. - Wilson, J. R.
Journal: J Biol Chem

Histone modification is well established as a fundamental mechanism driving the regulation of transcription, replication and DNA repair through the control of chromatin structure. Likewise, it is apparent that incorrect targeting of histone modifications contributes to misregulated gene expression and hence to developmental disorders and diseases of genomic instability such as cancer. The KMT2 family of SET domain methyltransferases, typified by MLL1, are responsible for histone H3 lysine-4 methylation, a marker of active genes. To ensure that this modification is correctly targeted, a multi-protein complex associates with the methyltransferase and directs activity. We have identified a novel interaction site on the core complex protein WDR5, and mapped the complementary site on its partner RbBP5. We have characterised this interaction by X-ray crystallography and show how it is fundamental to the assembly of the complex and to the regulation of methyltransferase activity. We show which region of RbBP5 contributes directly to MLL activation and combine our structural and biochemical data to produce a model to show how WDR5 and RbBP5 act cooperatively to stimulate activity.

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Regulation of the AGS3-G{alpha}i signaling complex by a seven-transmembrane span receptor.

Publication Date: 2010 Aug 31 PMID: 20716524
Authors: Oner, S. S. - An, N. - Vural, A. - Breton, B. - Bouvier, M. - Blumer, J. B. - Lanier, S. M.
Journal: J Biol Chem

G-protein signaling modulators (GPSM) play diverse functional roles through their interaction with G-protein subunits. AGS3 (GPSM1) contains four G-protein regulatory motifs (GPR) that directly bind Galphai free of Gbetagamma providing an unusual scaffold for the G-switch and signaling complexes, but the mechanism by which signals track into this scaffold are not well understood. We report the regulation of the AGS3-Galphai signaling module by a cell surface, seven-transmembrane receptor. AGS3 and Galphai1 tagged with Renilla luciferase or yellow fluorescent protein expressed in mammalian cells exhibited saturable, specific bioluminescence resonance energy transfer (BRET) indicating complex formation in the cell. Activation of alpha2-adrenergic receptors or mu-opioid receptors reduced AGS3-Rluc - Galphai1YFP energy transfer by over 30%. The agonist-mediated effects were inhibited by pertussis toxin and by co-expression of RGS4, but were not altered by Gbetagamma sequestration with the carboxyl terminus of GRK2. Galphai-dependent and agonist-sensitive BRET was also observed between AGS3 and cell-surface receptors typically coupled to Galphai and/or Galphao indicating that AGS3 is part of a larger signaling complex. Upon receptor activation, AGS3 reversibly dissociates from this complex at the cell cortex. Receptor coupling to both Galphabetagamma and GPR-Galphai offer additional flexibility for systems to respond and adapt to challenges and orchestrate complex behaviors.

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Editing of EBV-encoded BART6 microRNAs controls their dicer targeting and consequently affects viral latency.

Publication Date: 2010 Aug 17 PMID: 20716523
Authors: Iizasa, H. - Wulff, B. E. - Alla, N. R. - Maragkakis, M. - Megraw, M. - Hatzigeorgiou, A. - Iwakiri, D. - Takada, K. - Wiedmer, A. - Showe, L. - Lieberman, P. - Nishikura, K.
Journal: J Biol Chem

Certain primary transcripts of miRNA (pri-miRNAs) undergo RNA editing that converts adenosine to inosine. The Epstein-Barr Virus (EBV) genome encodes multiple microRNA genes of its own. Here we report that primary transcripts of ebv-miR-BART6 (pri-miR-BART6) are edited in latently EBV-infected cells. Editing of wild-type pri-miR-BART6 RNAs dramatically reduced loading of miR-BART6-5p RNAs onto the miRNA-induced silencing complex. Editing of a mutation-containing pri-miR-BART6 found in Daudi Burkitt lymphoma and nasopharyngeal carcinoma C666-1 cell lines suppressed processing of miR-BART6 RNAs. Most importantly, miR-BART6-5p RNAs silence Dicer through multiple target sites located in the 3'UTR of Dicer mRNA. The significance of miR-BART6 was further investigated in cells in various stages of latency. We found that miR-BART6-5p RNAs suppress the EBNA2 viral oncogene required for transition from immunologically less responsive type I and type II latency to the more immunoreactive type III latency as well as Zta and Rta viral proteins essential for lytic replication, revealing the regulatory function of miR-BART6 in EBV infection and latency. Mutation and A-to-I editing appear to be adaptive mechanisms that antagonize miR-BART6 activities.

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Metabolic depression and increased ROS production by isolated mitochondria at moderately low temperatures.

Publication Date: 2010 Aug 19 PMID: 20716522
Authors: Ali, S. S. - Marcondes, M. C. - Bajova, H. - Dugan, L. L. - Conti, B.
Journal: J Biol Chem

Temperature (T) reduction increases lifespan but the mechanisms are not understood. Since reactive oxygen species (ROS) contribute to aging we hypothesized that lowering T might decrease mitochondrial ROS production. We measured respiratory response and ROS production in isolated mitochondria at 32, 35, and 37 C. Lowering T decreased the rates of resting (state 4) and phosphorylating (state 3) respiration phases. Surprisingly, this respiratory slowdown was associated with an increase of ROS production and hydrogen peroxide release, and with elevation of the mitochondrial membrane potential. We also found that at lower T mitochondria produced more carbon-centered lipid radicals, a species known to activate uncoupling proteins. These data indicate that reduced mitochondrial ROS production is not one of the mechanisms mediating lifespan extension at lower T. They suggest instead that increased ROS leakage may mediate mitochondrial responses to hypothermia.

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A major fraction of glycosphingolipids in model and cellular cholesterol containing membranes are undetectable by their binding proteins.

Publication Date: 2010 Aug 17 PMID: 20716521
Authors: Mahfoud, R. - Manis, A. - Binnington, B. - Ackerley, C. - Lingwood, C. A.
Journal: J Biol Chem

Glycosphingolipids (GSLs) accumulate in cholesterol-enriched cell membrane domains, and provide receptors for protein ligands. Lipid-based aglycone interactions can influence GSL carbohydrate epitope presentation. To evaluate this relationship, Verotoxin binding its receptor GSL, globotriaosyl ceramide (Gb3), was analyzed in simple GSL/cholesterol, detergent resistant membrane vesicles by equilibrium density gradient centrifugation. Vesicles separated into two Gb3/cholesterol-containing populations. The lighter, minor fraction (<5% total GSL), bound VT1, VT2, IgG/IgM mAb antiGb3, HIVgp120 or B. simplicifolia lectin. Only IgM antiGb3, more tolerant of carbohydrate modification, bound both vesicle fractions. Post-embedding cryoimmuno-EM confirmed these results. This appears a general GSL-cholesterol property, since similar receptor-inactive vesicles were separated for other GSL-protein ligand systems; cholera toxin (CTx)-GM1, HIVgp120-galactosyl ceramide/sulfatide. Inclusion of galactosyl or glucosyl ceramide (GalCer, GlcCer) rendered VT1-unreactive Gb3/cholesterol vesicles, VT1 reactive. We found GalCer and GlcCer bind Gb3, suggesting GSL-GSL interaction can counter cholesterol masking of Gb3. The similar separation of Vero cell membrane-derived vesicles into minor binding, and major non-binding fractions when probed with VT1, CTx or anti-SSEA4 (a human GSL stem cell marker), demonstrates potential physiological relevance. Cell membrane GSL masking was cholesterol and actin dependent. Cholesterol depletion of Vero and HeLa cells enabled differential VT1B subunit labeling of available and cholesterol-masked plasma membrane Gb3 pools by fluorescence microscopy. Thus, the model GSL/cholesterol vesicle studies predicted two distinct membrane GSL formats, which were demonstrated within the plasma membrane of cultured cells. Cholesterol masking of most cell membrane GSLs may impinge many GSL receptor functions.

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Ubiquitin proteasome dependent degradation of the transcriptional coactivator PGC-1alpha via the N-terminal pathway.

Publication Date: 2010 Aug 15 PMID: 20713359
Authors: Trausch-Azar, J. - Leone, T. C. - Kelly, D. P. - Schwartz, A. L.
Journal: J Biol Chem

PGC-1alpha is a potent, inducible transcriptional coactivator which exerts control on mitochrondrial biogenesis and multiple cellular energy metabolic pathways. PGC-1alpha levels are controlled in a highly dynamic manner reflecting regulation at both transcriptional and post-transcriptional levels. Herein we demonstrate that PGC-1alpha is rapidly degraded in the nucleus (t(1/2) 0.3h) via the ubiquitin proteasome system. An N-terminal deletion mutant of 182 residues, PGC182, as well as a lysine-less mutant form, is nuclear and rapidly degraded (t(1/2) 0.5h) consistent with degradation via the N-terminus-dependent ubiquitin sub-pathway. Both PGC-1alpha and PGC182 degradation rates are increased in cells under low serum conditions. However, a naturally-occurring N-terminal splice variant of 270 residues, NT-PGC-1alpha is cytoplasmic and stable (t(1/2) >7h) providing additional evidence that PGC-1alpha is degraded in the nucleus. These results strongly suggest that the nuclear N-terminus-dependent ubiquitin proteasome pathway governs PGC-1alpha cellular degradation. In contrast, the cellular localization of NT-PCG-1alpha results in a longer-half-life and possible distinct temporal and potentially biological actions.

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Posttranscriptional upregulation of Tsc-22 by Ybx1, a target of miR-216a, mediates TGF-{beta}-induced collagen expression in kidney cells.

Publication Date: 2010 Aug 16 PMID: 20713358
Authors: Kato, M. - Wang, L. - Putta, S. - Wang, M. - Yuan, H. - Sun, G. - Lanting, L. - Todorov, I. - Rossi, J. J. - Natarajan, R.
Journal: J Biol Chem

Increased accumulation of extracellular matrix proteins and hypertrophy by Transforming growth factor-beta1 (TGF-beta) in renal mesangial cells (MC) are hallmark features of diabetic nephropathy (DN). Although the post-transcriptional regulation of key genes has been implicated in these events, details are not fully understood. Here we show that TGF-beta increased microRNA-216a (miR-216a) levels in mouse MC, with parallel downregulation of Ybx1, a miR-216a target and RNA binding protein. TGF-beta also enhanced protein levels of Tsc-22 (TGF-beta stimulated clone 22) and collagen type I alpha-2 (Col1a2) expression in MC through far-upstream enhancer E-boxes by interaction of Tsc-22 with an E-box regulator, Tfe3. Ybx1 colocalized with processing bodies (P-bodies) in MC and formed a ribonucleoprotein complex with Tsc-22 mRNA, and this complex formation was reduced by TGF-beta, miR-216a mimics or Ybx1 shRNA to increase Tsc-22 protein levels, but enhanced by miR-216a inhibitor oligonucleotides. Chromatin immunoprecipitation (ChIP) assays revealed that TGF-beta could increase the occupancies of Tsc-22 and Tfe3 on enhancer E-boxes of Col1a2. Co-IP assays revealed that TGF-beta promoted the interaction of Tsc-22 with Tfe3. These results demonstrate that post-transcriptional regulation of Tsc-22 mediated through Ybx1, a miR-216a target, plays a key role in TGF-beta-induced Col1a2 in MC related to the pathogenesis of DN.

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Maspin regulates endothelial cells adhesion and migration through an integrin signaling pathway.

Publication Date: 2010 Aug 16 PMID: 20713357
Authors: Qin, L. - Zhang, M.
Journal: J Biol Chem

Maspin has been identified as a potent angiogenesis inhibitor. However, the molecular mechanism responsible for its anti-angiogenic property is unclear. In this study, we examined the effect of maspin on endothelial cell (EC) adhesion and migration in a cell culture system. We found that maspin was expressed in blood vessels ECs and human umbilical vein endothelial cells (HUVECs). Maspin significantly enhanced HUVEC cell adhesion to various matrix proteins. This effect was dependent on the activation of integrin beta1, which subsequently led to distribution pattern changes of vinculin and F-actin. These results indicated that maspin affects cell adhesion and cytoskeleton reorganization through an integrin signal transduction pathway. Analysis of HUVECs following maspin treatment revealed increased ILK activities and phosphorylated FAK levels, consistent with increased cell adhesion. Interestingly, when HUVECs were induced to migrate by migration stimulatory factor bFGF, active Rac1 and cdc42 small GTPase levels were decreased dramatically at 30 minutes following maspin treatment. Using phosphorylated FAK at Y397 as an indicator of focal adhesion disassembly, maspin treated HUVECs had elevated FAK phosphorylation compared to the mock treated control. The results were a reduction in focal adhesion disassembly and the retardation in EC migration. This study uncovers a mechanism by which maspin exerts its effect on EC adhesion and migration through an integrin signal transduction pathway.

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Characterization of hMTr1, a human cap1 2'O-ribose methyltransferase.

Publication Date: 2010 Aug 16 PMID: 20713356
Authors: Belanger, F. - Stepinski, J. - Darzynkiewicz, E. - Pelletier, J.
Journal: J Biol Chem

Cellular eukaryotic mRNAs are capped at their 5' ends with a 7-methylguanosine nucleotide, a structural feature that has been shown to be important for conferring mRNA stability, stimulating mRNA biogenesis (splicing, poly[A] addition, nucleo-cytoplasmic transport), and increasing translational efficiency. Whereas yeast mRNAs have no additional modifications to the cap (called cap0), higher eukaryotes are methylated at the 2'O-ribose of the first or the first and second transcribed nucleotides - called cap1 and cap2, respectively. In the present study, we identify the methyltransferase responsible for cap1 formation in human cells, which we call hMTr1 (also known as FTSJD2 and ISG95). We show in vitro that hMTr1 catalyses specific methylation of the 2'O-ribose of the first nucleotide of a capped RNA transcript. Using siRNA mediated knockdown of hMTr1 in Hela cells, we demonstrate that hMTr1 is responsible for cap1 formation in vivo.

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Interdependent phosphorylation within the kinase domain T-loop regulates Chk2 activity.

Publication Date: 2010 Aug 16 PMID: 20713355
Authors: Guo, X. - Ward, M. D. - Tiedebohl, J. B. - Oden, Y. M. - Nyalwidhe, J. O. - Semmes, O. J.
Journal: J Biol Chem

Chk2 is a critical regulator of the cellular DNA damage repair response. Activation of Chk2 in response to IR-induced damage is initiated by phosphorylation of the Chk2 SCD at Ser19, Ser33, Ser35 and Thr68. This precedes autophosphorylation of Thr383/Thr387 in the T-loop region of the kinase domain an event that is a prerequisite for efficient kinase activity. We conducted an in depth analysis of phosphorylation within the T-loop region (residues 366-406). We report four novel phosphorylation sites at Thr372, Thr378, Thr389 and Tyr390. Substitution mutation Y390F was defective for kinase function. The substitution mutation T378A ablated the IR-induction of kinase activity. Interestingly, the substitution mutation T389A demonstrated 6-fold increase in kinase activity when compared to wild type Chk2. In addition, phosphorylation at Thr389 was a prerequisite to phosphorylation at Thr387 but not at Thr383. Semi-quantitative mass spectrometry analysis revealed IR-induced changes in the abundance and subcellular distribution of Chk2 phosphorylation species. We observed IR-induced increase in the abundance of Chk2 species harboring phosphorylation at Ser379, Thr389 and Thr383/Thr389. Almost all Chk2 species phosphorylated at Tyr390 were reduced after IR. There were a number of Chk2 single and double phosphorylation species, including Thr383 and Thr383/Thr389, which relocalized from the nucleus to chromatin following IR. However, only Chk2 hyper-phosphorylated species at Thr383/Thr387/Thr389 and Thr383/Thr387/Thr389/Tyr390 relocalized from almost exclusively chromatin to predominately nuclear expression. These results demonstrate that a complex interdependent network of phosphorylation events within the T-loop exchange region regulates dimerization/autophosphorylation, activation and chromatin egress of Chk2.

