Journal of Biological Chemistry

Functional Oligomerization of the Saccharomyces cerevisiae Isoprenylcysteine Carboxyl Methyltransferase, Ste14p.

Publication Date: 2010 Mar 3 PMID: 20202940
Authors: Griggs, A. M. - Hahne, K. - Hrycyna, C. A.
Journal: J Biol Chem

The isoprenylcysteine carboxyl methyltransferase (Icmt) from Saccharomyces cerevisiae, also designated Ste14p, is a 26 kDa integral membrane protein that contains six transmembrane spanning segments. This protein is localized to the endoplasmic reticulum membrane where it performs the methylation step of the CaaX post-translational processing pathway. Sequence analysis reveals a putative GXXXG dimerization motif located in TM1 of Ste14p, but it is not known whether Ste14p forms or functions as a dimer or higher order oligomer. We determined that Ste14p predominantly formed a homodimer in the presence of the cross-linking agent, bis-sulfosuccinimidyl suberate. Wild-type untagged Ste14p also co-immunoprecipitated and co-purified with N-terminally tagged His(10)-myc(3)-Ste14p (His-Ste14p). Furthermore, enzymatically inactive His-Ste14p variants L81F and E213Q both exerted a dominant-negative effect on methyltransferase activity when co-expressed and co-purified with untagged wild-type Ste14p. Together, these data, although indirect, suggest that Ste14p forms and functions as a homodimer or perhaps a higher oligomeric species.

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Characterization and structure determination of the Cdt1 binding domain of human minichromosome maintenance (Mcm) 6.

Publication Date: 2010 Mar 4 PMID: 20202939
Authors: Wei, Z. - Liu, C. - Wu, X. - Xu, N. - Zhou, B. - Liang, C. - Zhu, G.
Journal: J Biol Chem

The minichromosome maintenance (Mcm) 2-7 complex is the replicative helicase in eukaryotic species, and it plays essential roles in the initiation and elongation phases of DNA replication. During late M and early G1, the Mcm2-7 complex is loaded onto chromatin to form pre-replicative complex (pre-RC) in a Cdt1-dependent manner. However, the detailed molecular mechanism of this loading process is still elusive. In this study we demonstrate that the previously uncharacterized C-terminal domain of human Mcm6 is the Cdt1 binding domain (CBD), and we present its high-resolution nuclear magnetic resonance (NMR) structure. The structure of CBD exhibits a typical winged-helix fold that is generally involved in protein-nucleic acid interaction. Nevertheless, the CBD failed to interact with DNA in our studies indicating that it is specific for protein-protein interaction. The CBD-Cdt1 interaction involves the helix-turn-helix (HTH) motif of CBD. The results reported here provide insight into the molecular mechanism of Mcm2-7 chromatin loading and pre-RC assembly.

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Proenzyme structure and activation of astacin metallopeptidase.

Publication Date: 2010 Mar 4 PMID: 20202938
Authors: Guevara, T. - Yiallouros, I. - Kappelhoff, R. - Bissdorf, S. - Stoecker, W. - Gomis-Ruth, F. X.
Journal: J Biol Chem

Proteolysis is regulated by inactive (latent) zymogens, with a pro-segment preventing access of substrates to the active-site cleft of the enzyme. How latency is maintained often depends on the catalytic mechanism of the protease. For example, in several families of the metzincin metallopeptidases, a cysteine-switch mechanism involves a conserved pro-segment motif with a cysteine residue that coordinates the catalytic zinc ion. Another family of metzincins, the astacins, do not possess a cysteine switch, so latency is maintained by other means. We have solved the high-resolution crystal structure of proastacin from the European crayfish, Astacus astacus. Its pro-segment is the shortest structurally reported for a metallopeptidase and it has a unique structure. It runs through the active-site cleft in reverse orientation to a genuine substrate. Moreover, a conserved aspartate, projected by a wide loop of the pro-segment, coordinates the zinc ion instead of the catalytic solvent molecule found in the mature enzyme. Activation occurs through two-step limited proteolysis and entails major rearrangement of a flexible activation domain, which becomes rigid and creates the base of the substrate-binding cleft. Maturation also requires the newly formed N-terminus to be precisely trimmed so that it can participate in a buried solvent-mediated hydrogen-bonding network, which includes an invariant active-site residue. We describe a novel mechanism for latency and activation, which shares some common features both with other metallopeptidases and with serine peptidases.

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On the relevance of the Met-turn methionine in metzincins.

Publication Date: 2010 Mar 4 PMID: 20202937
Authors: Tallant, C. - Garcia-Castellanos, R. - Baumann, U. - Gomis-Ruth, F. X.
Journal: J Biol Chem

The metzincins are a clan of metallopeptidases consisting of families that share a series of structural elements. Among them is the Met-turn, a tight 1,4-turn found directly below the zinc-binding site, which is structurally and spatially conserved and invariantly shows a methionine at position three in all metzincins identified. The reason for this conservation has been a matter of debate since its discovery. We have studied this structural element in Methanosarcina acetivorans ulilysin, the structural prototype of the pappalysin family, by generating ten mutants that replaced methionine with proteogenic amino acids. We compared recombinant overexpression yields, autolytic and tryptic activation, proteolytic activity, thermal stability, and three-dimensional structure with those of the wild type. All forms were soluble and could be purified, although with varying yields, and three variants underwent autolysis, could be activated by trypsin, and displayed significant proteolytic activity. All variants were analyzed for the thermal stability of their zymogens. None of the mutants analyzed proved more stable or active than the wild type. Both bulky and small side chains, as well as hydrophilic ones, showed diminished thermal stability. Two mutants, leucine and cysteine, crystallized and showed three dimensional structures that were indistinguishable from the wild type. These studies reveal that the Met-turn acts as a plug that snugly inserts laterally into a core structure created by the protein segment engaged in zinc binding and thus contributes to its structural integrity, which is indispensable for function. Replacement of the methionine with residues that deviate in size, side-chain conformation, and chemical properties impairs the plug-core interaction and prejudices molecular stability and activity.

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Funtional and kinetic analysis of the phosphotransferase CapP conferring selective self-resistance to capuramycin antibiotics.

Publication Date: 2010 Mar 4 PMID: 20202936
Authors: Yang, Z. - Funabashi, M. - Nonaka, K. - Hosobuchi, M. - Shibata, T. - Pahari, P. - Van Lanen, S. G.
Journal: J Biol Chem

Capuramycin-related compounds, including A-500359s and A-503083s, are nucleoside antibiotics that inhibit the enzyme bacterial translocase I (MraY) involved in peptidoglycan cell wall biosynthesis. Within the biosynthetic gene cluster for the A-500359s exists a gene encoding a putative aminoglycoside 3-phosphotransferase (APH) that was previously demonstrated to be highly expressed during the production of A-500359s and confers selective resistance to capuramycins when expressed in heterologous hosts. A similar gene (capP) was identified within the biosynthetic gene cluster for the A-503083s, and CapP is now shown to similarly confer selective resistance to capuramycins. Recombinant CapP was produced and purified from Escherichia coli and the function of CapP is established as an ATP-dependent capuramycin phosphotransferase that regio-specifically transfers the gamma-phosphate to the 3"-hydroxyl of the unsaturated hexuronic acid moiety of A-503083 B. Kinetic analysis with the three major A-503083 congeners suggests CapP preferentially phosphorylates A-503083s containing an aminocaprolactam moiety attached to the hexuronic acid, and bi-substrate kinetic analysis was consistent with CapP employing a sequential kinetic mechanism similar to most known APHs. The purified CapP product lost its antibiotic activity against Mycobacterium smegmatis, and this loss in bioactivity is primarily due to a 272-fold increase in the IC50 in the MraY-catalyzed reaction. The results establish CapP-mediated phosphorylation as a mechanism of resistance to capuramycins, and now sets the stage to explore this strategy of resistance as a potential mechanism inherent to pathogens and provides the impetus for preparing second generation analogues as a preemptive strike to such resistance strategies.