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Adenosine Monophosphate Activated Protein Kinase Induces Cholesterol Efflux from Macrophage-Derived Foam Cells and Alleviates Atherosclerosis in Apolipoprotein E-Deficient Mice.

Publication Date: 2010 Aug 16 PMID: 20713354
Authors: Li, D. - Wang, D. - Wang, Y. - Ling, W. - Feng, X. - Xia, M.
Journal: J Biol Chem

Increasing evidence suggests that adenosine monophosphate activated protein kinase (AMPK) exerts protective effects for cardiovascular diseases apart from the regulation of energy homeostasis. However, the role of AMPK and its underlying mechanism on macrophage foam cell formation are poorly understood. In this study, we sought to investigate the potential effects of AMPK in modulating cholesterol deposition by using murine macrophage- derived foam cells. Incubation with 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) markedly attenuated the cholesterol ester accumulation in oxidized low density lipoprotein (OxLDL)loaded macrophages. Notably, AICAR treatment significantly increased ATP-binding cassette transporters G1 (ABCG1)mRNA and protein levels without affecting mRNA and protein expression of ABCA1, scavenger receptors, including scavenger receptor-A (SR-A), CD36, and scavenger receptor-BI (SR-BI), and cholesterol synthesis-related genes. The upregulation of ABCG1 by AICAR was independent on liver X receptor/retinoid X receptor (LXR/RXR) pathway but dependent on external signal regulated kinase (ERK) activation. AICAR elevates ABCG1 expression through a post-transcriptional mechanism that stabilizes the mRNA. Using a heterologous system with luciferase as a reporter, we further identify the ABCG1 mRNA 3' untranslated region (3-UTR) responsible for the regulatory effect of AICAR. Prevention of ABCG1 expression by small interfering RNA abolished the AICAR-mediated attenuation on foam cell formation. Furthermore, increased ABCG1 expression and reduced lipid accumulation were demonstrated in AICAR treated-macrophages isolated from apolipoprotein E-deficient mice (apoE-/- mice). AICAR treatment also inhibited atherosclerotic plaque formation in apoE-/- mice. Our findings elucidate a precise mechanism involved in the prevention of atherogenesis by AMPK.

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Phosphorylation and activation of androgen receptor by Aurora-A.

Publication Date: 2010 Aug 16 PMID: 20713353
Authors: Shu, S. K. - Liu, Q. - Coppola, D. - Cheng, J. Q.
Journal: J Biol Chem

Aurora-A kinase is frequently overexpressed/activated in various types of human malignancy including prostate cancer. In this study, we demonstrate elevated levels of Aurora-A in androgen-refractory LNCaP-RF but not androgen-sensitive LNCaP cells, which prompted us to examine whether Aurora-A regulates AR and whether elevated Aurora-A is involved in androgen-independent cell growth. We show that ectopic expression of Aurora-A induces AR transactivation activity in the presence and absence of androgen. Aurora-A interacts with AR and phosphorylates AR at Thr282 and Ser293 in vitro and in vivo. Aurora-A induces AR transactivation activity in a phosphorylation-dependent manner. Ectopic expression Aurora-A in LNCaP cells induces the PSA expression and cell survival whereas knockdown of Aurora-A sensitizes LNCaP-RF cells to apoptosis and cell growth arrest. These data indicate that AR is a substrate of Aurora-A and that elevated Aurora-A could contribute to androgen-independent cell growth by phosphorylation and activation of AR.

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RB/E2F1 complex mediates DNA damage responses through transcriptional regulation of ZBRK1.

Publication Date: 2010 Aug 16 PMID: 20713352
Authors: Liao, C. C. - Tsai, C. Y. - Chang, W. C. - Lee, W. H. - Wang, J. M.
Journal: J Biol Chem

RB plays an essential role in DNA damage-induced growth arrest and regulates the expression of several factors essential for DNA repair machinery. However, how RB coordinates DNA damage response through transcriptional regulation of genes involved in growth arrest remains largely unexplored. We examined whether RB can mediate the response to DNA damage through modulation of ZBRK1, a zinc finger-containing transcriptional repressor that can modulate the expression of GADD45A, a DNA damage response gene, to induce cell cycle arrest in response to DNA damage. We found that the ZBRK1 promoter contains an authentic E2F-recognition sequence that specifically binds E2F1, but not E2F4 or E2F6, together with the chromatin remodeling proteins CtIP and CtBP to form a repression complex that suppresses ZBRK1 transcription. Furthermore, loss of RB-mediated transcriptional repression led to an increase in ZBRK1 transcript levels, correlating with increased sensitivity to ultraviolet (UV) and methyl methanesulfonate (MMS) induced DNA damage. Taken together, these results suggest that the RB/E2F1 complex plays a critical role in ZBRK1 transcriptional repression, and loss of this repression may contribute to cellular sensitivity of DNA damage, ultimately leading to carcinogenesis.

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Oxidized cholesteryl esters and phospholipids in zebrafish larvae fed a high-cholesterol diet: macrophage binding and activation.

Publication Date: 2010 Aug 14 PMID: 20710028
Authors: Fang, L. - Harkewicz, R. - Hartvigsen, K. - Wiesner, P. - Choi, S. H. - Almazan, F. - Pattison, J. - Deer, E. - Sayaphupha, T. - Dennis, E. A. - Witztum, J. L. - Tsimikas, S. - Miller, Y. I.
Journal: J Biol Chem

A novel hypercholesterolemic zebrafish model has been developed to study early events of atherogenesis. This model utilizes optically transparent zebrafish larvae, fed a high-cholesterol diet (HCD), to monitor processes of vascular inflammation in live animals. Because lipoprotein oxidation is an important factor in the development of atherosclerosis, in this study, we characterized the oxidized lipid milieu in HCD-fed zebrafish larvae. Using liquid chromatography-mass spectrometry, we show that feeding a HCD for only 2 weeks resulted in up to 70-fold increases in specific oxidized cholesteryl esters, identical to those present in human minimally oxidized LDL (mmLDL) and in murine atherosclerotic lesions. The levels of oxidized phospholipids, such as 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphocholine, and of various lysophosphatidylcholines were also significantly elevated. Moreover, lipoproteins isolated from homogenates of HCD-fed larvae induced cell spreading as well as ERK1/2, Akt and JNK phosphorylation in murine macrophages. Removal of apoB-containing lipoproteins from the zebrafish homogenates with an anti-human LDL antibody, as well as reducing lipid hydroperoxides with ebselen, resulted in inhibition of macrophage activation. The TLR4 deficiency in murine macrophages prevented their activation with zebrafish lipoproteins. Using biotinylated homogenates of HCD-fed larvae, we demonstrated that their components bound to murine macrophages, and this binding was effectively competed by mmLDL, but not by native LDL. These data provide evidence that molecular lipid determinants of pro-atherogenic macrophage phenotypes are present in large quantities in hypercholesterolemic zebrafish larvae and support the use of the HCD-fed zebrafish as a valuable model to study early events of atherogenesis.

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Novel changes in NF-{kappa}B activity during progression and regression phases of hyperplasia: role of MEK, ERK AND p38.

Publication Date: 2010 Aug 14 PMID: 20710027
Authors: Chandrakesan, P. - Ahmed, I. - Anwar, T. - Wang, Y. - Sarkar, S. - Singh, P. - Peleg, S. - Umar, S.
Journal: J Biol Chem

Utilizing the Citrobacter rodentium (CR)-induced transmissible murine colonic hyperplasia (TMCH) model, we measured hyperplasia and NF-kappaB activation during progression (days 6 and 12 post-infection) and regression (days 20-34 post-infection) phases of TMCH. NF-kappaB activity increased at progression in conjunction with bacterial attachment and translocation to the colonic crypts and decreased 40% by day 20. NF-kappaB activity at days 27 and 34 however, remained 2-3 fold higher than uninfected control. Expression of the downstream target gene CXCL-1/KC in the crypts correlated with NF-kappaB activation kinetics. Phosphorylation of cellular IKKalpha/beta (Ser(176/180)) was elevated during progression and regression of TMCH. Phosphorylation (Ser(32/36)) and degradation of IkappaBalpha however, contributed to NF-kappaB activation only from days 6 to 20 but not at later time points. Phosphorylation of MEK1/2 (Ser(217/221)), ERK1/2 (Thr(202)/Tyr(204)) and p38 (Thr(180)/Tyr(182)) paralleled IKKalpha/beta kinetics at days-6 and 12 without declining with regressing hyperplasia. siRNAs to MEK, ERK and p38 significantly blocked NF-kappaB activity in vitro while MEK1/2-inhibitor (PD 98059) also blocked increases in MEK1/2, ERK1/2 and IKKalpha/beta thereby inhibiting NF-kappaB activity in vivo. Cellular and nuclear levels of Ser-536-phosphorylated (p65(536)) and Lys-310-acetylated p65 subunit accompanied functional NF-kappaB activation during TMCH. RSK-1 phosphorylation at Thr(359)/Ser(363) in cellular/nuclear extracts and co-immunoprecipitation with cellular p65-NF-kappaB overlapped with p65(536) kinetics. Dietary pectin (6%) blocked NF-kappaB activity by blocking increases in p65 abundance and nuclear translocation thereby down-regulating CXCL-1/KC expression in the crypts. Thus, NF-kappaB activation persists despite lack of bacterial attachment to colonic mucosa beyond peak hyperplasia. MEK/ERK/p38 pathway therefore seems to modulate sustained activation of NF-kappaB in colonic crypts in response to CR infection.

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Insights into an unusual NRPS biosynthesis: the identification and characterization of the GE81112 biosynthetic gene cluster.

Publication Date: 2010 Aug 14 PMID: 20710026
Authors: Binz, T. M. - Maffioli, S. I. - Sosio, M. - Donadio, S. - Mueller, R.
Journal: J Biol Chem

The GE81112 tetrapeptides (1-3) represent a structurally unique class of antibiotics, acting as specific inhibitors of prokaryotic protein synthesis. Here we report the cloning and sequencing of the GE81112 biosynthetic gene cluster from Streptomyces sp. L-49973 and the development of a genetic manipulation system for S. sp. L-49973. The biosynthetic gene cluster for the tetrapeptide antibiotic GE81112 (getA-N) was identified within a 61.7 kb region comprising 29 open reading frames (ORFs), 14 of which were assigned to the biosynthetic gene cluster. Sequence analysis revealed the GE81112 cluster to consist of 6 nonribosomal peptide synthetase (NRPS) genes encoding incomplete didomain NRPS modules, a single free standing NRPS domain, as well as genes encoding other biosynthetic and modifying proteins. The involvement of the cloned gene cluster in GE81112 biosynthesis was confirmed by inactivating the NRPS gene getE resulting in a GE81112 production abolished mutant. In addition, we characterized the NRPS A-domains from the pathway by expression in E. coli and in vitro enzymatic assays. The previously unknown stereochemistry of most chiral centers in GE81112 was established from a combined chemical and biosynthetic approach. Taken together, these findings have allowed us to propose a rational model for GE81112 biosynthesis. The results further open the door to developing new derivatives of these promising antibiotic compounds by genetic engineering.

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RyR1 expression and the cell boundary theorem.

Publication Date: 2010 Aug 20 PMID: 20709761
Authors: Rios, E.
Journal: J Biol Chem

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The cystic fibrosis transmembrane conductance regulator impedes proteolytic stimulation of the epithelial Na+ channel.

Publication Date: 2010 Aug 13 PMID: 20709758
Authors: Gentzsch, M. - Dang, H. - Dang, Y. - Garcia-Caballero, A. - Suchindran, H. - Boucher, R. C. - Stutts, M. J.
Journal: J Biol Chem

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) that prevent its proper folding and trafficking to the apical membrane of epithelial cells. Absence of cAMP-mediated Cl(-) secretion in CF airways causes poorly hydrated airway surfaces in CF patients and this condition is exacerbated by excessive Na(+) absorption. The mechanistic link between missing CFTR and increased Na(+) absorption in airway epithelia has remained elusive, although substantial evidence implicates hyperactivity of the epithelial Na(+) channel (ENaC). ENaC is known to be activated by selective endoproteolysis of the extracellular domains of its alpha- and gamma-subunits, and it was recently reported that ENaC and CFTR physically associate in mammalian cells. We confirmed this interaction in oocytes by co-immunoprecipitation and find that ENaC associated with wild type CFTR is protected from proteolytic cleavage and stimulation of open probability. In contrast, DeltaF508 CFTR, the most common mutant protein in CF patients, failed to protect ENaC from proteolytic cleavage and stimulation. In normal airway epithelial cells, ENaC was contained in the anti-CFTR immunoprecipitate. In CF airway epithelial cultures, the proportion of full length to total alpha-ENaC protein signal was consistently reduced compared to normal cultures. Our results identify limiting proteolytic cleavage of ENaC as a mechanism by which CFTR down regulates Na(+) absorption.

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Vacuolar cation/H+ antiporters of Saccharomyces cerevisiae.

Publication Date: 2010 Aug 13 PMID: 20709757
Authors: Cagnac, O. - Aranda-Sicilia, M. N. - Leterrier, M. - Rodriguez-Rosales, M. P. - Venema, K.
Journal: J Biol Chem

We previously demonstrated that Saccharomyces cerevisiae vnx1 mutant strains displayed an almost total loss of Na+ and K+/H+ antiporter activity in a vacuolar-enriched fraction. However, using different in vitro transport conditions we were able to reveal additional K+/H+ antiporter activity. By disrupting genes encoding transporters potentially involved in the vnx1 mutant strain, we determined that Vcx1p is responsible for this activity. This result was further confirmed by complementation of the vnx1vcx1 nhx1 triple mutant with Vcx1p and its inactivated mutant Vcx1p-H303A. Like the Ca2+/H+ antiporter activity catalyzed by Vcx1p, the K+/H+ antiporter activity was strongly inhibited by Cd2+, and to a lesser extend by Zn2+. Unlike previously observed for Nhx1 or Vnx1, Vcx1 overexpression only marginally improved the growth of yeast strain AXT3 in the presence of high concentrations of K+, and had no effect on hygromycin sensitivity. Subcellular localization showed that Vcx1 and Vnx1 are targeted to the vacuolar membrane, while Nhx1p is targeted to prevacuoles. The relative importance of Nhx1p, Vnx1p, and Vcx1p in the vacuolar accumulation of monovalent cations will be discussed.

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Cysteine is not the sulfur source for iron-sulfur cluster and methionine biosynthesis in the methanogenic archaeon Methanococcus maripaludis.

Publication Date: 2010 Aug 13 PMID: 20709756
Authors: Liu, Y. - Sieprawska-Lupa, M. - Whitman, W. B. - White, R. H.
Journal: J Biol Chem

Three multi-protein systems are known for iron-sulfur (Fe-S) cluster biogenesis in bacteria and eukaryotes: the NIF (nitrogen fixation), the ISC (iron-sulfur cluster), and the SUF (mobilization of sulfur) systems. In all three, cysteine is the physiological sulfur source, and the sulfur is transferred from cysteine desulfurase through a persulfidic intermediate to a scaffold protein. However, the biochemical nature of the sulfur source for Fe-S cluster assembly in archaea is not known, and many archaea lack homologs of cysteine desulfurases. Methanococcus maripaludis is a methanogenic archaeon that contains a high amount of protein-bound Fe-S clusters (45 nmol/mg protein). Cysteine in this archaeon is synthesized primarily via the tRNA-dependent SepRS/SepCysS pathway. When a DeltasepS mutant (a cysteine auxotroph) was grown with (34)S-labeled sulfide and unlabeled cysteine, < 8% of the cysteine, > 92% of the methionine, and > 87% of the sulfur in the Fe-S clusters in proteins was labeled, suggesting that the sulfur in methionine and Fe-S clusters was derived predominantly from exogenous sulfide instead of cysteine. Therefore, this investigation challenges the prior knowledge that cysteine is always the sulfur source for Fe-S cluster biosynthesis in vivo and suggests that Fe-S clusters are derived from sulfide in organisms living in sulfide-rich habitats.

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RNA binding protein muscleblind-like 3 (MBNL3) disrupts myocyte enhancer factor 2 (Mef2) beta-exon splicing.

Publication Date: 2010 Aug 13 PMID: 20709755
Authors: Lee, K. S. - Cao, Y. - Witwicka, H. E. - Tom, S. - Tapscott, S. J. - Wang, E. H.
Journal: J Biol Chem

Mammalian muscleblind-like (MBNL) proteins are regulators of alternative splicing and have been implicated in myotonic dystrophy (DM), the most common form of adult onset muscular dystrophy. MBNL3 functions as an inhibitor of muscle differentiation and is expressed in proliferating muscle precursor cells but not in differentiated skeletal muscle. Here we demonstrate that MBNL3 regulates the splicing pattern of the muscle transcription factor myocyte enhancer factor 2 (Mef2) by promoting exclusion of the alternatively spliced beta-exon. Expression of the transcriptionally more active (+)beta isoform of Mef2D was sufficient to overcome the inhibitory effects of MBNL3 on muscle differentiation. These data suggest that MBNL3 antagonizes muscle differentiation by disrupting Mef2 beta-exon splicing. MBNL3 regulates Mef2D splicing by directly binding to intron 7 downstream of the alternatively spliced exon in the pre-mRNA. The RNA binding activity of MBNL3 requires the Cys3His zinc finger domains. Using a cell culture model of myotonic dystrophy and DM patient tissue, we have evidence that expression of CUG expanded RNAs can lead to an increase in MBNL3 expression and a decrease in Mef2D beta-exon splicing. These studies suggest that elevating MBNL3 activity in myogenic cells could lead to muscle degeneration disorders such as myotonic dystrophy.

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Overlapping binding sites of structurally different antiarrhythmics flecainide and propafenone in the subunit interface of potassium channel Kv2.1.

Publication Date: 2010 Aug 13 PMID: 20709754
Authors: Madeja, M. - Steffen, W. - Mesic, I. - Garic, B. - Zhorov, B. S.
Journal: J Biol Chem

Kv2.1 channels, which are expressed in brain, heart, pancreas, and other organs and tissues, are important targets for drug design. Flecainide and propafenone are known to block Kv2.1 channels more potently than other Kv channels. Here we sought to explore structural determinants of this selectivity. We demonstrated that flecainide reduced the K+ currents through Kv2.1 channels expressed in Xenopus laevis oocytes in a voltage- and time-dependent manner. By systematically exchanging various segments of Kv2.1 with those from Kv1.2, we determined flecainide-sensing residues in the P-helix and inner helix S6. These residues are not exposed to the inner pore, a conventional binding region of open-channel blockers. The flecainide-sensing residues also contribute to propafenone binding suggesting overlapping receptors for the drugs. Indeed, propafenone and flecainide compete for binding in Kv2.1. We further used Monte Carlo-energy minimizations to map the drugs receptors. Flecainide docking in the Kv1.2-based homology model of Kv2.1 predicts the ligand ammonium group in the central cavity and the benzamide moiety in a niche between S6 and P-helix. Propafenone also binds in the niche. Its carbonyl group accepts an H-bond from the P-helix, the amino group donates an H-bond to the P-loop turn, while the propyl group protrudes in the pore and blocks the access to the selectivity filter. Thus, besides the binding region in the central cavity, certain K+ channel ligands can expand in the subunit interface whose residues are less conserved between K+ channels and hence may be targets for design of highly desirable subtype-specific K+ channel drugs.

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Selenium Compounds Activate ATM-dependent DNA damage response via the mismatch repair protein hMLH1 in Colorectal Cancer Cells.

Publication Date: 2010 Aug 13 PMID: 20709753
Authors: Qi, Y. - Schoene, N. W. - Lartey, F. M. - Cheng, W. H.
Journal: J Biol Chem

Epidemiological and animal studies indicate that selenium supplementation suppresses risk of colorectal and other cancers. The majority of colorectal cancers are characterized by a defective DNA mismatch repair (MMR). Here, we have employed the MMR-deficient HCT 116 colorectal cancer cells and the MMR-proficient HCT 116 cells with hMLH1 complementation to investigate the role of hMLH1 in selenium-induced DNA damage response, a tumorigenesis barrier. The ataxia telangiectasia mutated (ATM) protein responds to clastogens and initiates DNA damage response. We show that hMLH1 complementation sensitizes HCT 116 cells to methylseleninic acid, methylselenocysteine and sodium selenite via reactive oxygen species, and facilitates the selenium-induced oxidative 8-oxoguanine damage, DNA breaks, G2/M checkpoint response, and ATM pathway activation. Pretreatment of the hMLH1-complemented HCT 116 cells with the antioxidant N-acetylcysteine or 2,2,6,6-tetramethylpiperidine-1-oxyl or the ATM kinase inhibitor KU55933 suppresses the hMLH1-dependent DNA damage responses to selenium exposure. Selenium treatment stimulates the association between hMLH1 and hPMS2 proteins, a heterodimer critical for functional MMR, in a manner dependent on ATM and reactive oxygen species. Taken together, the results suggest a new role of selenium in mitigating tumorigenesis by targeting the MMR pathway, whereby lack of hMLH1 renders the HCT 116 colorectal cancer cells resistant to selenium-induced DNA damage response.

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The active Site Metal Ion in UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) switches between Fe(II) and Zn(II) depending on cellular conditions.

Publication Date: 2010 Aug 13 PMID: 20709752
Authors: Gattis, S. G. - Hernick, M. - Fierke, C. A.
Journal: J Biol Chem

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the deacetylation of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine to form UDP-3-O-myristoyl-glucosamine and acetate in Gram-negative bacteria. This second, and committed, step in lipid A biosynthesis is a target for antibiotic development. LpxC was previously identified as a mononuclear Zn(II) metalloenzyme; however, LpxC is 6-8-fold more active with the oxygen-sensitive Fe(II) cofactor (Hernick et al, Biochemistry (2010), 49, 2246-2255). To analyze the native metal cofactor bound to LpxC, we developed a pull-down method to rapidly purify tagged-LpxC under anaerobic conditions. The metal bound to LpxC purified from E. coli grown in minimal medium is mainly Fe(II). However, the ratio of Fe/Zn bound to LpxC varies with the metal content of the medium. Furthermore, the Fe/Zn ratio bound to native LpxC, determined by activity assays, has a similar dependence on the growth conditions. LpxC has significantly higher affinity for Zn(II) compared to Fe(II) with KD values of 60 +/- 20 pM and 110 +/- 40 nM, respectively. However, in vivo concentrations of readily exchangeable Fe are significantly higher than Zn, suggesting that Fe(II) is the thermodynamically favored metal cofactor for LpxC under cellular conditions. These data indicate that LpxC expressed in E. coli grown in standard medium predominantly exists as the Fe(II)-enzyme. However, the metal cofactor in LpxC can switch between Fe and Zn in response to perturbations in available metal ions. This alteration may be important for regulating the LpxC activity upon changes in environmental conditions and may be a general mechanism of regulating the activity of metalloenzymes.

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Regulation of the unfolded protein response by eIF2B{delta} isoforms.

Publication Date: 2010 Aug 13 PMID: 20709751
Authors: Martin, L. - Kimball, S. R. - Gardner, L. B.
Journal: J Biol Chem

Cells respond to a variety of stresses, including unfolded proteins in the endoplasmic reticulum (ER), by phosphorylating a subunit of translation initiation factor eIF2, eIF2alpha. eIF2alpha phosphorylation inactivates the eIF2B complex. The inactivation of eIF2B not only suppresses the initiation of protein translation, but paradoxically up-regulates the translation and expression of transcription factor ATF-4. Both of these processes are important for the cellular response to ER stress, also termed the unfolded protein response. Here we demonstrate that cellular response resulting from eIF2alpha phosphorylation is attenuated in several cancer cell lines. The deficiency of the unfolded protein response in these cells correlates with the expression of a specific isoform of a regulatory eIF2B subunit, eIF2Bdelta variant 1. Replacement of total eIF2Bdelta with variant 1 renders cells insensitive to eIF2alpha phosphorylation: specifically they neither up-regulate ATF-4 and ATF-4 targets nor suppress protein translation. Expression of variant 2 eIF2Bdelta in ER stress response-deficient cells restores the stress response. Our data suggest that variant 1 does not interact with the eIF2 complex, a requisite for eIF2B inhibition by eIF2alpha phosphorylation. Together, these data delineate a novel physiological mechanism to regulate the ER stress response with a large potential impact on a variety of diseases which result in ER stress.

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ADIPONECTIN INDUCED ERK AND AKT PHOSPHORYLATION PROTECTS AGAINST PANCREATIC BETA CELL APOPTOSIS AND INCREASES INSULIN GENE EXPRESSION AND SECRETION.

Publication Date: 2010 Aug 13 PMID: 20709750
Authors: Wijesekara, N. - Krishnamurthy, M. - Bhattacharjee, A. - Suhail, A. - Sweeney, G. - Wheeler, M. B.
Journal: J Biol Chem

The functional impact of adiponectin on pancreatic beta cells is so far poorly understood. Although adiponectin receptors (AdipoR1/2) were identified, their involvement in adiponectin induced signaling and other molecules involved are not clearly defined. Therefore, we investigated the physiological role of adiponectin in beta cells and the signaling mediators involved. MIN6 beta cells and mouse islets were stimulated with globular [2.5 mug/mL] or full-length [5 mug/mL] adiponectin under serum starvation, and cell viability, proliferation, apoptosis, insulin gene expression and secretion were measured. Lysates were subjected to western blot analysis to determine phosphorylation of AMPK, Akt or ERK. Functional significance of signaling was confirmed using dominant negative mutants or pharmacological inhibitors. Participation of AdipoRs was assessed by overexpression or siRNA. Adiponectin failed to activate AMPK after 10 min, 1 h and 24 h stimulation. ERK was significantly phosphorylated after 24 h treatment with adiponectin, while Akt was activated at all time points examined. 24 h stimulation with adiponectin significantly increased cell viability by decreasing cellular apoptosis and this was prevented by dominant negative Akt, wortmannin (PI3K inhibitor) and U0126 (MEK inhibitor). Moreover, adiponectin regulated insulin gene expression and glucose-stimulated insulin secretion, which was also prevented by wortmannin and U0126 treatment. Interestingly, adiponectin-induced changes in Akt and ERK phosphorylation and caspase-3 may occur independent of the level of AdipoR expression. This study demonstrates a lack of AMPK involvement and implicates Akt and ERK in adiponectin signaling, leading to protection against apoptosis and stimulation of insulin gene expression and secretion in pancreatic beta cells.

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Involvement of the SRF coactivator megakaryoblastic leukemia in the activin-regulated dendritic complexity of rat cortical neurons.

Publication Date: 2010 Aug 17 PMID: 20709749
Authors: Ishikawa, M. - Nishijima, N. - Shiota, J. - Sakagami, H. - Tsuchida, K. - Mizukoshi, M. - Fukuchi, M. - Tsuda, M. - Tabuchi, A.
Journal: J Biol Chem

Dynamic changes in neuronal morphology and transcriptional regulation play crucial roles in the neuronal network and function. Accumulating evidence suggests that the megakaryoblastic leukemia (MKL) family members, which function not only as actin-binding proteins but also as serum response factor (SRF) transcriptional coactivators, regulate neuronal morphology. However, the extracellular ligands and signaling pathways, which activate MKL-mediated morphological changes in neurons, remain unresolved. Here, we demonstrate that in addition to MKL1, MKL2, highly enriched in the forebrain, strongly contributes to the dendritic complexity, and this process is triggered by stimulation with activin, a member of the transforming growth factor beta (TGF-beta) superfamily. Activin promoted dendritic complexity in a SRF- and MKL-dependent manner without drastically affecting MKL localization and protein levels. In contrast, activin promoted the nuclear export of suppressor of cancer cell invasion (SCAI), which is a corepressor for SRF and MKL. Furthermore, overexpression of SCAI blocked activin-induced SRF-transcriptional responses and dendritic complexity. Collectively, these results strongly suggest that activin-SCAI-MKL signaling is a novel pathway that regulates the dendritic morphology of rat cortical neurons by excluding SCAI from the nucleus and activating MKL/SRF-mediated gene expression.

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Splice variants of SmgGDS control small GTPase prenylation and membrane localization.

Publication Date: 2010 Aug 13 PMID: 20709748
Authors: Berg, T. J. - Gastonguay, A. J. - Lorimer, E. L. - Kuhnmuench, J. R. - Li, R. - Fields, A. P. - Williams, C. L.
Journal: J Biol Chem

Ras and Rho small GTPases possessing a C-terminal polybasic region (PBR) are vital signaling proteins whose misregulation can lead to cancer. Signaling by these proteins depends on their ability to bind guanine nucleotides and their prenylation with a geranylgeranyl or farnesyl isoprenoid moiety and subsequent trafficking to cellular membranes. There is little previous evidence that cellular signals can restrain non-prenylated GTPases from entering the prenylation pathway, leading to the general belief that PBR-possessing GTPases are prenylated as soon as they are synthesized. Here, we present evidence that challenges this belief. We demonstrate that insertion of the dominant negative mutation to inhibit GDP/GTP exchange diminishes prenylation of Rap1A and RhoA, enhances prenylation of Rac1, and does not detectably alter prenylation of K-Ras. Our results indicate that the entrance and passage of these small GTPases through the prenylation pathway is regulated by two splice variants of SmgGDS, a protein that has been reported to promote GDP/GTP exchange by PBR-possessing GTPases and to be upregulated in several forms of cancer. We show that the previously-characterized 558 residue SmgGDS splice variant (SmgGDS-558) selectively associates with prenylated small GTPases and facilitates trafficking of Rap1A to the plasma membrane, whereas the less well characterized 607 residue SmgGDS splice variant (SmgGDS-607) associates with non-prenylated GTPases and regulates the entry of Rap1A, RhoA, and Rac1 into the prenylation pathway. These results indicate that guanine nucleotide exchange and interactions with SmgGDS splice variants can regulate the entrance and passage of PBR-possessing small GTPases through the prenylation pathway.