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Tight coupling of partial reactions in the acetyl-CoA decarbonylase/synthase (ACDS) multienzyme complex from Methanosarcina thermophila: Acetyl C-C bond fragmentation at the A cluster promoted by protein conformational changes.

Publication Date: 2010 Mar 4 PMID: 20202935
Authors: Gencic, S. - Duin, E. C. - Grahame, D. A.
Journal: J Biol Chem

Direct synthesis and cleavage of acetyl-CoA is carried out by the bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) enzyme in anaerobic bacteria, and by the acetyl-CoA decarbonylase/synthase(ACDS) multienzyme complex in Archaea. In both systems, a Ni- and Fe/S-containing active site metal center, the A cluster, catalyzes acetyl C-C bond formation/breakdown. Carbonyl group exchange of [1-(14)C]acetyl-CoA with unlabeled CO, a hallmark of CODH/ACS, is weakly active in ACDS, and exchange with CO(2) was up to 350 times faster, indicating tight coupling of CO release at the A cluster to CO oxidation to CO(2) at the C cluster in CO dehydrogenase. The basis for tight coupling was investigated by analysis of three recombinant A cluster proteins, ACDS beta subunit from Methanosarcina thermophila, acetyl-CoA synthase of Carboxydothermus hydrogenoformans (ACS(Ch)), and truncated ACS(Ch) lacking its 317 aa N-terminal domain. A comparison of acetyl-CoA synthesis kinetics, CO exchange, acetyltransferase, and A cluster Ni(1+)-CO EPR characteristics demonstrated a direct role of the ACS N-terminal domain in promoting acetyl C-C bond fragmentation. Protein conformational changes, related to "open/closed" states previously identified crystallographically, were indicated to have direct effects on the coordination geometry and stability of the A cluster Ni(2+)-acetyl intermediate, controlling Ni(2+)-acetyl fragmentation and Ni(2+)(CO)(CH(3)) condensation. EPR spectral changes likely reflect variations in the Ni(1+)-CO equatorial coordination environment in closed buried-hydrophobic, and open solvent-exposed states. The involvement of subunit-subunit interactions in ACDS, versus interdomain contacts in ACS, ensures that CO is not released from the ACDS beta subunit in the absence of appropriate interactions with the alpha(2)epsilon(2) CO dehydrogenase component. The resultant high efficiency CO transfer explains the low rate of CO exchange relative to CO(2).

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Formation of heteromeric Kv2 channels in mammalian brain neurons.

Publication Date: 2010 Mar 4 PMID: 20202934
Authors: Kihira, Y. - Hermanstyne, T. O. - Misonou, H.
Journal: J Biol Chem

The formation of heteromeric tetramers is a common feature of voltage-gated potassium (Kv) channels. This results in the generation of a variety of tetrameric Kv channels, which exhibit distinct biophysical and biochemical characteristics. Kv2 delayed rectifier channels, however, are unique exceptions. It has previously shown that mammalian Kv2.1 and Kv2.2 are localized in distinct domains of neuronal membrane and are not capable of forming heteromeric channels each other. In the present paper, we report a novel form of rat Kv2.2, Kv2.2long, which have not previously been recognized. Our data indicate that Kv2.2long is the predominant form of Kv2.2 expressed in cortical pyramidal neurons. We also found, in contrast to the previous findings, that rat Kv2.1 and Kv2.2long are colocalized in the somata and the proximal dendrites of cortical pyramidal neurons and are capable of forming functional heteromeric delayed rectifier channels. Our results suggest that the delayed rectifier currents, which regulate action potential firing, are encoded by heteromeric Kv2 channels in cortical neurons.

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Novel mode of phosphorylation-triggered reorganization of the nuclear lamina during nuclear egress of human cytomegalovirus.

Publication Date: 2010 Mar 4 PMID: 20202933
Authors: Milbradt, J. - Webel, R. - Auerochs, S. - Sticht, H. - Marschall, M.
Journal: J Biol Chem

The nucleo-cytoplasmic egress of viral capsids is a rate-limiting step in the replication of the human cytomegalovirus (HCMV). As reported recently, an HCMV-specific nuclear egress complex (NEC) is composed of viral and cellular proteins, in particular protein kinases with the capacity to induce destabilization of the nuclear lamina. Viral protein kinase pUL97 and cellular PKC hereby play important roles by phosphorylating several types of nuclear lamins. By using pUL97 mutants we show that the lamin-phosphorylating activity of pUL97 is associated with a reorganization of nuclear lamin A/C. Either pUL97 or PKC has the potential to induce distinct, punctate lamina-depleted areas at the periphery of the nuclear envelope, which were detectable in transiently transfected and HCMV-infected cells. Using recombinant HCMV that produces GFP-labeled viral capsids, the direct transition of viral capsids through these areas could be visualized. This process was sensitive to an inhibitor of pUL97/PKC activity. The pUL97-mediated phosphorylation of lamin A/C at serine 22 generated a novel binding motif for the peptidyl-prolyl cis/trans isomerase (PPIase) Pin1. In HCMV-infected fibroblasts, the physiological localization of Pin1 was altered, leading to recruitment of Pin1 to viral replication centers and to the nuclear lamina. The local increase of Pin1 PPIase activity may promote conformational modulation of lamins. Thus, we postulate a novel phosphorylation-triggered mechanism for the reorganization of the nuclear lamina in HCMV-infected cells.

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An intersubunit interaction between S4-S5 linker and S6 is responsible for the slow off-gating component in shaker K+ channels.

Publication Date: 2010 Mar 4 PMID: 20202932
Authors: Batulan, Z. - Haddad, G. A. - Blunck, R.
Journal: J Biol Chem

Voltage-gated ion channels are controlled by the membrane potential, which is sensed by peripheral, positively charged voltage sensors. The movement of the charged residues in the voltage sensor may be detected as gating currents. In Shaker K+ channels, the gating currents are asymmetric; while the ON-gating currents are fast, the OFF-gating currents contain a slow component. This slow component is caused by a stabilization of the activated state of the voltage sensor and has been suggested to be linked to ion permeation or C-type inactivation. The molecular determinants responsible for the stabilization, however, remain unknown. Here, we identified an interaction between R394, E395 and L398 on the C-termini of the S4-S5 linker and Y485 on the S6 of neighboring subunit, which is responsible for the development of the slow OFF-gating component. Mutation of residues involved in this intersubunit interaction modulated the strength of the associated interaction. Impairment of the interaction still led to pore opening but did not exhibit slow gating kinetics. Development of this interaction occurs under physiological ion conduction and is correlated with pore opening. We, thus, suggest that the above residues stabilize the channel in the open state.

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Communication between tandem cAMP-binding domains in the regulatory subunit of protein kinase A (PKA)-I{alpha} as revealed by domain-silencing mutations.

Publication Date: 2010 Mar 4 PMID: 20202931
Authors: McNicholl, E. T. - Das, R. - Sildas, S. - Taylor, S. S. - Melacini, G.
Journal: J Biol Chem

Protein kinase A (PKA) is the main receptor for the universal cAMP second messenger. PKA is a tetramer with two catalytic (C) and two regulatory (R) subunits, each including two tandem cAMP-binding domains, i.e. CBD-A and -B. Structural investigations of RIalpha have revealed that while CBD-A plays a pivotal role in the cAMP-dependent inhibition of C, the main function of CBD-B is to regulate the access of cAMP to site A. In order to further understand the mechanism underlying the cross-talk between CBD-A and -B, we report here the NMR investigation of a construct of R, RIalpha (119-379), which unlike previous fragments characterized by NMR, spans in full both CBDs. Our NMR studies were also extended to two mutants, R209K and the corresponding R333K, which severely reduce the affinity of cAMP for CBD-A and -B, respectively. The comparative NMR analysis of wild-type RIalpha (119-379) and of the two domain silencing mutations has led to the definition at an unprecedented level of detail of both intra- and inter-domain allosteric networks, revealing several striking differences between the two CBDs. First, the two domains, although homologous in sequence and structure, exhibit remarkably different responses to the R/K mutations especially at the beta2-3 allosteric "hot-spot". Second, while the two CBDs are reciprocally coupled at the level of local unfolding of the hinge, the A-to-B and B-to-A pathways are dramatically asymmetrical at the level of global unfolding. Such an asymmetric inter-domain cross-talk ensures efficiency and robustness in both the activation and de-activation of PKA.

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Mapping of domains on HIV envelope protein mediating association with calnexin and protein disulfide isomerase.

Publication Date: 2010 Mar 4 PMID: 20202930
Authors: Papandreou, M. J. - Barbouche, R. - Guieu, R. - Rivera, S. - Fantini, J. - Khrestchatisky, M. - Jones, I. M. - Fenouillet, E.
Journal: J Biol Chem

The cell-catalysts calnexin (CNX) and protein disulfide-isomerase (PDI) cooperate in establishing the disulfide bonding of the HIV envelope glycoprotein. Following HIV-binding to lymphocytes, cell-surface PDI also reduces Env to induce the fusogenic conformation. We sought to define the contact points between Env and these catalysts to illustrate their potential as therapeutic targets. In lysates of Env-expressing cells, 15% of the gp160 precursor, but not gp120, coprecipitated with CNX while only 0.25% of gp160 and gp120 coprecipitated with PDI. Under in-vitro conditions which mimic the Env/PDI interaction during virus/cell contact, PDI readily associated with Env. The domains of Env interacting in-cellulo with CNX or in-vitro with PDI were then determined using anti-Env antibodies whose binding site was occluded by CNX or PDI. Antibodies against domains V1/V2, C2 and the C-terminus of V3 did not bind CNX-associated Env while those against C1, V1/V2 and the CD4-binding domain did not react with PDI-associated Env. In addition, a mixture of the latter antibodies interfered with PDI-mediated Env reduction. Thus, Env interacts with intracellular CNX and extracellular PDI via discreet, largely non-overlapping, regions. The sites of interaction explain the mode of action of compounds that target these two catalysts and may enable the design of further new competitive agents.

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Lysosomal degradation of {alpha}-synuclein in vivo.

Publication Date: 2010 Mar 3 PMID: 20200163
Authors: Mak, S. K. - McCormack, A. L. - Manning-Bog, A. B. - Cuervo, A. M. - Di Monte, D. A.
Journal: J Biol Chem

Pathologic accumulation of alpha-synuclein is a feature of human parkinsonism and other neurodegenerative diseases. This accumulation may be counteracted by mechanisms of protein degradation that have been investigated in vitro but remain to be elucidated in animal models. In this study, lysosomal clearance of alpha-synuclein in vivo was indicated by the detection of alpha-synuclein in the lumen of lysosomes isolated from the mouse midbrain. When neuronal alpha-synuclein expression was enhanced as a result of toxic injury (i.e. treatment of mice with the herbicide paraquat) or transgenic protein overexpression, the intralysosomal content of alpha-synuclein was also significantly increased. This effect was paralleled by a marked elevation of the lysosome-associated membrane protein type 2A (LAMP-2A) and the lysosomal heat shock cognate protein of 70 kDa (hsc70), two essential components of chaperone-mediated autophagy (CMA). Immunofluorescence microscopy revealed an increase in punctate (lysosomal) LAMP-2A staining that co-localized with alpha-synuclein within nigral dopaminergic neurons of paraquat-treated and alpha-synuclein overexpressing animals. The data provide in vivo evidence of lysosomal degradation of alpha-synuclein under normal conditions and, quite importantly, under conditions of enhanced protein burden. In the latter, increased lysosomal clearance of alpha-synuclein was mediated, at least in part, by CMA induction. It is conceivable that these neuronal mechanisms of protein clearance play an important role in neurodegenerative processes characterized by abnormal alpha-synuclein buildup.

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G{alpha}q acts as an adaptor protein in PKC{zeta}-mediated ERK5 activation by GPCR.

Publication Date: 2010 Mar 3 PMID: 20200162
Authors: Garcia-Hoz, C. - Sanchez-Fernandez, G. - Diaz-Meco, M. T. - Moscat, J. - Mayor, F. - Ribas, C.
Journal: J Biol Chem

Gq-coupled G protein-coupled receptors (GPCR) mediate the actions of a variety of messengers that are key regulators of different cellular functions. These receptors can regulate a highly interconnected network of biochemical routes that control the activity of several members of the mitogen-activated protein kinase (MAPK) family. The ERK5 MAPK has been shown to be activated by Gq-coupled GPCR via unknown mechanisms. We find that the atypical protein kinase C PKCzeta, previously reported to interact with the ERK5 activator MEK5 and to be involved in EGF-mediated ERK5 stimulation, plays a crucial role in the activation of the ERK5 pathway by Gq-coupled GPCR. Stimulation of ERK5 by Gq-coupled GPCR is abolished upon pharmacological inhibition of PKCzeta as well as in embryonic fibroblasts obtained from PKCzeta-deficient mice. Both PKCzeta and MEK5 associate to Galphaq upon GPCR activation thus forming a ternary complex that seems essential for the activation of ERK5. These data put forward a novel function of Galphaq as a scaffold protein involved in the modulation of the ERK5 cascade by GPCR that could be relevant in Gq-mediated physiological functions.

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Stromal hyaluronan interaction with epithelial CD44variants promotes prostate cancer invasiveness by augmenting expression and function of HGF and androgen receptor.