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Co-Assembly of Kv4 {alpha} Subunits with K+ Channel Interacting Protein 2 Stabilizes Protein Expression and Promotes Surface Retention of Channel Complexes.

Publication Date: 2010 Aug 13 PMID: 20709747
Authors: Foeger, N. C. - Marionneau, C. - Nerbonne, J. M.
Journal: J Biol Chem

Members of the K(+) Channel Interacting Protein (KChIP) family bind the distal amino- (N-) termini of members of the Shal subfamily of voltage-gated K(+) channel (Kv4) pore-forming (alpha) subunits to generate rapidly activating, rapidly inactivating neuronal A-type (I(A)) and cardiac transient outward (I(to)) currents. In heterologous cells, KChIP co-expression increases cell surface expression of Kv4 alpha subunits and Kv4 current densities, findings interpreted to suggest that Kv4/KChIP complex formation enhances forward trafficking of channels (from the endoplasmic reticulum or the Golgi complex) to the surface membrane. The results of experiments here, however, demonstrate that KChIP2 increases cell surface Kv4.2 protein expression (~40 fold) by an order of magnitude more than the increase in total protein (~2 fold) or in current densities (~3 fold), suggesting that mechanisms at the cell surface regulate the functional expression of Kv4.2 channels. Additional experiments demonstrated that KChIP2 decreases the turnover rate of cell surface Kv4.2 protein by inhibiting endocytosis and/or promoting recycling. Unexpectedly, the experiments here also revealed that Kv4.2/KChIP2 complex formation stabilizes not only (total and cell surface) Kv4.2, but also KChIP2 protein expression. This reciprocal protein stabilization, as well as Kv4/KChIP2 complex formation, are lost with deletion of the distal (10 amino acids) Kv4.2 N-terminus. Taken together, these observations demonstrate that KChIP2 differentially regulates total and cell surface Kv4.2 protein expression and Kv4 current densities.

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SSS1p is required to complete protein translocon activation.

Publication Date: 2010 Aug 13 PMID: 20709746
Authors: Wilkinson, B. M. - Brownsword, J. K. - Mousley, C. J. - Stirling, C. J.
Journal: J Biol Chem

Protein translocation across the endoplasmic reticulum membrane occurs at the Sec61 translocon. This has two essential subunits, the channel-forming multi-spanning membrane protein Sec61p/Sec61alpha and the tail-anchored Sss1p/Sec61gamma which has been proposed to clamp the channel. We have analysed the function of Sss1p using a series of domain mutants and found that both the cytosolic and transmembrane clamp domains of Sss1p are essential for protein translocation. Our data reveal that the cytosolic domain is required for Sec61p interaction but that the transmembrane clamp domain is required to complete activation of the translocon after precursor targeting to Sec61p.

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The Epsin family of endocytic adaptors promotes fibrosarcoma migration and invasion.

Publication Date: 2010 Aug 13 PMID: 20709745
Authors: Coon, B. G. - Burgner, J. - Camonis, J. H. - Aguilar, R. C.
Journal: J Biol Chem

Abnormalities in the process of endocytosis are classically linked to malignant transformation through the deficient downregulation of signaling receptors. The present study describes a non-classical mechanism that does not require internalization by which endocytic proteins affect cell migration and basement membrane invasion. Specifically, we found that the endocytic adaptor epsin binds and regulates the biological properties of the signaling molecule RalBP1 (Ral Binding Protein 1). Epsin interacted with the N-terminus of RalBP1 via its characteristic Epsin N-Terminal Homology (ENTH) domain. A combination of siRNA-mediated knock-down and transfection of siRNA-resistant constructs in fibrosarcoma cells demonstrated that impairment of the epsin-RalBP1 interaction led to cell migration and basement membrane invasion defects. We found the ENTH domain was necessary and sufficient to sustain normal cell migration and invasion. Because all epsin endocytic motifs reside in the C-terminal part of the molecule, these results suggest that this novel regulatory circuit does not require endocytosis. In addition, cells depleted of epsin-RalBP1 complex displayed deficient activation of Rac1 and Arf6 suggesting a signaling function for this novel interaction. Further, overexpression of either epsin or RalBP1 enhanced migration and invasion of fibrosarcoma cells. Collectively, our results indicate that epsin regulates RalBP1 function in Rac1- and Arf6-dependent pathways to ultimately affect cell migration and invasion. We propose that the observed upregulation of both epsin and RalBP1 in certain cancers contributes to their invasive characteristics.

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Conversion of mouse epiblast stem cells to an earlier pluripotency state by small molecules.

Publication Date: 2010 Aug 12 PMID: 20705612
Authors: Zhou, H. - Li, W. - Zhu, S. - Joo, J. Y. - Do, J. T. - Xiong, W. - Kim, J. B. - Zhang, K. - Scholer, H. R. - Ding, S.
Journal: J Biol Chem

Epiblast stem cells (EpiSCs) are pluripotent cells derived from the post-implantation epiblast in vitro. EpiSCs are incapable of contributing to chimerism, indicating that EpiSCs are less pluripotent and represent a later developmental pluripotency state than inner cell mass (ICM)-stage murine ESCs. Using chemical approach, we found that blockage of the TGFbeta pathway or inhibition of demethylase LSD1 with small molecule inhibitors induced dramatic morphological changes of EpiSCs towards mESC phenotypes with activation of ICM-specific gene expression. However, full conversion of EpiSCs to a mESC-like state with competence to chimeric contribution can only be readily generated with a combination of inhibitors of LSD1, ALK5, MEK, and GSK3. Our results demonstrate that appropriate synergy of epigenetic and signaling modulations may convert cells at the later developmental pluripotency state to an earlier mESC-like pluripotency state, providing new insights into pluripotency regulation.

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Proinflammatory cytokines enhance estrogen-dependent expression of the multidrug transporter gene ABCG2 through estrogen receptor and NF{kappa}B cooperativity at adjacent response elements.

Publication Date: 2010 Aug 12 PMID: 20705611
Authors: Pradhan, M. - Bembinster, L. A. - Baumgarten, S. C. - Frasor, J.
Journal: J Biol Chem

Constitutive activation of NFkappaB in estrogen receptor (ER) positive breast cancer is associated with tumor recurrence and development of antiestrogen resistance. Furthermore, a gene expression signature containing common targets for ER and NFkappaB has been identified and found to be associated with the more aggressive Luminal B intrinsic subtype of ER positive breast tumors. Here we describe a novel mechanism by which ER and NFkappaB cooperate to up-regulate expression of one important gene from this signature, ABCG2, which encodes a transporter protein associated with the development of drug-resistant breast cancer. We and others have confirmed that this gene is primarily regulated by estrogen in an ER and estrogen response element (ERE)- dependent manner. We find that whereas proinflammatory cytokines have little effect on this gene in the absence of E2, they can potentiate ER activity in an NFkappaB-dependent manner. ER allows the NFkappaB family member, p65, to access a latent NFkappaB response element located near the ERE in the gene promoter. NFkappaB recruitment to the gene, in turn, is required to stabilize ER occupancy at the functional ERE. The result of this cooperative binding of ER and p65 at adjacent response elements leads to a major increase in both ABCG2 mRNA and protein expression. These findings indicate that estrogen and inflammatory factors can modify each other's activity through modulation of transcription factor accessibility and/or occupancy at adjacent response elements. This novel transcriptional mechanism could have important implications in breast cancer where inflammation and estrogen can both promote cancer progression.

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Raman spectroscopy analysis of botryococcene hydrocarbons from the green microalga Botryococcus braunii.

Publication Date: 2010 Aug 12 PMID: 20705610
Authors: Weiss, T. L. - Chun, H. J. - Okada, S. - Vitha, S. - Holzenburg, A. - Laane, J. - Devarenne, T. P.
Journal: J Biol Chem

Botryococcus braunii, B race is a unique green microalga that produces large amounts of liquid hydrocarbons known as botryococcenes that can be used as a fuel for internal combustion engines. The simplest botryococcene (C(30)) is metabolized by methylation to give intermediates of C(31), C(32), C(33), and C(34), with C(34) being the predominant botryococcene in some strains. In the present work we have used Raman spectroscopy to characterize the structure of botryococcenes in an attempt to identify and localize botryococcenes within B. braunii cells. The spectral region from 1600 - 1700 cm(-1) showed nu(C=C) stretching bands specific for botryococcenes. Distinct botryococcene Raman bands at 1640 and 1647 cm(-1) were assigned to the stretching of the C=C bond in the botryococcene branch and the exomethylene C=C bonds produced by the methylations, respectively. A Raman band at 1670 cm(-1) was assigned to the backbone C=C bond stretching. Density function theory (DFT) calculations were used to determine the Raman spectra of all botryococcenes in order to compare computed theoretical values to those observed. The analysis showed that the nu(C=C) stretching bands at 1647 and 1670 cm(-1) are actually composed of several closely spaced bands arising from the six individual C=C bonds in the molecule. We also used confocal Raman microspectroscopy to map the presence and location of methylated botryococcenes within a colony of B. braunii cells based on the methylation-specific 1647 cm(-1) botryococcene Raman shift.

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Gata proteins work together with friend of gata (Fog) and C-terminal binding protein (Ctbp) co-regulators to control adipogenesis.

Publication Date: 2010 Aug 11 PMID: 20705609
Authors: Jack, B. H. - Crossley, M.
Journal: J Biol Chem

Gata transcription factors have been implicated in controlling adipogenesis in Drosophila and in mammals. In mammals, both Gata2 and Gata3 have been shown to be present in pre-adipocytes, and their silencing allows the onset of adipogenesis. Over-expression of Gata proteins blocks adipogenesis in cellular assays. Gata factors have been found to operate through recruiting cofactors of the Friend of Gata (Fog) family. Fog proteins, in turn, recruit co-regulators, including C-terminal binding proteins (Ctbps). We have investigated whether Fogs and Ctbps influence adipogenesis. We found both Fog1 and Fog2 are expressed in cells prior to adipogenesis but are down-regulated as adipogenesis proceeds. Over-expression of Fog1 or Fog2 interferes with adipogenesis. Mutant versions of Fog2 unable to bind Ctbp or Gata proteins are impaired in their inability to inhibit adipogenesis. Finally, a mutant version of Gata2, unable to associate with Fogs, also displays abnormal activity and causes enhanced cell proliferation. These results implicate Fogs and Ctbps as partners of Gata proteins in the control of adipocyte proliferation and differentiation.

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Interfacial kinetic and binding properties of mammalian group IVB phospholipase A2 (cPLA2{beta} and comparison to the other cPLA2 isoforms.

Publication Date: 2010 Aug 11 PMID: 20705608
Authors: Ghomashchi, F. - Naika, G. S. - Bollinger, J. G. - Aloulou, A. - Lehr, M. - Leslie, C. C. - Gelb, M. H.
Journal: J Biol Chem

The cytosolic (group IV) phospholipase A(2) (cPLA(2s)) family contains 6 members. We have prepared recombinant proteins for human alpha, mouse beta , human gamma,humandelta,human epsilon and mouse zeta cPLA(2s) and have studied their interfacial kinetic and binding properties in vitro. Mouse cPLA(2) beta action on phosphatidylcholine vesicles is activated by anionic phosphoinositides and cardiolipin but displays a requirement for Ca(2+) only in the presence of cardiolipin. This activation pattern is explained by the effects of anionic phospholipids and Ca(2+) on the interfacial binding of mouse cPLA(2)beta; and its C2 domain to vesicles. Ca(2+)-dependent binding of mouse cPLA(2)beta to cardiolipin-containing vesicles requires a patch of basic residues near the Ca(2+)-binding surface loops of the C2 domain, but binding to phosphoinositide-containing vesicles does not depend on any specific cluster of basic residues. Human cPLA(2)delta also displays Ca(2+)- and cardiolipin-enhanced interfacial binding and activity. The lysophospholipase, phospholipase A(1) and phospholipase A(2) activities of the full set of mammalian cPLA(2s) were quantified. The relative level of these activities is very different among the isoforms, and human cPLA(2)delta stands out as having relatively high phospholipase A(1) activity. We also tested the susceptibility of all cPLA(2) family members to a panel of previously reported inhibitors of human cPLA(2)alpha and analogs of these compounds. This led to the discovery of a potent and selective inhibitor of mouse cPLA(2)beta. These in vitro studies help determine the regulation and function of the cPLA(2) family members.

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Turning the RING domain protein MdmX into an active ubiquitin-protein ligase.

Publication Date: 2010 Aug 12 PMID: 20705607
Authors: Iyappan, S. - Wollscheid, H. P. - Rojas-Fernandez, A. - Marquardt, A. - Tang, H. C. - Singh, R. K. - Scheffner, M.
Journal: J Biol Chem

The related RING domain proteins MdmX and Mdm2 are best known for their role as negative regulators of the tumor suppressor p53. However, while Mdm2 functions as a ubiquitin ligase for p53, MdmX does not have appreciable ubiquitin ligase activity. In this study, we performed a mutational analysis of the RING domain of MdmX and identified two distinct regions that when replaced by the respective regions of Mdm2, turn MdmX into an active ubiquitin ligase for p53. Mdm2 and MdmX form homodimers as well as heterodimers with each other. One of the regions identified localizes to the dimer interface indicating that subtle conformational changes in this region either affect dimer stability and/or the interaction with the ubiquitin-conjugating enzyme UbcH5b. The second region contains the nucleolar localization signal of Mdm2 but is also assumed to be involved in the interaction with UbcH5b. Here, we show that this region has a significant impact on the ability of respective MdmX mutants to functionally interact with UbcH5b in vitro supporting the notion that this region serves two distinct functional purposes, nucleolar localization and ubiquitin ligase activity. Finally, evidence is provided to suggest that the RING domain of Mdm2 not only binds to UbcH5b but moreover, acts as an allosteric activator of UbcH5b.

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Elucidation of a novel extracellular calcium binding site on metabotropic glutamate receptor 1 alpha(mGluR1{alpha}) that controls receptor activation.

Publication Date: 2010 Aug 12 PMID: 20705606
Authors: Jiang, Y. - Huang, Y. - Wong, H. C. - Zhou, Y. - Wang, X. - Yang, J. - Hall, R. A. - Brown, E. M. - Yang, J. J.
Journal: J Biol Chem

Metabotropic glutamate receptor 1alpha (mGluR1alpha) exerts important effects on numerous neurological processes. Although mGluR1alpha is known to respond to extracellular Ca2+ ([Ca2+]o), and the crystal structures of the extracellular domains (ECDs) of several mGluRs have been determined, the calcium-binding site(s) and structural determinants of Ca2+-modulated signaling in the Glu receptor family remain elusive. Here, we identify a novel Ca2+-binding site in the mGluR1alpha ECD using a recently developed computational algorithm. This predicted site (comprising D318, E325, D322, and the carboxylate side chain of the receptor's agonist, Glu) is situated in the hinge region in the ECD of mGluR1alpha, adjacent to the reported Glu-binding site with D318 involved in both Glu- and calcium-binding. Mutagenesis studies indicate that binding of Glu and Ca2+ to their distinct but partially-overlapping binding sites synergistically modulate mGluR1alpha activation of intracellular Ca2+ ([Ca2+]i) signaling. Mutating the Glu-binding site completely abolishes Glu signaling, while leaving its Ca2+-sensing capability largely intact. Mutating predicted Ca2+-binding residues abolishes or significantly reduces the sensitivity of mGluR1alpha not only to [Ca2+]o and [Gd3+]o, but also, in some cases, to Glu. The dual activation of mGluR1alpha by [Ca2+]o and Glu has important implications for the activation of other mGluR subtypes and related receptors. It also opens up new avenues for developing allosteric modulators of mGluR function that target specific human diseases.

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The effect of trehalose on the properties of mutant {gamma}PKC, which causes spinocerebellar ataxia type 14 (SCA14), in neuronal cell lines and cultured Purkinje cells.

Publication Date: 2010 Aug 12 PMID: 20705605
Authors: Seki, T. - Abe-Seki, N. - Kikawada, T. - Takahashi, H. - Yamamoto, K. - Adachi, N. - Tanaka, S. - Hide, I. - Saito, N. - Sakai, N.
Journal: J Biol Chem

Several missense mutations in the protein kinase Cgamma (gammaPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that the mutant gammaPKC found in SCA14 is susceptible to aggregation, which induces apoptotic cell death. The disaccharide trehalose has been reported to inhibit aggregate formation and to alleviate symptoms in cellular and animal models of Huntington disease, Alzheimer disease and prion disease. Here, we show that trehalose can be incorporated into SH-SY5Y cells and reduces the aggregation of mutant gammaPKC-GFP, thereby inhibiting apoptotic cell death in SH-SY5Y cells and primary cultured Purkinje cells (PCs). Trehalose acts by directly stabilizing the conformation of mutant gammaPKC without affecting protein turnover. Trehalose was also found to alleviate the improper development of dendrites in PCs expressing mutant gammaPKC-GFP without aggregates but not in PCs with aggregates. In PCs without aggregates, trehalose improves the mobility and translocation of mutant gammaPKC-GFP, probably by inhibiting oligomerization and thereby alleviating the improper development of dendrites. These results suggest that trehalose counteracts various cellular dysfunctions that are triggered by mutant gammaPKC in both neuronal cell lines and primary cultured PCs by inhibiting oligomerization and aggregation of mutant gammaPKC.

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Abcg2 transports and transfers heme to albumin through its large extracellular loop.

Publication Date: 2010 Aug 12 PMID: 20705604
Authors: Desuzinges-Mandon, E. - Arnaud, O. - Martinez, L. - Huche, F. - Di Pietro, A. - Falson, P.
Journal: J Biol Chem

ABCG2 is an ATP-binding cassette (ABC) transporter preferentially expressed by immature human hemato-poietic progenitors. Due to its role in drug resistance, its expression has been correlated to a protection role against protoporhyrin IX (PPIX) accumulation in stem cells under hypoxic conditions. We show here that zinc mesoporphyrin (ZnMP), a validated fluorescent heme analog, is transported by ABCG2. We also show that the ABCG2 large extracellular loop ECL3 constitutes a porphyrin-binding domain, which strongly interacts with heme, hemin, PPIX, ZnPPIX, CoPPIX and much less efficiently with pheophorbide a, but not with vitamin B12. Kd values are in the range 0.5-3.5 muM, with heme displaying the highest affinity. Non-porphyrin substrates of ABCG2, such as mitoxantrone, doxo/daunorubicin and riboflavin, do not bind to ECL3. Single-point mutations H583A and C603A inside ECL3 prevent the binding of hemin but hardly affect that of iron-free PPIX. The extracellular location of ECL3 downstream from the transport sites suggests that, after membrane trans-location, hemin is transferred to ECL3, which is strategically positioned to release the bound porphyrin to extracellular partners. We show here that human serum albumin could be one of these possible partners as it removes hemin bound to ECL3 and interacts with ABCG2, with a Kd of about 3 muM.

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A new role for PTEN in regulating transient receptor potential canonical channel 6- mediated Ca2+ entry, endothelial permeability and angiogenesis.

Publication Date: 2010 Aug 12 PMID: 20705603
Authors: Kini, V. - Chavez, A. - Mehta, D.
Journal: J Biol Chem

Phosphatase and tensin homologue (PTEN) is a dual lipid-protein phosphatase that catalyzes the conversion of PIP3 to PIP2 and thereby inhibits PI3K-Akt-dependent cell proliferation and migration, and tumor vascularization. We have uncovered a previously unrecognized role for PTEN in regulating Ca2+ entry through transient receptor potential canonical channel 6 (TRPC6) that does not require PTENs phosphatase activity. We show that PTENs tail-domain residues 394-403 permit PTEN to associate with TRPC6 and the inflammatory mediator thrombin promotes this association. Deletion of PTEN residues 394-403 prevents TRPC6 activity whereas PTEN mutant, C124S, which lacks phosphatase activity, had no effect. Moreover, thrombin failed to increase endothelial monolayer permeability in endothelial cells transducing Delta394-403 PTEN mutant. Paradoxically, we also show that thrombin failed to induce endothelial cell migration and tube formation in cells transducing Delta394-403 PTEN mutant. Our results demonstrate that PTEN through association between its 394-403 peptide sequence and TRPC6 increases endothelial permeability and directly promotes angiogenesis, thus indicating PTEN to have a role beyond suppressing PI3K signaling.

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SIRT1 activation by small molecules - kinetic and biophysical evidence for direct interaction of enzyme and activator.

Publication Date: 2010 Aug 11 PMID: 20702418
Authors: Dai, H. - Kustigian, L. - Carney, D. - Case, A. - Considine, T. - Hubbard, B. P. - Perni, R. B. - Riera, T. V. - Szczepankiewicz, B. - Vlasuk, G. P. - Stein, R. L.
Journal: J Biol Chem

SIRT1 is a protein deacetylase that has emerged as a therapeutic target for the development of activators to treat diseases of aging. SIRT1 activating compounds (STACs) have been developed that produce biological effects consistent with direct SIRT1 activation. At the molecular level, the mechanism by which STACs activate SIRT1 remains elusive. In the studies reported herein, the mechanism of SIRT1 activation is examined using representative compounds chosen from a collection of STACs. These studies reveal that activation of SIRT1 by STACs is strongly dependent on structural features of the peptide substrate. Significantly, and in contrast to studies reporting that peptides must bear a fluorophore for their deacetylation to be accelerated, we find that some STACs can accelerate the SIRT1-catalyzed deacetylation of specific unlabeled peptides composed only of natural amino acids. These results, together with others of this study, are at odds with a recent claim that complex formation between STACs and fluorophore-labeled peptides plays a role in the activation of SIRT1 [Pacholec et al., (2010) JBC 285, 8340]. Rather, the data suggest that STACs interact directly with SIRT1, and activate SIRT1-catalyzed deacetylation through an allosteric mechanism.

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Porphyromonas gingivalis peptidoglycans induce excessive activation of the innate immune system in silkworm larvae.

Publication Date: 2010 Aug 11 PMID: 20702417
Authors: Ishii, K. - Hamamoto, H. - Imamura, K. - Adachi, T. - Shoji, M. - Nakayama, K. - Sekimizu, K.
Journal: J Biol Chem

Porphyromonas gingivalis, a pathogen that causes inflammation in human periodontal tissue, killed silkworm (Bombyx mori, Lepidoptera) larvae when injected into the blood (hemolymph). Silkworm lethality was not rescued by antibiotic treatment, and heat-killed bacteria were also lethal. Heat-killed bacteria of mutant P. gingivalis strains lacking virulence factors also killed silkworms. Silkworms died after injection of peptidoglycans purified from P. gingivalis (pPG), and pPG toxicity was blocked by treatment with mutanolysin, a peptidoglycan-degrading enzyme. pPG induced silkworm hemolymph melanization at the same dose as that required to kill the animal. pPG injection increased caspase activity in silkworm tissues. pPG-induced silkworm death was delayed by injecting melanization-inhibiting reagents (a serine protease inhibitor and 1-phenyl-2-thiourea), antioxidants (N-acetyl-L-cysteine, glutathione, and catalase), and a caspase inhibitor (Ac-DEVD-CHO). Thus, pPG induces excessive activation of the innate immune response, which leads to the generation of reactive oxygen species and apoptotic cell death in the host tissue.

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Diversity of innate immune recognition mechanism for bacterial polymeric meso-diaminopimelic acid-type peptidoglycan in insects.

Publication Date: 2010 Aug 13 PMID: 20702416
Authors: Yu, Y. - Park, J. W. - Kwon, H. M. - Hwang, H. O. - Jang, I. H. - Masuda, A. - Kurokawa, K. - Nakayama, H. - Lee, W. J. - Dohmae, N. - Zhang, J. - Lee, B. L.
Journal: J Biol Chem

In Drosophila, the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathway. The Toll signaling pathway responds mainly to the lysine-type peptidoglycan of Gram-positive bacteria and fungal beta-1,3-glucan, whereas the Imd pathway responds to meso-diaminopimelic acid (DAP)-type peptidoglycan of Gram-negative bacteria and certain Gram-positive bacilli. Recently we determined activation mechanism of Toll signaling pathway biochemically using a large beetle, Tenebrio molitor. However, DAP-type peptidoglycan recognition mechanism and its signaling pathway are still unclear in the fly and beetle. Here, we show that polymeric DAP-type peptidoglycan, but not its monomeric form, formed a complex with Tenebrio peptidoglycan recognition protein-SA, and this complex activated the three-step proteolytic cascade to produce processed Spatzle, a Toll receptor ligand, and induced Drosophila defensin-like antimicrobial peptide in Tenebrio larvae similarly to polymeric lysine-type peptidoglycan. Monomeric DAP-type peptidoglycan induced Drosophila diptericin-like antimicrobial peptide in Tenebrio hemocytes. In addition, both polymeric and monomeric DAP-type peptidoglycans induced expression of Tenebrio peptidoglycan recognition protein-SC2, which is DAP-type peptidoglycan-selective N-acetylmuramyl L-alanine amidase that functions as a DAP-type peptidoglycan scavenger, appearing to function as a negative regulator of the DAP-type peptidoglycan signaling by cleaving DAP-type peptidoglycan and enduring it incapable of inducing antimicrobial peptides in Tenebrio larvae. Taken together, these results demonstrate that molecular recognition mechanism for polymeric DAP-type peptidoglycan is different between Tenebrio larvae and Drosophila adults, providing biochemical evidences of biological diversity of innate immune responses in insects.

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MTA1 coregulator regulates LPS response via MyD88-dependent signaling.

Publication Date: 2010 Aug 11 PMID: 20702415
Authors: Pakala, S. B. - Divijendra Natha Reddy, S. - Bui-Nguyen, T. M. - Rangparia, S. S. - Bommana, A. - Kumar, R.
Journal: J Biol Chem

Although metastatic tumor antigen 1 (MTA1) contributes to the responsiveness of macrophages to LPS, the underlying mechanism remains unknown. Here we investigated the role of MTA1 in the regulation of expression and function of MyD88, a proximal component of NF-kB signaling. We discovered that MyD88 is a chromatin target of MTA1 and that of NF-kB signaling in LPS stimulated macrophages. We found that MTA1 is required for MyD88-dependent stimulation of NF-kB signaling and expression of proinflammatory cytokines like IL-1 beta, MIP2 and TNF-alpha as MTA1 depletion leads to a substantial reduction in the expression of NF-kB target genes. In addition, LPS-mediated stimulation of MyD88 transcription was accompanied by an enhanced recruitment of MTA1, RNA polymerase II and p65RelA complex to the NF-kB consensus sites in the MyD88 promoter. Interestingly, both the recruitment of MTA1 and MyD88 expression are effectively blocked by NF-kB inhibitor parthenolide. Selective knockdown of MyD88 by a dominant negative mutant of MyD88 or selective siRNA impairs the ability of LPS to stimulate the NF-kB target genes. These findings reveal an inherent coregulatory role of MTA1 upon MyD88 expression and suggest that MTA1 regulation of MyD88 may constitute, at-least, one of the mechanisms by which MTA1 stimulates LPS-induced NF-kB signaling in stimulated macrophages.

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The p97 ATPase dislocates MHC class I heavy chain in US2 expressing cells via an Ufd1-Npl4 independent mechanism.

Publication Date: 2010 Aug 11 PMID: 20702414
Authors: Soetandyo, N. - Ye, Y.
Journal: J Biol Chem

The human cytomegalovirus (HCMV) protein US2 hijacks the ER-associated degradation (ERAD) machinery to dispose of newly synthesized MHC class I heavy chain at the endoplasmic reticulum (ER). This process requires retrotranslocation of the heavy chain molecules from the ER membrane into the cytosol, but the mechanism underlying the dislocation reaction has been unclear. Here we establish an in vitro permeabilized cell assay that recapitulates the retrotranslocation of MHC heavy chain in US2-expressing cells. Using this assay, we demonstrate that the dislocation process requires ATP and ubiquitin, as expected. The retrotranslocation also involves the p97 ATPase. However, the mechanism by which p97dislocates MHC class I heavy chain in US2 cells is distinct from that in US11 cells; the dislocation reaction in US2 cells is independent of the p97 cofactor Ufd1-Npl4. Our results suggest that different retrotranslocation mechanisms may employ distinct p97 ATPase complexes to destruct ERAD substrates.

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A novel pathway for inducible nitric oxide synthase (iNOS) activation through inflammasomes.

Publication Date: 2010 Aug 11 PMID: 20702413
Authors: Buzzo, C. L. - Campopiano, J. C. - Massis, L. M. - Lage, S. L. - Cassado, A. A. - Leme-Souza, R. - Dias da Cunha, L. - Russo, M. - Zamboni, D. S. - Amarante-Mendes, G. P. - Bortoluci, K. R.
Journal: J Biol Chem

Innate immune recognition of flagellin is shared by transmembranic TLR5 and cytosolic Nlrc4 (Nucleotide Oligomerization Domain (NOD)-Like Receptors (NLR) - family CARD (caspase activation recruitment domain) domain containing 4)/ Naip5 (Neuronal apoptosis inhibitory protein 5). TLR5 activates inflammatory genes through MYD88 pathway whereas Nlrc4 and Naip5 assemble multiprotein complexes called inflammasomes, culminating in caspase-1 activation, IL-1beta/IL-18 secretion and pyroptosis. Although both TLR5 and Naip5/Nlrc4 pathways cooperate to clear infections, little is known about the relative anti-pathogen effector mechanisms operating through each of them. Here we show that the cytosolic flagellin (FLA-BSDot) was able to activate iNOS, an enzyme previously associated with TLR5 pathway. Using Nlrc4- or Naip5-deficient macrophages, we found that both receptors are involved in iNOS activation by FLA-BSDot. Moreover, distinct from extracellular flagellin (FLA-BS), iNOS activation by intracellular flagellin is completely abrogated in the absence of caspase-1. Interestingly, IL-1beta and IL-18 do not seem to be important for FLA-BSDot-mediated iNOS production. Together, our data defined an additional anti-pathogen effector mechanism operated through Naip5 and Nlrc4 inflammasomes and illustrated a novel signaling transduction pathway that activates iNOS.

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Pro-inflammatory genes as biomarkers and therapeutic targets in oral squamous cell carcinoma.