Publication Date: 2010 Mar 3 PMID: 20200161
Authors: Ghatak, S. - Hascall, V. C. - Markwald, R. R. - Misra, S.
Journal: J Biol Chem

The main aim of our study is to determine the significance of the stromal microenvironment in the malignant behavior of prostate cancer. The stromal-derived growth factors/cytokines and hyaluronan act in autocrine/paracrine ways with their receptors, including receptor-tyrosine kinases and CD44-variants (CD44v), to potentiate and support tumor epithelial cell survival. Overexpression of hyaluronan, CD44v9 variants, and stromal-derived growth factors/cytokines are specific features in many cancers including prostate cancer. Androgen/androgen receptor interaction has a critical role in regulating prostate cancer growth. Our previous study showed 1) that increased synthesis of hyaluronan in normal epithelial cells promotes expression of CD44 variats; 2) that hyaluronan interaction with CD44v6-v9 promotes activation of receptor-tyrosine kinase, which stimulates PI3kinase-induced cell survival pathways, and 3) that CD44v6/short-hairpin-RNA reduces colon tumor growth in vivo. Our results now show that hepatocyte growth factor synthesized by myofibroblasts associated with prostate cancer cells induces activation of HGF-receptor/cMet and stimulates hyaluronan/CD44v9 signaling. This, in turn, stabilizes the androgen receptor functions in prostate cancer cells. The stromal-derived HGF induces a lipid raft-associated signaling complex that contains CD44v9, cMet/PI3Kinase, HSP90 and androgen receptor. CD44v9/short-hairpin-RNA reverses the assembly of these components in the complex and inhibits androgen receptor function. Our results provide new insight into the hyaluronan/CD44v9 regulated androgen receptor function and the consequent malignant activities in prostate cancer cells. The present study describes a physiologically relevant in vitro model for studying the molecular mechanisms by which stromal-derived HGF and hyaluronan influence androgen receptor and CD44 functions in the secretory epithelia during prostate carcinogenesis.

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Down-regulation of PROS1 gene expression by 17{beta}-estradiol via estrogen receptor {alpha} (ER{alpha})/Sp1 interaction recruiting receptor interacting protein 140 and corepressors-HDAC3 complex.

Publication Date: 2010 Mar 3 PMID: 20200160
Authors: Suzuki, A. - Sanda, N. - Miyawaki, Y. - Fujimori, Y. - Yamada, T. - Takagi, A. - Murate, T. - Saito, H. - Kojima, T.
Journal: J Biol Chem

Pregnant women show a low level of protein S (PS) in plasma, which is known to be a risk factor for deep venous thrombosis (DVT). 17beta-estradiol (E2), an estrogen whose concentration increases in the late stages of pregnancy, regulates the expression of various genes via the estrogen receptor (ER). Here, we investigated the molecular mechanisms behind the reduction in PS levels caused by E2 in HepG2-ERalpha cells, which stably express ERalpha, and thus the genomic ER signaling pathway modulating the ligand-dependent repression of the PSalpha gene (PROS1). We observed that E2 repressed the production of mRNA and antigen of PS. A luciferase-reporter assay revealed that E2 down-regulated the PROS1 promoter's activity, and that this E2-dependent repression disappeared on the deletion or mutation of two adjacent GC-rich motifs in the promoter. An electrophoretic mobility shift assay and DNA pull-down assay revealed that the GC-rich motifs were associated with Sp1, Sp3 and ERalpha. In a chromatin immunoprecipitation assay, we found ERalpha/Sp protein-promoter interaction to be involved in the E2-dependent repression of PROS1 transcription. Furthermore, we demonstrated that E2 treatment recruited receptor interacting protein 140 (RIP140) and nuclear receptor corepressor (NCoR)/silencing mediator of retinoid and thyroid hormone receptors (SMRT)-histone deacetylase 3 (HDAC3) complex to the PROS1 promoter, which would hypo-acetylate chromatin. Taken together, it was suggested that E2 might repress PROS1 transcription depending upon ERalpha/Sp1 recruiting transcriptional repressors in HepG2-ERalpha cells, and consequently that high levels of E2 leading to reduced levels of plasma PS would be a risk factor for DVT in pregnant women.

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The lammer kinase homolog, Lkh1, regulates Tup transcriptional repressors through phosphorylation in schizosaccharomyces pombe.

Publication Date: 2010 Mar 3 PMID: 20200159
Authors: Kang, W. H. - Park, Y. H. - Park, H. M.
Journal: J Biol Chem

Disruption of the fission yeast LAMMER kinase, Lkh1, gene resulted in diverse phenotypes including adhesive filamentous growth and oxidative stress sensitivity, but an exact cellular function had not been assigned to Lkh1. Through an in vitro pull-down approach, a transcriptional repressor, Tup12, was identified as an Lkh1 binding partner. Interactions between Lkh1 and Tup11 or Tup12 were confirmed by in vitro and in vivo binding assays. Tup proteins were phosphorylated by Lkh1 in a LAMMER motif dependent manner. The LAMMER motif was also necessary for substrate recognition in vitro, and cellular function in vivo. Transcriptional activity assays using promoters negatively regulated by Tup11 and Tup12, showed six- or two-times higher activity in the Deltalkh1 mutant than the wild-type, respectively. Northern analysis revealed de-repressed expression of the fbp1(+) mRNA in Deltalkh1 and in Deltatup11Deltatup12 mutant cells under repressed conditions. Deltalkh1 and Deltatup11Deltatup12 mutant cells showed flocculation, which was reversed by co-expression of Tup11 and 12 with Ssn6. Here, we presented a new aspect of the LAMMER kinase by demonstrating that the activity of global transcriptional repressors, Tup11 and Tup12, were positively regulated by Lkh1-mediated phosphorylation.

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Order and disorder in the domain organisation of the plasmid partition protein KorB.

Publication Date: 2010 Mar 3 PMID: 20200158
Authors: Rajasekar, K. - Tul Muntaha, S. - Tame, J. R. - Kommareddy, S. - Morris, G. - Wharton, C. W. - Thomas, C. M. - White, S. A. - Hyde, E. I. - Scott, D. J.
Journal: J Biol Chem

The plasmid partition protein KorB has a dual role: it is essential for the correct segregation of the low copy number broad host range RK2 plasmid, while also being an important regulator of transcription. KorB belongs to the ParB family of proteins and partitioning in RK2 has been studied as a simplified model of bacterial chromosome segregation. Structural information on full-length ParB proteins is limited, mainly due to the inability to grow crystals suitable for diffraction studies. We show, using CD and NMR, that KorB has regions of significant intrinsic disorder and hence it adopts a multiplicity of conformations in solution. The biophysical data are consistent with bioinformatic predictions based on the amino acid sequence that the N-terminal region and also the region between the central DNA-binding domain and the C-terminal dimerisation domain are intrinsically disordered. We have used small angle X-ray scattering data (SAXS) to determine the ensemble of solution conformations for KorB, and selected deletion mutants, based on models of the known domain structures. This conformational range of KorB is likely to be biologically required for DNA partitioning and for binding to diverse set of partner proteins.

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NMR analysis of the structure, dynamics, and unique oligomerization properties of the human chemokine CTACK/CCL27.

Publication Date: 2010 Mar 3 PMID: 20200157
Authors: Jansma, A. L. - Kirkpatrick, J. P. - Hsu, A. R. - Handel, T. M. - Nietlispach, D.
Journal: J Biol Chem

Chemokines have two essential interactions in vivo - with G protein-coupled receptors (GPCRs) which activate intracellular signaling pathways, and with glycosaminoglycans (GAGs) which are involved in cell surface localization and transport. While it has been shown that chemokines bind and activate their respective GPCRs as monomers, many chemokines oligomerize upon GAG-binding, and both the ability to oligomerize and to bind GAGs is required for in vivo function. In this study, we investigated the structure, dynamics and oligomerization behavior of Cutaneous T cell-Attracting ChemoKine (CTACK, a.k.a. CCL27) by NMR. 15N relaxation and translational self-diffusion rates indicate that CCL27 oligomerizes, but in contrast to many other chemokines that form relatively discrete oligomers, CCL27 transitions between monomer, dimer and tetramer species over a relatively narrow concentration range. A 3D structure determination was pursued under conditions where CCL27 is primarily dimeric, revealing the standard motif for a chemokine monomer. Analysis of chemical shift perturbations of 1H-15N HSQC spectra, relaxation-dispersion experiments and filtered NOEs suggest that CCL27 does not adopt a discrete CXC or CC dimer motif. Instead, CCL27 has uncommon oligomerization behavior, where several equilibria involving relatively low affinity interactions between different interfaces seem to be simultaneously at work. However, interaction with heparin avidly promotes oligomerization under conditions where CCL27 is monomeric by itself. We hypothesize that the plasticity in the oligomerization state may enable CCL27 to adopt different oligomeric structures, depending on the nature of the GAG binding partner, thereby providing a mechanism for increased diversity and specificity in GAG-binding and GAG-related functions.

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Insights into regulated ligand binding sites from the structure of ZO-1 SH3-guanylate kinase module.

Publication Date: 2010 Mar 3 PMID: 20200156
Authors: Lye, M. F. - Fanning, A. S. - Su, Y. - Anderson, J. M. - Lavie, A.
Journal: J Biol Chem

Tight junctions are dynamic components of epithelial and endothelial cells that regulate the paracellular transport of ions, solutes and immune cells. The assembly and permeability of these junctions is dependent on the Zonula Occludens (ZO) proteins, members of the MAGUK protein family, which are characterized by a core SH3-GUK module that coordinates multiple protein-protein interactions. The structure of the ZO-1 SH3-GUK domain confirms that the interdependent folding of the SH3 and GUK domains is a conserved feature of MAGUKs, but differences in the orientation of the GUK domains in three different MAGUKs reveal inter-domain flexibility of the core unit. Using pull down assays we show that an effector loop, the U6 region in ZO-1, forms a novel intramolecular interaction with the core module. This interaction is divalent-cation dependant and overlaps with the binding site for the regulatory molecule calmodulin on the GUK domain. These findings provide insight into the previously observed ability of U6 region to regulate TJ assembly in vivo, and the structural basis for the complex protein interactions of the MAGUK family.

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Analysis of a TIR-less splice variant of TRIF reveals an unexpected mechanism of TLR3-mediated signaling.

Publication Date: 2010 Mar 3 PMID: 20200155
Authors: Han, K. J. - Yang, Y. - Xu, L. G. - Shu, H. B.
Journal: J Biol Chem

Recognition of viral RNA by Toll-like receptor 3 (TLR3) triggers activation of the transcription factors NF-kappaB and IRF3 and induction of type I interferons (IFNs). TRIF is a TIR domain containing adapter protein critically involved in TLR3-mediated signaling. It has been shown that TRIF interacts with TLR3 through their respective TIR domains. In this report, we identified a splice variant of TRIF lacking the TIR domain, which is designated as TRIS. Overexpression of TRIS activates NF-kappaB, ISRE as well as the IFN-beta promoter, whereas knockdown of TRIS inhibited TLR3-mediated signaling, suggesting that TRIS is involved in TLR3-mediated signaling. Furthermore, we identified an N-terminal TBK1-binding motif of TRIS or TRIF that was important for its interaction with TBK1 and ability to activate ISRE. Activation of ISRE by TRIS also needs its dimerization or oligomerization mediated by its C-terminal RHIM motif. Finally, we demonstrated that TRIS was associated with TRIF upon TLR3 activation by poly(I:C). These findings reveal an unexpected mechanism of TLR3-mediated signaling.

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Molecular dynamics simulations show that conformational selection governs the binding preferences of imatinib for several tyrosine kinases.

Publication Date: 2010 Mar 3 PMID: 20200154
Authors: Aleksandrov, A. - Simonson, T.
Journal: J Biol Chem

Tyrosine kinases transmit cellular signals through a complex mechanism, involving their phosphorylation an switching between inactive and active conformations. The cancer drug imatinib binds tightly to several homologous kinases, including Abl, but weakly to others, including Src. Imatinib specifically targets Abl's inactive, so-called ;DFG-out' conformation, which differs from Src's preferred, ;DFG-in' conformation in the orientation of a conserved activation loop. However, recent Xray structures showed that Src can also adopt the DFG-out conformation and uses it to bind imatinib. The Src/Abl binding free energy difference can thus be decomposed into two contributions. Contribution (i) measures the different protein:imatinib interactions when either kinase is in its DFG-out conformation. Contribution (ii) depends on imatinib's ability to select or induce this conformation; i.e., on the relative stabilities of the DFG-out and DFG-in conformations of each kinase. Neither contribution has been measured experimentally. We use molecular dynamics simulations to show that (i) is very small, 0.2+/-0.6 kcal/mol: imatinib interactions are very similar in the two kinases-including long-range electrostatic interactions with the imatinib positive charge. Contribution (ii), deduced using the experimental binding free energy difference, is much larger: 4.4+/-0.9 kcal/mol. Thus, conformational selection, easy in Abl, difficult in Src, underpins imatinib specificity. Contribution (ii) has a simple interpretation: it closely approximates the stability difference between the DFG-out and DFG-in conformations of apo-Src. Additional calculations show that conformational selection also governs the relative binding of imatinib to the kinases c-Kit and Lck. These results should help clarify the current framework for engineering kinase signalling.

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The thap-zinc finger protein thap1 associates with coactivator HCF-1 and O-GLcNAc transferase: A link between DYT6 and DYT3 dystonias.

Publication Date: 2010 Mar 3 PMID: 20200153
Authors: Mazars, R. - Gonzalez-de-Peredo, A. - Cayrol, C. - Lavigne, A. C. - Vogel, J. L. - Ortega, N. - Lacroix, C. - Gautier, V. - Huet, G. - Ray, A. - Monsarrat, B. - Kristie, T. M. - Girard, J. P.
Journal: J Biol Chem

THAP1 is a sequence-specific DNA-binding factor which regulates cell proliferation through modulation of target genes such as the cell cycle-specific gene RRM1. Mutations in the THAP1 DNA-binding domain, an atypical zinc finger (THAP-zf), have recently been found to cause DYT6 dystonia, a neurological disease characterized by twisting movements and abnormal postures. In this study, we report that THAP1 shares sequence characteristics, in vivo expression patterns and protein partners with THAP3, another THAP-zf protein. Proteomic analyses identified HCF-1, a potent transcriptional coactivator and cell cycle regulator, and O-GlcNAc transferase (OGT), the enzyme which catalyzes the addition of O-GlcNAc, as major cellular partners of THAP3. THAP3 interacts with HCF-1 through a consensus HCF-1-binding motif (HBM), a motif that is also present in THAP1. Accordingly, THAP1 was found to bind HCF-1 in vitro and to associate with HCF-1 and OGT in vivo. THAP1 and THAP3 belong to a large family of HCF-1-binding factors since seven other members of the human THAP-zf protein family were identified, which harbor evolutionary conserved HBMs and bind to HCF-1. Chromatin immunoprecipitation (ChIP) assays and RNA interference experiments showed that endogenous THAP1 mediates the recruitment of HCF-1 to the RRM1 promoter during endothelial cell proliferation and that HCF-1 is essential for transcriptional activation of RRM1. Together, our findings suggest HCF-1 is an important cofactor for THAP1. Interestingly, our results also provide an unexpected link between DYT6 and DYT3 (X-linked dystonia-parkinsonism) dystonias since the gene encoding the THAP1/DYT6 protein partner OGT maps within the DYT3 critical region on Xq13.1.