Publication Date: 2010 Aug 11 PMID: 20702412
Authors: Kishan Rao, S. - Pavicevic, Z. - Du, Z. - Kim, J. G. - Fan, M. - Jiao, Y. - Rosebush, M. - Samant, S. - Gu, W. - Pfeffer, L. M. - Nosrat, C. A.
Journal: J Biol Chem

Oral squamous cell carcinoma (OSCC) is a major health problem worldwide, and patients have a particularly poor 5-year survival rate. Thus, identification of the molecular targets in OSCC and subsequent innovative therapies are greatly needed. Prolonged exposure to alcohol, tobacco and pathogenic agents are known risk factors, and have suggested that chronic inflammation may represent a potential common denominator in the development of OSCC. Microarray analysis of gene expression in OSCC cell lines with high basal NF-kB activity and OSCC patient samples identified dysregulation of many genes involved in inflammation, wound healing, angiogenesis and growth regulation. In particular IL-8, CCL5, STAT1 and VEGF gene expression was up-regulated in OSCC. Moreover, IL-8 protein levels were significantly higher in OSCC cell lines, as compared to normal human oral keratinocytes. Targeting IL-8 expression by siRNA significantly reduced the survival of OSCC cells indicating that it plays an important role in OSCC development and/or progression. Inhibiting the inflammatory pathway by aspirin and the proteasome/NF-kB pathway by bortezomib resulted in marked reduction in cell viability in OSCC lines. Taken together our studies indicate a strong link between inflammation and OSCC development and reveal IL-8 as a potential mediator. Treatment based on prevention of general inflammation and/or the NF-kB pathway shows promise in OSCCs.

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Structure and metal-loading of a soluble periplasm cupro-protein.

Publication Date: 2010 Aug 10 PMID: 20702411
Authors: Waldron, K. J. - Firbank, S. J. - Dainty, S. J. - Perez-Rama, M. - Tottey, S. - Robinson, N. J.
Journal: J Biol Chem

A copper-trafficking pathway is found to enable Cu(2+)-occupancy of a soluble periplasm-protein, CucA, even when competing Zn(2+) is abundant in the periplasm. Here we solve the structure of CucA (a new cupin) and find that binding of Cu(2+)-, but not Zn(2+)-, quenches the fluorescence of Trp(165) which is adjacent to the metal site. Using this fluorescence-probe we establish that CucA becomes partly occupied by Zn(2+) following exposure to equimolar Zn(2+) and Cu(2+). Cu(2+)-CucA is more thermodynamically stable than Zn(2+)-CucA, but k((Zn-->Cu)exchange) is slow raising questions about how the periplasm contains solely the Cu(2+)-form. We discover that a copper-trafficking pathway involving two copper transporters (CtaA and PacS) and a metallochaperone (Atx1) is obligatory for Cu(2+)-CucA to accumulate in the periplasm. There is negligible CucA protein in the periplasm of DeltactaA cells but the abundance of cucA transcripts is unaltered. Crucially, DeltactaA cells over-accumulate low M(r) copper-complexes in the periplasm and purified apo-CucA can readily acquire Cu(2+) from DeltactaA periplasm extracts, but in vivo apo-CucA fails to come into contact with these periplasmic copper-pools. Instead, copper traffics via a cytoplasmic pathway which is coupled to CucA translocation to the periplasm.

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The caspase-8 dimerisation/dissociation balance is a highly potent regulator of caspase-8, -3, -6 signalling.

Publication Date: 2010 Aug 11 PMID: 20702410
Authors: Wurstle, M. L. - Laussmann, M. A. - Rehm, M.
Journal: J Biol Chem

Apoptosis is driven by positive feedback activation between asparate-specific cysteinyl proteases (caspases). These feedback loops ensure the swift and efficient elimination of cells upon initiation of apoptosis execution. At the same time, the signaling network must be insensitive to erroneous, mild caspase activation to avoid unwanted, excessive cell death. Sub-lethal caspase activation in fact was shown to be a requirement for the differentiation of multiple cell types but might also occur accidentally during short, transient cellular stress conditions. Here we carried out an in silico comparison of the molecular mechanisms that so far have been identified to impair the amplification of caspase activities via the caspase-8, -3, -6 loop. In a systems model resembling HeLa cervical cancer cells, the dimerisation/dissociation balance of caspase-8 potently suppressed the amplification of caspase responses, surprisingly outperforming or matching known caspase-8 and -3 inhibitors such as bi-functional apoptosis repressor (BAR) or x-linked inhibitor of apoptosis protein (XIAP). These findings were further substantiated in global sensitivity analyses based on combinations of protein concentrations from the sub- to super-physiological range in order to screen the full spectrum of biological variability that can be expected within cell populations and between distinct cell types. Additional modeling showed that the combined effects of XIAP and caspase-8 dimerisation/dissociation processes can provide resistance also to larger inputs of active caspases. Our study therefore highlights a central and so far underappreciated role of caspase-8 dimerisation/dissociation in avoiding unwanted cell death by lethal amplification of caspase responses via the caspase-8, -3, -6 loop.

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Tyrosine phosphorylation of {beta}3 integrin regulates kindlin-2 binding and integrin activation.

Publication Date: 2010 Aug 11 PMID: 20702409
Authors: Bledzka, K. - Bialkowska, K. - Nie, H. - Qin, J. - Byzova, T. - Wu, C. - Plow, E. F. - Ma, Y. Q.
Journal: J Biol Chem

Kindlins are essential for integrin activation in cellular systems and do so by working in a cooperative fashion with talin via their direct interaction with beta integrin cytoplasmic tails. Kindlins interact with the membrane-distal NxxY motif, which is distinct from the talin binding site within the membrane-proximal NxxY motif. The tyrosine residues in both motifs can be phosphorylated, and it has been suggested that this modification of the membrane-proximal NxxY motif negatively regulates interaction with talin head domain. However, the influence of tyrosine phosphorylation of the membrane-distal NxxY motif on kindlin binding is unknown. Using phosphomimetic mutations and phosphorylated peptides, we show that phosphorylation of the membrane-distal NITY759 motif in the beta3 cytoplasmic tail disrupts kindlin-2 recognition. Phosphorylation of this membrane distal tyrosine also disables the ability of kindlin-2 to co-activate the integrin. In direct binding studies, peptides corresponding to the non-phosphorylated beta3 cytoplasmic tail interacted well with kindlin-2 whereas the Tyr759-phosphorylated peptide failed to bind kindlin-2 with measurable affinity. These observations indicate that transitions between the phosphorylated and non-phosphorylated state of integrin beta3 cytoplasmic tail determine reactivity with kindlin-2 and govern the role of kindlin-2 in regulating integrin activation.

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CCAAT/enhancer-binding protein {beta} (C/EBP{beta}) and nuclear factor kappa B (NF-{kappa}B) mediate high level expression of chemokine genes CCL3 and CCL4 by human chondrocytes in response to interleukin-1{beta} (IL-1{beta}).

Publication Date: 2010 Aug 11 PMID: 20702408
Authors: Zhang, Z. - Bryan, J. L. - Delassus, E. - Chang, L. W. - Liao, W. - Sandell, L. J.
Journal: J Biol Chem

A large set of chemokines is highly up-regulated in human chondrocytes in response to IL-1beta (Sandell, L.J. et al. 2008. Osteoarthritis Cartilage 16, 1560-1571). To investigate the mechanism of transcriptional regulation, deletion constructs of selected chemokine gene promoters, the human CCL3 (MIP-1alpha) and CCL4 (MIP-1beta), were transfected into human chondrocytes with or without IL-1beta. The results show that an IL-1beta-responsive element is located between -300 to -140bp of CCL3 promoter, and -222 to -100bp of CCL4 promoter. As both of these elements contain C/EBPbeta motifs, the function of C/EBPbeta was examined. IL-1beta stimulated the expression of C/EBPbeta, and the direct binding of C/EBPbeta to the C/EBPbeta motif was confirmed by EMSA and ChIP analyses. The -300bp CCL3 promoter and -222bp CCL4 promoter were strongly up-regulated by co-transfection with C/EBPbeta expression vector. Mutation of the C/EBPbeta motif and reduction of C/EBPbeta expression by siRNA decreased the up-regulation. Additionally, another cytokine-related transcriptional factor, NF-kappaB, was also shown to be involved in the up-regulation of chemokines in response to IL-1beta and the binding site was identified. The regulation of C/EBPbeta and NF-kappaB was confirmed by the inhibition by C/EBPbeta and NF-kappaB and by transfection with C/EBPbeta and NF-kappaB expression vectors in the presence or absence of IL-1beta. Taken together, our results suggest that C/EBPbeta and NF-kappaB are both involved in the IL-1beta-responsive up-regulation of chemokine genes in human chondrocytes. Time course experiments indicated that C/EBPbeta gradually and steadily induces chemokine up-regulation, whereas the NF-kappaB activity was highest at the early stage of chemokine up-regulation.

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Regulation of response regulator autophosphorylation through interdomain contacts.

Publication Date: 2010 Aug 11 PMID: 20702407
Authors: Barbieri, C. M. - Mack, T. R. - Robinson, V. L. - Miller, M. T. - Stock, A. M.
Journal: J Biol Chem

DNA-binding response regulators of the OmpR/PhoB subfamily alternate between inactive and active conformational states, with the latter having enhanced DNA-binding affinity. Phosphorylation of an aspartate residue in the receiver domain, usually via phosphotransfer from a cognate histidine kinase, stabilizes the active conformation. Many of the available structures of inactive OmpR/PhoB family proteins exhibit extensive interfaces between the N-terminal receiver and C-terminal DNA-binding domains. These interfaces invariably involve the alpha4-beta5-alpha5 face of the receiver domain, the locus of the largest differences between inactive and active conformations and the surface that mediates dimerization of receiver domains in the active state. Structures of receiver domain dimers of DrrB, DrrD and MtrA have been determined and phosphorylation kinetics analyzed. Analysis of phosphotransfer from small molecule phosphodonors has revealed large differences in autophosphorylation rates among OmpR/PhoB response regulators. Response regulators with substantial domain interfaces exhibit slow rates of phosphorylation. Rates are greatly increased in isolated receiver domain constructs. Such differences are not observed between autophosphorylation rates of full-length and isolated receiver domain of a response regulator that lacks interdomain interfaces nor are they observed in histidine kinase-mediated phosphotransfer. These findings suggest that domain interfaces restrict receiver domain conformational dynamics, stabilizing an inactive conformation that is catalytically incompetent for phosphotransfer from small molecule phosphodonors. Inhibition of phosphotransfer by domain interfaces provides an explanation for the observation that some response regulators cannot be phosphorylated by small molecule phosphodonors in vitro and provides a potential mechanism for insulating some response regulators from small molecule-mediated phosphorylation in vivo.

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Regulation of MRP2/ABCC2 and BSEP/ABCB11 expression in sandwich cultured human and rat hepatocytes exposed to inflammatory cytokines TNF-alpha, IL-6 and IL-1beta.

Publication Date: 2010 Aug 11 PMID: 20702406
Authors: Diao, L. - Li, N. - Brayman, T. G. - Hotz, K. J. - Lai, Y.
Journal: J Biol Chem

In the present study, MRP2/ABCC2 and BSEP/ABCB11 expression were investigated in sandwich cultured (SC) human and rat hepatocytes exposed to the proinflammatory cytokines. The investigation was also done in lipopolysaccharide (LPS)-treated rats. In SC human hepatocytes, both absolute protein and mRNA levels of MRP2/ABCC2 were significantly down-regulated by TNF-alpha, IL-6 or IL-1beta. In contrast to a mRNA decrease which was observed for BSEP/ABCB11, the protein amount was significantly increased by IL-6 or IL-1beta. A discrepancy between the change in BSEP/ABCB11 mRNA and protein levels was encountered in SC human hepatocytes treated with proinflammatory cytokines. In SC rat hepatocytes, Mrp2/Abcc2 mRNA was down-regulated by TNF-alpha and IL-6, while the protein level was decreased by all three cytokines. Down-regulations of both Bsep/Abcb11 mRNA and protein levels were found in SC rat hepatocytes exposed to TNF-alpha or IL-1beta. Administration of LPS triggered the release of the proinflammatory cytokines and caused the decrease of Mrp2/Abcc2 and Bsep/Abcb11 protein in liver at 24 hr post treatment; however, the Mrp2 and Bsep protein levels rebounded at 48 hr post LPS treatment. In total, our results indicate that proinflammatory cytokines regulate the expression of MRP2/Mrp2 and BSEP/Bsep, and for the first time, demonstrate the differential effects on BSEP/Bsep expression between SC human and rat hepatocytes. Furthermore, the agreement between transporter regulation in vitro in SC rat hepatocytes and in vivo in LPS-treated rats during the acute response phase (ARP) demonstrates the utility of in vitro SC hepatocyte models for predicting in vivo effects.

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Ablation of succinate production from glucose metabolism in the procyclic trypanosomes induces metabolic switches to the Gly3p/DHAP shuttle and to proline metabolism.

Publication Date: 2010 Aug 11 PMID: 20702405
Authors: Ebikeme, C. - Hubert, J. - Biran, M. - Gouspillou, G. - Morand, P. - Plazolles, N. - Guegan, F. - Diolez, P. - Franconi, J. M. - Portais, J. C. - Bringaud, F.
Journal: J Biol Chem

Trypanosoma brucei is a parasitic protist that undergoes a complex life cycle during transmission from its mammalian host (bloodstream forms) to the midgut of its insect vector (procyclic form). In both parasitic forms, most glycolytic steps take place within specialized peroxisomes, called glycosomes. Here we studied metabolic adaptations in procyclic trypanosome mutants affected in their maintenance of the glycosomal redox balance. T. brucei can theoretically use three strategies to maintain the glycosomal NAD+/NADH balance, (i) the glycosomal succinic fermentation branch, (ii) the glycerol-3-phosphate (Gly3P) / dihydroxyacetone-phosphate (DHAP) shuttle that transfers reducing equivalents to the mitochondrion and (iii) the glycosomal glycerol production pathway. We showed a hierarchy in the use of these glycosomal NADH-consuming pathways by determining metabolic perturbations and adaptations in single and double mutant cell lines using a combination of NMR, IC-MS/MS and HPLC approaches. Although functional, the Gly3P/DHAP shuttle is primarily used when the preferred succinate fermentation pathway is abolished in the pepck mutant cell line. In the absence of these two pathways (Deltapepck/RNAiFAD-GPDH.i mutant), glycerol production is used, but with a 16-fold reduced glycolytic flux. In addition, the pepck mutant cell line shows a 3.3-fold reduced glycolytic flux compensated by an increase of proline metabolism. The inability of the Deltapepck mutant to maintain a high glycolytic flux demonstrates that the Gly3P/DHAP shuttle is not adapted to the procyclic trypanosome context. In contrast, this shuttle was shown earlier to be the only way used by the bloodstream forms of T. brucei to sustain their high glycolytic flux.

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Novel inhibitors of plasmodium falciparum dihydroorotate dehydrogenase with anti-malarial activity in the mouse model.

Publication Date: 2010 Aug 11 PMID: 20702404
Authors: Booker, M. L. - Bastos, C. M. - Kramer, M. L. - Barker, R. H. Jr - Skerlj, R. - Bir Sidhu, A. - Deng, X. - Celatka, C. - Cortese, J. F. - Guerrero Bravo, J. E. - Krespo Llado, K. N. - Serrano, A. E. - Angulo-Barturen, I. - Jimenez-Diaz, M. B. - Viera, S. - Garuti, H. - Wittlin, S. - Papastogiannidis, P. - Lin, J. W. - Janse, C. J. - Khan, S. M. - Duraisingh, M. - Coleman, B. - Goldsmith, E. J. - Phillips, M. A. - Munoz, B. - Wirth, D. F. - Klinger, J. D. - Wiegand, R. - Sybertz, E.
Journal: J Biol Chem

The apicomplexan parasite Plasmodium falciparum is the causative agent of the most deadly form of human malaria. This organism is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in the pyrimidine biosynthetic pathway and represents a potential target for anti-malarial therapy. A high-throughput screen and subsequent medicinal chemistry program identified a series of N-alkyl-5-(1H-benzimidazol-1-yl)thiophene-2-carboxamides with low nanomolar in vitro potency against DHODH from P. falciparum, P. vivax, and P. berghei. The compounds were selective for the parasite enzymes over human DHODH, and X-ray structural data on the analog Genz-667348 elucidated the inhibitor-binding mode, demonstrating that species selectivity could be attributed to amino acid differences in the inhibitor-binding site between the human and malarial enzymes. Compounds from this series demonstrated in vitro potency against the 3D7 and Dd2 strains of P. falciparum that correlated well with enzyme inhibitory activity. Several of the most potent compounds demonstrated good tolerability and oral exposure in the mouse, as well as ED(50) values in the 4-day murine P. berghei efficacy model of 13-21 mg/kg/day with oral twice-daily dosing. In particular, treatment with Genz-667348 at 100 mg/kg/day resulted in sterile cure, as defined by absence of recrudescence during a 30-day period following the cessation of dosing. This compound exhibited comparable activity in the P. falciparum humanized NOD-scid mouse model. Two recent analogs of Genz-667348 are currently undergoing pilot toxicity testing to determine suitability as clinical development candidates.

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Role of H-1 and H-2 subunits of soybean seed ferritin in oxidative deposition of iron in protein.

Publication Date: 2010 Aug 11 PMID: 20702403
Authors: Deng, J. - Liao, X. - Yang, H. - Hua, Z. - Masuda, T. - Goto, F. - Yoshihara, T. - Zhao, G.
Journal: J Biol Chem

Naturally occurring phytoferritin is a heteropolymer consisting of two different H-type subunits, H-1 and H-2. Prior to this study, however, the function of the two subunits in oxidative deposition of iron in ferritin was unknown. The data show that, upon aerobic addition of 48 to 200 Fe2+/shell to apoferritin, iron oxidation occurs only at the diiron ferroxidase center of rH-1. In additional to the diiron ferroxidase mechanism, such oxidation is catalyzed by the extension peptide (a specific domain found in phytoferritin) of rH-2, because the H-1 subunit is able to remove Fe3+ from the center to the inner cavity better than the H-2 subunit. These findings support the idea that the H-1 and H-2 subunits play different roles in iron mineralization in protein. Interestingly, at medium iron loading (200 iron/shell), wide-type soybean seed ferritin (WT SSF) exhibits a stronger activity in catalyzing iron oxidation than rH-1 and rH-2, demonstrating that a synergistic interaction exists between the H-1 and H-2 subunits in SSF during iron mineralization. Such synergistic interaction becomes considerably stronger at high iron loading (400 iron/shell) as indicated by the observation that the iron oxidation activity of WT SSF is about 10 times larger than those of rH-1 and rH-2. This helps elucidate the widespread occurrence of heteropolymeric ferritins in plant.

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Kinetics of re-establishing H3 K79 methylation marks in global human chromatin.

Publication Date: 2010 Aug 9 PMID: 20699226
Authors: Sweet, S. M. - Li, M. - Thomas, P. M. - Durbin, K. R. - Kelleher, N. L.
Journal: J Biol Chem

We employ a stable isotope strategy wherein both histones and their methylations are labeled in synchronized human cells. This allows us to differentiate between old and new methylations on pre-existing vs. newly-synthesized histones. The strategy is implemented on K79 methylation in an isoform-specific manner for histones H3.1, H3.2 and H3.3. While levels of H3.3 K79 monomethylation are higher than that of H3.2/H3.1, the rate of establishing the K79 methylation is the same for all three isoforms. Surprisingly, we find that pre-existing "old" histones continue to be K79 mono- and di-methylated at a rate equal to the newly-synthesized histones. These observations imply that some degree of positional "scrambling" of K79 methylation occurs through the cell cycle.

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Lipid raft association and cholesterol sensitivity of P2X1-4 receptors for ATP; chimeras and point mutants identify intracellular amino terminal residues involved in lipid regulation of P2X1 receptors.

Publication Date: 2010 Aug 10 PMID: 20699225
Authors: Allsopp, R. C. - Lalo, U. - Evans, R. J.
Journal: J Biol Chem

Cholesterol rich lipid rafts act as signalling microdomains and can regulate receptor function. We have shown in HEK293 cells that recombinant P2X1,2,3 and 4 receptors (ATP-gated ion channels) are expressed in lipid rafts. Localisation to flotillin rich lipid rafts was reduced by the detergent Triton X 100. This sensitivity to Triton X 100 was concentration dependent, and varied between P2X receptor subtypes demonstrating differential association of P2X1-4 receptors with lipid rafts. The importance of rafts to ATP evoked P2X receptor responses was determined in patch clamp studies. The cholesterol depleting agents methyl beta cyclodextrin or filipin disrupt lipid rafts, and reduced P2X1 receptor currents by >90%. In contrast ATP evoked P2X2,3&4 receptor currents were unaffected by lipid raft disruption. To determine the molecular basis of cholesterol sensitivity we generated chimeric receptors replacing portions of the cholesterol sensitive P2X1 receptor with the corresponding region from the insensitive P2X2 receptor. These chimeras identified the importance of the intracellular amino-terminal region between the conserved protein kinase C site and the first transmembrane segment (TM1) for the sensitivity to cholesterol depletion. Mutation of any of the variant residues between P2X1 and P2X2 receptors in this region (residues 20-23 and 27-29) to cysteine in the P2X1 receptor removed cholesterol sensitivity. Cholesterol depletion did not change the ATP sensitivity or cell surface expression of P2X1 receptors. This suggests that cholesterol is normally needed to facilitate the opening/gating of ATP bound P2X1 receptor channels and mutations in the pre-TM1 region removes this requirement.

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The novel importin-alpha family member KPNA7, is required for normal fertility and fecundity in the mouse.

Publication Date: 2010 Aug 10 PMID: 20699224
Authors: Hu, J. - Wang, F. - Yuan, Y. - Zhu, X. - Wang, Y. - Zhang, Y. - Kou, Z. - Wang, S. - Gao, S.
Journal: J Biol Chem

Nuclear importing system and nuclear factors play important roles in mediating nuclear reprogramming and zygotic gene activation(ZGA). However, the components and mechanisms that mediate nuclear-specific targeting of the nuclear proteins during nuclear reprogramming and ZGA remain largely unknown. Here we identified a novel member of the importin-alpha family, AW146299(Kpna7), which is predominantly expressed in mouse oocytes and zygotes and localizes to the nucleus or spindle. Mutation of Kpna7 gene caused reproductivity reduction and sex imbalance by inducing preferential fetal lethality in females. Parthenogenesis analysis showed that cell cycle of activated one-cell embryos is loss of control and ahead of schedule, but finally failed to develop into blastocyst stage. Further RT-PCR and epigenetic modification analysis showed that knocking out of Kpna7 induced abnormalities of gene expression (Dppa2, Dppa4 and Piwil2) and epigenetic modifications (down-regulation of histone H3K27me3). Biochemical analysis showed that KPNA7 interacts with KPNB1 (importin-beta1). In summary, we identified a novel Kpna7 gene that is required for normal fertility and fecundity.

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Yeast 18S rRNA is directly involved in the ribosmeal response to stringent AUG selection during translation initiation.

Publication Date: 2010 Aug 10 PMID: 20699223
Authors: Nemoto, N. - Singh, C. R. - Udagawa, T. - Wang, S. - Thorson, E. - Winter, Z. - Ohira, T. - Ii, M. - Valasek, L. - Brown, S. J. - Asano, K.
Journal: J Biol Chem

In eukaryotes, the 40S ribosomal subunit serves as the platform of initiation factor assembly, to place itself precisely on the AUG start codon. Structural arrangement of the 18S rRNA determines the overall shape of the 40S subunit. Here we present genetic evaluation of yeast 18S rRNA function using ten point-mutations altering the polysome profile. All the mutants reduce the abundance of the mutant 40S, making it limiting for translation initiation. Two of the isolated mutations, G875A, altering the core of the platform domain that binds eIF1 and eIF2, and A1193U, changing the h31 loop located beneath the P-site tRNA(i)(Met), show phenotypes indicating defective regulation of AUG selection. Evidence is provided that these mutations reduce the interaction with the components of the pre-initiation complex (PIC), thereby inhibiting its function at different steps. These results indicate that the 18S rRNA mutations impair the integrity of scanning-competent PIC, thereby altering the 40S subunits; response to stringent AUG selection. Interestingly, nine of the mutations alter the body/platform domains of 18S rRNA, potentially affecting the bridges to the 60S subunit, but do not change the level of 18S rRNA intermediates. Based on these results, we also discuss the mechanism of the selective degradation of the mutant 40S subunits.

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Molecular mapping of the RNA Cap 2'-O-methyltransferase activation interface between SARS coronavirus nsp10 and nsp16.

Publication Date: 2010 Aug 27 PMID: 20699222
Authors: Lugari, A. - Betzi, S. - Decroly, E. - Bonnaud, E. - Hermant, A. - Guillemot, J. C. - Debarnot, C. - Borg, J. P. - Bouvet, M. - Canard, B. - Morelli, X. - Lecine, P.
Journal: J Biol Chem

The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) was identified in 2003 as a previously unknown coronavirus responsible of a sudden outbreak spreading rapidly from Asia, with a final number of cases around 8,000 and a 10% mortality. Several protein-protein interactions within the SARS-CoV proteome have been identified, one of them being strong and reciprocal between non-structural proteins nsp10 and nsp16. In this work, we have mapped key residues on the nsp10 surface involved in this interaction using Reverse Yeast two-Hybrid (RY2H). Alanine-scanning mutagenesis, bioinformatics and molecular modelling studies were used to identify several 'hot spots' such as V42, M44, L45, A71, K93, G94 and Y96, forming a continuous protein-protein surface of about 830 A(2), bearing very conserved amino acids within coronaviruses. As nsp16 carries RNA cap 2[prime]O-MTase activity only in the presence of its interacting partner nsp10 (1), functional consequences of mutations on this surface were evaluated biochemically. Most changes which disrupted the nsp10/nsp16 interaction without structural perturbations, as judged by heteronuclear NMR experiments, were shown to abrogate stimulation of nsp16 cap 2'O-MTase activity. More strikingly, we show that Y96 plays a central role in the interaction and nsp16 stimulation: the Y96A mutation abrogates stimulation of nsp16 2'O-MTase activity while Y96F over stimulates it. Together with nsp16 2'O-MTase activity, the nsp10/nsp16 interface may represent an attractive target for antivirals against human and animal pathogenic coronaviruses.

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Anti-apoptotic protein, BCL2, downregulates DNA end joining in cancer cells.

Publication Date: 2010 Aug 10 PMID: 20699221
Authors: Satish Kumar, T. - Kari, V. - Choudhary, B. - Nambiar, M. - Akila, T. S. - Raghavan, S. C.
Journal: J Biol Chem

Cancer cells are often associated with secondary chromosomal rearrangements such as deletions, inversions and translocations, which could be the consequence of unrepaired/misrepaired DNA double-strand breaks. Nonhomologous DNA end joining (NHEJ) is one of the most common pathways to repair DSBs in higher eukaryotes. By using oligomeric DNA substrates mimicking various endogenous DSBs in a cell-free system, we have studied end joining (EJ) in different cancer cell lines. We find that efficiency of EJ varies among cancer cells; however, there was no remarkable difference in the mechanism and expression of EJ proteins. Interestingly, cancer cells with lower level of EJ possessed elevated expression of BCL2 and vice versa. Removal of BCL2 by immunoprecipitation or protein fractionation led to elevated EJ. More importantly, we show that overexpression of BCL2 or addition of purified BCL2 led to the downregulation of EJ. Further, we find that BCL2 interacts with KU proteins both in vitro and in vivo. Hence, our results suggest that EJ in cancer cells could be negatively regulated by the anti-apoptotic protein, BCL2, and this may contribute towards increased chromosomal abnormalities in cancer.

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Influenza A virus polymerase inhibits type I interferon induction by binding to interferon {beta} promoter stimulator 1.

Publication Date: 2010 Aug 13 PMID: 20699220
Authors: Iwai, A. - Shiozaki, T. - Kawai, T. - Akira, S. - Kawaoka, Y. - Takada, A. - Kida, H. - Miyazaki, T.
Journal: J Biol Chem

Type I interferons (IFNs) are known to be critical factors in the activation of host antiviral responses, and are also important in protection from influenza A virus infection. Especially, the RIG-I- and IPS-1-mediated intracellular type I IFN inducing pathway is essential in the activation of antiviral responses in cells infected by influenza A virus. Previously, it has been reported that influenza A virus NS1 is involved in the inhibition of this pathway. We show in this report that the influenza A virus utilizes another critical inhibitory mechanism in this pathway. In fact, the viral polymerase complex exhibited an inhibitory activity on IFNbeta promoter activation mediated by RIG-I and IPS-1, and this activity was not competitive with the function of NS1. Co-immunoprecipitation analysis revealed that each polymerase subunit bound to IPS-1 in mammalian cells, and each subunit inhibited the activation of IFNbeta promoter by IPS-1 independently. In addition, by a combinational expression of each polymerase subunit, IPS-1 induced activation of IFNbeta promoter was more efficiently inhibited by the expression of PB2 or PB2 containing complex. Moreover, the expression of PB2 inhibited the transcription of the endogenous IFNbeta gene induced after influenza A virus infection. These findings demonstrate that the viral polymerase plays an important role for regulating host anti-viral response through the binding to IPS-1 and inhibition of IFNbeta production.

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A Novel Pax-like Protein Involved in Transcriptional activation of Cyst Wall Protein Genes in Giardia lamblia.

Publication Date: 2010 Aug 10 PMID: 20699219
Authors: Wang, Y. T. - Pan, Y. J. - Cho, C. C. - Lin, B. C. - Su, L. H. - Huang, Y. C. - Sun, C. H.
Journal: J Biol Chem

Giardia lamblia differentiates into infectious cysts to survive outside of the host. It is of interest to identify factors involved in up-regulation of cyst wall proteins (CWPs) during this differentiation. Pax proteins are important regulators of development and cell differentiation in Drosophila and vertebrates. No member of this gene family has been reported to date in yeast, plants, or protozoan parasites. We have identified a pax-like gene (pax1) encoding a putative paired domain in the G. lamblia genome. Epitope-tagged Pax1 localized to nuclei during both vegetative growth and encystation. Recombinant Pax1 specifically bound to the AT-rich initiator elements of the encystation-induced cwp1-3 and myb2 genes. Interestingly, overexpression of Pax1 increased the cwp1-3 and myb2 gene expression and cyst formation. Deletion of the C-terminal paired domain or mutation of the basic amino acids of the paired domain resulted in a decrease of transactivation function of Pax1. Our results indicate that Pax family has been conserved during evolution and Pax1 could up-regulate the key encystation-induced genes to regulate differentiation of the protozoan eukaryote, G. lamblia.