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A slow, tight-binding inhibitor of InhA, the enoyl-ACP reductase from Mycobacterium tuberculosis.

Publication Date: 2010 Mar 3 PMID: 20200152
Authors: Luckner, S. R. - Liu, N. - Am Ende, C. W. - Tonge, P. J. - Kisker, C.
Journal: J Biol Chem

InhA, the enoyl-ACP reductase in Mycobacterium tuberculosis is an attractive target for the development of novel drugs against tuberculosis, a disease that kills more than two million people each year. InhA is the target of the current first line drug isoniazid (INH) for the treatment of tuberculosis infections. Compounds that directly target InhA and do not require activation by the mycobacterial catalase-peroxidase KatG are promising candidates for treating infections caused by INH resistant strains. Previously we reported the synthesis of several diphenyl ethers with nM affinity for InhA. However, these compounds are rapid reversible inhibitors of the enzyme, and based on the knowledge that long drug-target residence times are an important factor for in vivo drug activity, we set out to generate a slow onset inhibitor of InhA using structure based drug design. 2-(o-Tolyloxy)-5-hexylphenol (PT70) is a slow, tight binding inhibitor of InhA with a K(1) value of 22 pM. PT70 binds preferentially to the InhA NAD(+) complex and has a residence time of 24 minutes on the target, which is 14,000 times longer than that of the rapid-reversible inhibitor from which it is derived. The 1.8 A crystal structure of the ternary complex between InhA, NAD(+) and PT70 reveals the molecular details of enzyme-inhibitor recognition and supports the hypothesis that slow onset inhibition is coupled to ordering of an active site loop, which leads to the closure of the substrate binding pocket.

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Sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) transduces signals that mediate dopamine inhibition of sodium-phosphate co-transport in mouse kidney.

Publication Date: 2010 Mar 3 PMID: 20200151
Authors: Weinman, E. J. - Biswas, R. - Steplock, D. - Douglas, T. S. - Cunningham, R. - Shenolikar, S.
Journal: J Biol Chem

Dopamine inhibited phosphate transport in isolated renal brush border membrane (BBM) vesicles and in cultured renal proximal tubule cells from wild-type but not from NHERF-1 null mice. Co-immunoprecipitation experiments established that NHERF-1 associated with D1-like receptors. In wild-type mice, dopamine stimulated cAMP accumulation and PKC activity in renal proximal tubule cells, an effect that was abolished by SCH-23390, a D1-like receptor antagonist. In NHERF-1 null kidney tissue, however, dopamine failed to stimulate either cAMP accumulation or PKC activity. Infection of proximal tubule cells from NHERF-1 null mice with adenovirus-GFP-NHERF-1 restored the ability of dopamine to stimulate cAMP and PKC. Finally, in 32P-labeled wild-type proximal tubule cells and in OK cells, dopamine increased NHERF-1 phosphorylation at serine77 of the PDZ I domain of NHERF-1; a site previously shown to attenuate binding of cellular targets including the sodium-dependent phosphate transporter-2a (Npt2a). Together, these studies establish that NHERF-1 plays a key role in dopamine signaling and is also a downstream target of D1-like receptors in the mouse kidney. These studies suggest a novel role for the PDZ adapter protein NHERF-1 in coordinating dopamine signals that inhibit renal phosphate transport.

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Perception of the Arabidopsis danger signal peptide 1 involves the pattern recognition receptor AtPEPR1 and its close homologue AtPEPR2.

Publication Date: 2010 Mar 3 PMID: 20200150
Authors: Krol, E. - Mentzel, T. - Chinchilla, D. - Boller, T. - Felix, G. - Kemmerling, B. - Postel, S. - Arents, M. - Jeworutzki, E. - Al-Rasheid, K. A. - Becker, D. - Hedrich, R.
Journal: J Biol Chem

Plasma membrane-borne pattern recognition receptors (PRRs), which recognize microbe-associated molecular patterns (MAMPs) and endogenous damage-associated molecular patterns (DAMPs), provide the first line of defence in innate immunity. In plants, leucine-rich repeat (LRR) receptor kinases fulfil this role, as exemplified by FLS2 and EFR, the receptors for the MAMPs flagellin and elongation factor Tu. Here we examined the perception of the DAMP peptide 1 (AtPep1), an endogenous peptide of Arabidopsis identified earlier and shown to be perceived by the LRR protein kinase PEPR1. Using seedling growth inhibition, elicitation of an oxidative burst and induction of ethylene biosynthesis, we show that wild type plants and the pepr1 and pepr2 mutants, affected in PEPR1 and in its homologue PEPR2, are sensitive to AtPep1, but that the double mutant pepr1/pepr2 is completely insensitive. As a central body of our study, we provide electrophysiological evidence that at the level of the plasma membrane, AtPep1 triggers a receptor-dependent transient depolarization through activation of plasma membrane anion channels, and that this effect is absent in the double mutant pepr1/pepr2. The double mutant also fails to respond to AtPep2 and AtPep3, two distant homologues of AtPep1 on the basis of homology screening, implying that the PEPR1 and PEPR2 are responsible for their perception too. Our findings provide a basic framework to study the biological role of AtPep1-related danger signals and their cognate receptors.

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Engineered interfaces of an AAA+ ATPase reveal a new nucleotide-dependent coordination mechanism.

Publication Date: 2010 Mar 2 PMID: 20197281
Authors: Joly, N. - Buck, M.
Journal: J Biol Chem

Homo-hexameric ring AAA+ ATPases are found in all kingdoms of life and are involved in all cellular processes. To accommodate the large spectrum of substrates, the conserved AAA+ core has become specialised through the insertion of specific substrate binding motifs. Given their critical roles in cellular function, understanding the nucleotide driven mechanisms of action is of wide range importance. For one type member AAA+ protein (Phage Shock Protein F, PspF) we identified and established the functional significance of strategically placed arginine and glutamate residues that form interacting pairs in response to nucleotide binding. We show that these interactions are critical for cis and trans subunit communication which support coordination between subunits for nucleotide-dependent substrate remodelling. Using alleles specific suppression approach for ATPase and substrate remodelling we demonstrate that the targeted residues directly interact and are unlikely to make any other pair wise critical interactions. We then propose a mechanistic rational by which the nucleotide bound state of adjacent subunits can be sensed without direct involvement of R-finger. As the structural AAA+ core is conserved, we propose that the functional networks established here could serve as a template to identify similar residue pairs in other AAA+ proteins.

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Agonist-selective dynamic compartmentalization of human mu opioid receptor as revealed by resolutive frap analysis.

Publication Date: 2010 Mar 2 PMID: 20197280
Authors: Sauliere-Nzeh Ndong, A. - Millot, C. - Corbani, M. - Mazeres, S. - Lopez, A. - Salome, L.
Journal: J Biol Chem

Techniques for analyzing the membrane diffusion of molecules are the most promising methods for investigating the compartmentalization of G-protein-coupled receptors, particularly as relevant to receptor signaling processes. Here, we report fluorescence recovery after photobleaching (FRAP) measurements performed at variable spot radius for human mu opioid (hMOP) receptors on SH-SY5Y neuroblastoma cells in the presence of ligands. Although an antagonist did not affect the behaviour of the receptors as compared to the basal state, two different agonists, DAMGO and morphine, caused markedly different changes to receptor diffusion. Like receptors in the absence of ligand, receptors bound to morphine exhibited diffusion confined to joined semi-permeable domains, but with smaller domain size and diffusion coefficient. This effect was inhibited by pertussis toxin, strongly suggesting that this dynamic behavior is associated with early steps of signaling. In the presence of DAMGO, half of the receptors displayed free long-range diffusion and the other half were confined to smaller isolated domains. Hypertonic sucrose buffer suppressed this effect which we attribute to receptor entry into clathrin-coated pits. It is likely that the observation of distinct receptor dynamics in the presence of DAMGO and morphine involves the agonist-selective phosphorylation of the receptor.