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Ctr1 IS AN APICAL COPPER TRANSPORTER IN MAMMALIAN INTESTINAL EPITHELIAL CELLS IN VIVO THAT IS CONTROLLED AT THE LEVEL OF PROTEIN STABILITY.

Publication Date: 2010 Aug 10 PMID: 20699218
Authors: Nose, Y. - Wood, L. K. - Kim, B. E. - Prohaska, J. R. - Fry, R. S. - Spears, J. W. - Thiele, D. J.
Journal: J Biol Chem

Copper (Cu) is an essential trace element that functions in a diverse array of biochemical processes that include mitochondrial respiration, neurotransmitter biogenesis, connective tissue maturation and reactive oxygen chemistry. The Ctr1 protein is a high affinity Cu(+) importer that is structurally and functionally conserved in yeast, plants, fruit flies and humans and which, in all of these organisms is localized to the plasma membrane and intracellular vesicles. While intestinal epithelial cell-specific deletion of Ctr1 in mice demonstrated a critical role for Ctr1 in dietary Cu absorption, some controversy exists over the localization of Ctr1 in intestinal epithelial cells in vivo. In this report we assess the localization of Ctr1 in intestinal epithelial cells through two independent mechanisms. We demonstrate by immunohistochemistry in intestinal sections that Ctr1 localizes to the apical membrane in intestinal epithelial cells of the mouse, rat and pig. Moreover, biotinylation of intestinal luminal proteins from mice raised on a control or Cu-deficient diet showed elevated levels of both total and apical membrane Ctr1 protein in response to transient dietary copper limitation. Experiments in cultured HEK293T cells demonstrate that alterations in the levels of the glycosylated form of Ctr1 in response to copper availability is a time-dependent, copper-specific post-translational response. Taken together these results demonstrate apical localization of Ctr1 in intestinal epithelia across three mammalian species and suggest that increased Ctr1 apical localization in response to dietary Cu limitation may represent an adaptive response to homeostatically modulate Ctr1 availability at the site of intestinal copper absorption.

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Rod/Zw10 complex is required for PIASy-dependent centromeric SUMOylation.

Publication Date: 2010 Aug 9 PMID: 20696768
Authors: Ryu, H. - Azuma, Y.
Journal: J Biol Chem

SUMO conjugation of cellular proteins is essential for proper progression of mitosis. PIASy, a SUMO E3 ligase, is required for mitotic SUMOylation of chromosomal proteins, yet the regulatory mechanism behind the PIASy-dependent SUMOylation during mitosis has not been determined. Using a series of truncated PIASy proteins, we have found that the N-terminus of PIASy is not required for SUMO modification in vitro but is essential for mitotic SUMOylation in Xenopus egg extracts. We demonstrate that swapping the N-terminus of PIASy protein with the corresponding region of other PIAS family members abolishes chromosomal binding and mitotic SUMOylation. We further show that the N-terminal domain of PIASy is sufficient for centromeric localization. We identified that the N-terminal domain of PIASy interacts with the Rod/Zw10 complex, and immunofluorescence further reveals that PIASy colocalizes with Rod/Zw10 in the centromeric region. We show that the Rod/Zw10 complex interacts with the first 47 residues of PIASy which were particularly important for mitotic SUMOylation. Finally, we show that depletion of Rod compromises the centromeric localization of PIASy and SUMO2/3 in mitosis. Together, we demonstrate a fundamental mechanism of PIASy to localize in the centromeric region of chromosome to execute centromeric SUMOylation during mitosis.

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Regulation of the matriptase-prostasin cell surface proteolytic cascade by hepatocyte growth factor activator inhibitor-1 (HAI-1) during epidermal differentiation.

Publication Date: 2010 Aug 9 PMID: 20696767
Authors: Chen, Y. W. - Wang, J. K. - Chou, F. P. - Chen, C. Y. - Rorke, E. A. - Chen, L. M. - Chai, K. X. - Eckert, R. L. - Johnson, M. D. - Lin, C. Y.
Journal: J Biol Chem

Matriptase, a membrane-tethered serine protease, plays essential roles in epidermal differentiation and barrier function, largely mediated via its activation of prostasin, a glycosylphosphatidylinositol (GPI)-anchored serine protease. Matriptase activity is tightly regulated by its inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1) such that free active matriptase is only briefly available to act on its substrates. In the current study we provide evidence for how matriptase activates prostasin under this tight control by HAI-1. When primary human keratinocytes are induced to differentiate in a skin organotypic culture model, both matriptase and prostasin are constitutively activated and then inhibited by HAI-1. These processes also occur in HaCaT human keratinocytes when matriptase activation is induced by exposure of the cells to a pH 6.0 buffer. Using this acid-inducible activation system we demonstrate that prostatin activation is suppressed by matriptase knockdown and by blocking matriptase activation with sodium chloride, suggesting that prostatin activation is dependent on matriptase in this system. Kinetics studies further reveal that the timing of autoactivation of matriptase, prostasin activation and the inhibition of both enzymes by HAI-1 binding are closely correlated. These data suggest that during epidermal differentiation the matriptase-prostasin proteolytic cascade is tight regulation by two mechanisms: 1) prostasin activation is temporally coupled to matriptase autoactivation and 2) HAI-1 rapidly inhibits not only active matriptase but also active prostasin, resulting in an extremely brief window of opportunity for both active matriptase and active prostasin to act on their substrates.

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Nucleoplasmin binds histone H2A-H2B dimers through its distal face.

Publication Date: 2010 Aug 9 PMID: 20696766
Authors: Ramos, I. - Martin-Benito, J. - Finn, R. - Bretana, L. - Aloria, K. - Arizmendi, J. M. - Ausio, J. - Muga, A. - Valpuesta, J. M. - Prado, A.
Journal: J Biol Chem

Nucleoplasmin (NP) is a pentameric chaperone that regulates the condensation state of chromatin extracting specific basic proteins from sperm chromatin and depositing H2A-H2B histone dimers. It has been proposed that histones could bind to either the lateral or distal face of the pentameric structure. Here we combine different biochemical and biophysical techniques to show that natural, hyperphosphorylated NP can bind five H2A-H2B dimers, and that the amount of bound ligand depends on the overall charge (phosphorylation level) of the chaperone. Three-dimensional reconstruction of NP/H2A-H2B complex carried out by electron microscopy reveals that histones interact with the chaperone distal face. Limited proteolysis and mass spectrometry indicate that the interaction results in protection of the histone fold and most of the H2A and H2B C-terminal tails. This structural information helps to understand the function of NP as a histone chaperone.

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Semaphorin 5A and plexin-B3 inhibit human glioma cell motility through RhoGDI{alpha}-mediated inactivation of Rac1 GTPase.

Publication Date: 2010 Aug 9 PMID: 20696765
Authors: Li, X. - Lee, A. Y.
Journal: J Biol Chem

Semaphorins and plexins are implicated in the progression of various types of cancer, though the molecular basis has not been fully elucidated. Here we report the expression of plexin-B3 in glioma cells, which upon stimulation by its ligand Sema5A results in significant inhibition of cell migration and invasion. A search for the underlying mechanism revealed direct interaction of plexin-B3 with RhoGDP dissociation inhibitor alpha (RhoGDIalpha), a negative regulator of RhoGTPases that blocks guanine nucleotide exchange and sequesters them away from the plasma membrane. Glioma cells challenged with Sema5A indeed showed a marked reduction in Rac1-GTP level by 60%, with a concomitant disruption of lamellipodia. The inactivation of Rac1 was corroborated to contribute to the impediment of glioma cell invasion by Sema5A, as supported by the abolishment of effect upon forced expression of a constitutively active Rac1 mutant. Further, silencing the endogenous expression of RhoGDIalpha in glioma cells was found to be sufficient in abrogating the downregulation of Rac1-GTP and the ensuing suppression of glioma cell motility induced by Sema5A. Mechanistically, we provide evidence that Sema5A promotes Rac1 recruitment to RhoGDIalpha and reduces its membrane localization in a plexin-B3 dependent manner, thereby preventing Rac1 activation. This represents a novel signaling of semaphorin and plexin in the control of cell motility by indirect inactivation of Rac1 through RhoGDIalpha.

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The LBC RHO GEF/alpha-catulin axis functions in serotonin-induced vascular smooth muscle cell mitogenesis and rhoa/rock activation.

Publication Date: 2010 Aug 9 PMID: 20696764
Authors: Bear, M. D. - Li, M. - Liu, Y. - Giel-Moloney, M. A. - Fanburg, B. L. - Toksoz, D.
Journal: J Biol Chem

Abstract. Serotonin (5-hydroxytryptamine, 5-HT) is mitogenic for several cell types including pulmonary arterial smooth muscle cells (PASMC), and is associated with the abnormal vascular smooth muscle remodeling present in pulmonary arterial hypertension. RhoA/ROCK function is required for 5-HT-induced PASMC mitogenesis, and 5-HT activates RhoA; however, the signaling steps are poorly defined. Rho guanine nucleotide exchange factors (Rho GEFs) transduce extracellular signals to Rho, and we found that 5-HT treatment of PASMC led to increased membrane-associated Lbc Rho GEF, suggesting modulation by 5-HT. Lbc knock-down by siRNA attenuated 5-HT-induced thymidine uptake in PASMC, indicating a role in PASMC mitogenesis. 5-HT triggered Rho-dependent SRF-mediated reporter activation in PASMC, and this was reduced by Lbc depletion. Lbc knock-down reduced 5-HT-induced RhoA/ROCK activation and RhoA membrane association, but not p42/44 ERK MAP kinase activation, suggesting that Lbc is an intermediary between 5-HT and RhoA/ROCK, but not ERK. 5-HT stimulation of PASMC led to increased association between Lbc, RhoA and the alpha-catulin scaffold. Furthermore, alpha-catulin knock-down attenuated 5-HT-induced PASMC thymidine uptake. These results for the first time define a Rho GEF involved in vascular smooth muscle cell growth and serotonin signaling, and suggest that Lbc Rho GEF family members play distinct roles. Thus, the Lbc/alpha-catulin axis participates in 5-HT-induced PASMC mitogenesis and RhoA/ROCK signaling, and may be an interventional target in diseases involving vascular smooth muscle remodeling.

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Modulation of agrin-induced acetylcholine receptor clustering by extracellular regulated kinases 1 and 2 in cultured myotubes.

Publication Date: 2010 Aug 9 PMID: 20696763
Authors: Rimer, M.
Journal: J Biol Chem

Agrin released by motoneurons induces and/or maintains acetylcholine receptor (AChR) clustering and other aspects of postsynaptic differentiation at the vertebrate neuromuscular junction. Agrin acts by binding and activating a receptor complex containing LDL receptor protein 4 (Lrp4) and muscle specific kinase (MuSK). Two critical downstream components of this signaling cascade, Dox-7 and rapsyn, have been identified. However, additional intracellular essential elements remain unknown. Prior observations by others and us suggested antagonistic interactions between agrin and neuregulin-1 (Nrg-1) signaling in cultured myotubes and developing muscle fibers in vivo. A hallmark of Nrg-1 signaling in skeletal muscle cells is the activation of extracellular regulated kinases 1 and 2 (Erk1/2). Erk1/2 are also activated in most cells by phorbol 12-myristate 13-acetate (PMA), a classical inhibitor of agrin-induced AChR clustering in myotubes. Here, it was investigated whether agrin activates Erk1/2 directly and whether such activation modulates agrin-induced AChR clustering. Agrin induced a rapid but transient activation of Erk1/2 in myotubes that was Lrp4/MuSK-dependent. However, blocking this Erk1/2 activation did not prevent but potentiated AChR clustering induced by agrin. Erk1/2 activation was dispensable for Nrg-1-mediated inhibition of agrin(')s AChR clustering activity, but was indispensable for such activity by PMA. Together, these results suggest agrin-induced activation of Erk1/2 is a negative modulator of agrin signaling in skeletal muscle cells.

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The critical role of 7,8-Didemethyl-8-hydroxy-5-deazariboflavin for photoreactivation in Chlamydomonas reinhardtii.

Publication Date: 2010 Aug 9 PMID: 20696762
Authors: Petersen, J. L. - Ronan, P. J.
Journal: J Biol Chem

DNA photolyases use two non-covalently bound chromophores to catalyze photoreactivation, the blue light-dependent repair of DNA that has been damaged by ultraviolet light. Flavin adenine dinucleotide (FAD) is the catalytic chromophore for all photolyases and is essential for photoreactivation. The identity of the second chromophore is often 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO). Under standard light conditions, the second chromophore is considered non-essential for photoreactivation because DNA photolyase bound to only FAD is sufficient to catalyze the repair of UV-damaged DNA. phr1 is a photoreactivation deficient strain of Chlamydomonas. In this work, the PHR1 gene of Chlamydomonas was cloned through molecular mapping and shown to encode a protein similar to known FO synthases. Additional results revealed that the phr1 strain was deficient in an FO-like molecule and that this deficiency, as well as the phr1 photoreactivation deficiency, could be rescued by transformation with DNA constructs containing the PHR1 gene. Further, expression of a PHR1 cDNA in Escherichia coli produced a protein that generated a molecule with characteristics similar to FO. Together, these results indicate that the Chlamydomonas PHR1 gene encodes an FO synthase and that optimal photoreactivation in Chlamydomonas requires FO, a molecule known to serve as a second chromophore for DNA photolyases.

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A new Kv1.2 channelopathy underlying cerebellar ataxia.

Publication Date: 2010 Aug 9 PMID: 20696761
Authors: Xie, G. - Harrison, J. - Clapcote, S. J. - Huang, Y. - Zhang, J. Y. - Wang, L. Y. - Roder, J. C.
Journal: J Biol Chem

A forward genetic screen of mice treated with the mutagen ENU identified a mutant mouse with chronic motor incoordination. This mutant, named Pingu (Pgu), carries a missense mutation, an I402T substitution in the S6 segment of the voltage-gated potassium channel Kcna2. The gene Kcna2 encodes the voltage-gated potassium channel alpha-subunit Kv1.2, which is abundantly expressed in the large axon terminals of basket cells that make powerful axo-somatic synapses onto Purkinje cells. Patch-clamp recordings from cerebellar slices revealed an increased frequency and amplitude of spontaneous GABAergic inhibitory postsynaptic currents and reduced action potential firing frequency in Purkinje cells, suggesting that an increase in GABA release from basket cells is involved in the motor incoordination in Pgu mice. In line with immunochemical analyses showing a significant reduction in the expression of Kv1 channels in the basket cell terminals of Pgu mice, expression of homomeric and heteromeric channels, containing the Kv1.2 (I402T) alpha-subunit in cultured CHO cells, revealed subtle changes in biophysical properties but a dramatic decrease in functional Kv1 channels. Pharmacological treatment with acetazolamide or transgenic complementation with wild-type Kcna2 cDNA partially rescued the motor incoordination in Pgu mice. These results suggest that independent of known mutations in Kcna1 encoding Kv1.1, Kcna2 mutations may be important molecular correlates underlying human cerebellar ataxic disease.

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