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Novel components of an active mitochondrial K+/H+ exchange.

Publication Date: 2010 Mar 2 PMID: 20197279
Authors: Zotova, L. - Aleschko, M. - Sponder, G. - Baumgartner, R. - Reipert, S. - Prinz, M. - Schweyen, R. J. - Nowikovsky, K.
Journal: J Biol Chem

Defects of the mitochondrial K(+)/H(+) exchanger (KHE) result in increased matrix K(+) content, swelling and autophagic decay of the organelle. We have identified the yeast Mdm38 and its human homologue LETM1, the candidate gene for seizures in Wolf-Hirschhorn Syndrom, as essential components of the KHE. In a genome-wide screen for multi-copy suppressors of the pet(-) (reduced growth on non-fermentable substrate) phenotype of mdm38Delta mutants, we now characterized the mitochondrial carriers PIC2 and MRS3 as moderate, and MRS7 and YDL183c as strong suppressors. Like Mdm38p, Mrs7p and Ydl183cp are mitochondrial inner-membrane proteins and constituents of approximately 500 kDa protein complexes. Triple mutant strains (mdm38Delta mrs7Delta ydl183cDelta) exhibit a remarkably stronger pet(-) phenotype than mdm38Delta and a general growth reduction. They totally lack KHE activity, show a dramatic drop of mitochondrial membrane potential and heavy fragmentation of mitochondria and vacuoles. Nigericin, an ionophore with KHE activity, fully restores growth of the triple mutant, indicating that loss of KHE activity is the underlying cause of its phenotype. Mdm38p or overexpression of Mrs7p, Ydl183cp or LETM1 in the triple mutant rescues growth and KHE activity. A LETM1 human homologue, HCCR-1/LETMD1, described as an oncogene, partially suppresses the yeast triple mutant phenotype. Accordingly, we propose that Ydl183p and the Mdm38 homologues Mrs7, LETM1 and HCCR-1 are involved in the formation of an active KHE system.

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Allosteric communication between cAMP binding sites in the RI subunit of PKA revealed by NMR.

Publication Date: 2010 Mar 2 PMID: 20197278
Authors: Byeon, I. J. - Dao, K. K. - Jung, J. - Keen, J. - Leiros, I. - Doskeland, S. O. - Martinez, A. - Gronenborn, A. M.
Journal: J Biol Chem

The activation of protein kinase A involves the synergistic binding of cAMP to two cAMP binding sites on the inhibitory R subunit, causing release of the C subunit, which subsequently can carry out catalysis. We used NMR to structurally characterize in solution the RIalpha(98-381) subunit, a construct comprising both cyclic nucleotide binding (CNB) domains, in the presence and absence of cAMP and map the effects of cAMP binding at single residue resolution. Several conformationally disordered regions in free RIalpha become structured upon cAMP binding, including the interdomain alphaC:A and alphaC':A helices that connect CNB domains A and B and are primary recognition sites for the C-subunit. NMR titration experiments with cAMP, B-site selective 2-Cl-8-hexylamino-cAMP and A-site selective N6-monobutyryl-cAMP revealed that cyclic nucleotide binding to either the B- or A site affected the interdomain helices. The NMR resonances of this interdomain region exhibited chemical shift changes upon ligand binding to a single site, either site B or A, with additional changes occurring upon binding to both sites. Such distinct, stepwise conformational changes in this region reflect the synergistic interplay between the two sites, and may underlie the positive cooperativity of cAMP activation of the kinase. Furthermore, nucleotide binding to the A-site also affected residues within the B domain. The present NMR study provides the first structural evidence of unidirectional allosteric communication between the sites. Trp262, which lines the CNB A site, but resides in the sequence of domain B, is an important structural determinant for inter-site communication.

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Deficiency of suppressor enhancer lin12 1 like (SEL1L) in mice leads to systemic endoplasmic reticulum stress and embryonic lethality.

Publication Date: 2010 Mar 2 PMID: 20197277
Authors: Francisco, A. B. - Singh, R. - Li, S. - Vani, A. K. - Yang, L. - Munroe, R. J. - Diaferia, G. - Cardano, M. - Biunno, I. - Qi, L. - Schimenti, J. C. - Long, Q.
Journal: J Biol Chem

Stress in the endoplasmic reticulum (ER) plays an important causal role in the pathogenesis of several chronic diseases such as Alzheimer's disease, Parkinson's disease and diabetes mellitus. Insight into the genetic determinants responsible for ER homeostasis will greatly facilitate the development of therapeutic strategies for the treatment of these debilitating diseases. Suppressor Enhancer Lin12 1 Like (SEL1L) is an ER membrane protein and was thought to be involved in the quality control of secreted proteins. Here we show that mice homozygous mutant for SEL1L were embryonic lethal. Electron microscopy studies revealed a severely dilated ER in the fetal liver of mutant embryos, indicative of alteration in ER homeostasis. Consistent with this, several ER stress responsive genes were significantly up-regulated in the mutant embryos. Mouse embryonic fibroblast cells deficient in SEL1L exhibited activated unfolded protein response at the basal state, impaired ER-associated protein degradation and reduced protein secretion. Furthermore, markedly increased apoptosis was observed in the forebrain and dorsal root ganglions of mutant embryos. Taken together, our results demonstrate an essential role for SEL1L in protein quality control during mouse embryonic development.

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Low density lipoprotein receptor-related protein (LRP1) regulates Rac1 and RhoA reciprocally to control Schwann cell adhesion and migration.

Publication Date: 2010 Mar 2 PMID: 20197276
Authors: Mantuano, E. - Jo, M. - Gonias, S. L. - Campana, W. M.
Journal: J Biol Chem

LDL receptor-related protein (LRP1) is expressed by Schwann cells in vivo mainly after injury to the peripheral nervous system (PNS). Schwann cells in primary culture, which provide a model of Schwann cells in the injured PNS, also express abundant LRP1. Herein, we show that LRP1 gene-silencing or treatment with receptor-associated protein (RAP) promotes Schwann cell adhesion and inhibits cell migration on fibronectin. LRP1 gene silencing also resulted in the formation of prominent focal adhesions and actin stress fibers. These changes, which were induced by loss of LRP1 expression or activity, were explained mechanistically by an increase in activated RhoA, coupled with a decrease in activated Rac1. Known LRP1 ligands, including matrix metalloprotease-9, tissue-type plasminogen activator, and alpha2-macroglobulin activated Rac1 in LRP1-expressing Schwann cells. An inhibitor of Rac1 activation promoted Schwann cell adhesion. Conversely, in cells in which LRP1 was silenced, a Rho kinase inhibitor promoted migration and inhibited adhesion. These results demonstrate that direct binding of ligands to LRP1 controls activation of small Rho family GTPases. The effects of LRP1 gene-silencing and RAP implicate autocrine pathways involving endogenously-produced LRP1 ligands. Regulation of Schwann cell migration by LRP1 may be important in PNS injury.

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Conformational changes of the HIV-1 envelope protein during membrane fusion were inhibited by the replacement of its membrane-spanning domain.

Publication Date: 2010 Mar 2 PMID: 20197275
Authors: Kondo, N. - Miyauchi, K. - Meng, F. - Iwamoto, A. - Matsuda, Z.
Journal: J Biol Chem

To help understand the dynamic nature of membrane fusion induced by the HIV-1 envelope protein, we developed a new cell-based real-time assay system employing a pair of novel reporter proteins. The reporter proteins consist of a pair of split Renilla luciferase (spRL) fused to split green fluorescent protein (spGFP). The spGFP modules were chosen not only to compensate weak self-association of spRL, but also to provide visual reporter signals during membrane fusion. Use of this reporter together with a membrane permeable substrate for RL achieved a simple real-time monitoring of membrane fusion using live cells. We analyzed the HIV-1 envelope mutants whose membrane-spanning domains (MSD) were replaced with that of glycophorin A or vesicular stomatitis virus G -protein. These mutants showed a slower kinetics of membrane fusion. The analysis of membrane fusion in the presence of fusion inhibitors, soluble CD4 and C34, revealed that these replacements prolonged the period during which the mutants were sensitive to the inhibitors, as compared with the wild type. These results suggest that the mutations within the MSD exerted an allosteric effect on the HIV-1 envelope protein, probably affecting the receptor-induced conformational changes of the ectodomain of the protein.

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Topological location and structural importance of the NBCe1-A residues mutated in pRTA.

Publication Date: 2010 Mar 2 PMID: 20197274
Authors: Zhu, Q. - Kao, L. - Azimov, R. - Newman, D. - Liu, W. - Pushkin, A. - Abuladze, N. - Kurtz, I.
Journal: J Biol Chem

NBCe1-A electrogenically cotransports Na+ and HCO3- across the basolateral membrane of renal proximal tubule cells. Eight missense mutations and 3 nonsense mutations in NBCe1-A cause severe proximal renal tubular acidosis (pRTA). In this study, the topologic properties and structural importance of the 8 endogenous residues mutated in pRTA and the in situ topology of NBCe1-A were examined by the substituted cysteine accessibility method. Of the 55 analyzed individually introduced cysteines, 8 were labeled with both membrane permeant (biotin maleimide (BM)) and impermeant (MTS-TAMRA) sulfhydryl reagents, 4 with only BM, and 3 with only MTS-TAMRA. The location of the labeled and unlabeled introduced cysteines clearly indicates that the transmembrane region of NBCe1-A contains 14 transmembrane segments (TMs). In this in situ based NBCe1-A topology, residues mutated in pRTA (pRTA residues) are assigned as: Ser-427, TM1; Thr-485 and Gly-486, TM3; Arg-510 and Leu-522, TM4; Ala-799, TM10 and Arg-881, TM12. Substitution of pRTA residues with cysteines impaired the membrane trafficking of R510C and R881C, the remaining membrane processed constructs had various impaired transport function. Surprisingly, none of the membrane-processed constructs was accessible to labeling with BM and MTS-TAMRA, nor were they functionally sensitive to the inhibition by MTSEA. Functional analysis of Thr-485 with different amino acid substitutions indicated it resides in a unique region important for NBCe1-A function. These findings demonstrate that the pRTA residues in NBCe1-A are buried in the protein complex/lipid bilayer where they perform important structural roles.

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The IBB-domain modulates the avidity of importin {beta} for the nuclear pore complex.

Publication Date: 2010 Mar 1 PMID: 20197273
Authors: Lott, K. - Bhardwaj, A. - Mitrousis, G. - Pante, N. - Cingolani, G.
Journal: J Biol Chem

Importin beta mediates active passage of cellular substrates through the nuclear pore complex (NPC). Adaptors such as importin alpha and snurportin (SNP1) associate with importin beta via an importin beta binding (IBB)-domain. The intrinsic structural flexibility of importin beta allows its concerted interactions with IBB-domains, phenylalanine-glycine nucleoporins (FG-nups) and the GTPase Ran during transport. In this paper, we provide evidences that the nature of the IBB-domain modulates the affinity of the import complex for the NPC. In permeabilized cells, importin beta imports a cargo fused to the snurportin IBB (sIBB) with ~70% reduced energy requirement as compared to the classical importin alpha IBB (alphaIBB). At the molecular level, this is explained by ~200-fold reduced affinity of importin beta for Nup62, when bound to the sIBB. Consistently, in vivo, the importin beta:sIBB complex has greatly reduced persistence inside the central channel of the NPC. We propose that by controlling the degree of strain in the tertiary structure of importin beta, the IBB-domain modulates the affinity of the import complex for nups, thus dictating its persistence inside the NPC.

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Activation of cAMP-dependent signaling induces oxidative modification of the cardiac Na+-K+ pump and inhibits its activity.

Publication Date: 2010 Mar 1 PMID: 20194511
Authors: White, C. N. - Liu, C. C. - Garcia, A. - Hamilton, E. J. - Chia, K. K. - Figtree, G. A. - Rasmussen, H. H.
Journal: J Biol Chem

Cellular signaling can inhibit the membrane Na-K pump via protein kinase C (PKC)-dependent activation of NADPH oxidase and a downstream oxidative modification, glutathionylation, of the beta1 subunit of the pump alpha/beta heterodimer. It is firmly established that cAMP-dependent signaling also regulates the pump and we have now examined the hypothesis that such regulation can be mediated by glutathionylation. Exposure of rabbit cardiac myocytes to the adenylyl cyclase activator forskolin increased the co-immunoprecipitation of NADPH oxidase subunits p47phox and p22phox, required for its activation; and increased superoxide-sensitive fluorescence. Forskolin also increased glutathionylation of the Na-K pumps beta1 subunit and decreased its co-immunoprecipitation with the alpha1 subunit, findings similar to those already established for PKC-dependent signaling. The decrease in co-immunoprecipitation indicates a decrease in the alpha1/beta1 subunit interaction known to be critical for pump function. In agreement with this, forskolin decreased ouabain-sensitive eletrogenic Na+-K+ pump current (arising from the 3:2 Na:K exchange ratio) of voltage-clamped, internally perfused myocytes. The decrease was abolished by the inclusion of superoxide dismutase, inhibitory peptide for the epsilon-isoform of PKC or inhibitory peptide for NADPH oxidase in patch pipette solutions that perfuse the intracellular compartment. Pump inhibition was also abolished by inhibitors of protein kinase A and phospholipase C. We conclude that cAMP- and PKC-dependent inhibition of the cardiac Na-K pump occurs via a shared downstream oxidative signaling pathway involving NADPH oxidase activation and glutathionylation of the pumps beta1 subunit.

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Evolution and multifarious horizontal transfer of an alternative biosynthetic pathway for the alternative polyamine sym-homospermidine.

Publication Date: 2010 Mar 1 PMID: 20194510
Authors: Shaw, F. L. - Elliott, K. A. - Kinch, L. N. - Fuell, C. - Phillips, M. A. - Michael, A. J.
Journal: J Biol Chem

Polyamines are small flexible organic polycations found in almost all cells. They likely existed in the last universal common ancestor of all extant life and yet relatively little is understood about their biological function, especially in bacteria and archaea. Unlike eukaryotes, where the predominant polyamine is spermidine, bacteria may contain instead an alternative polyamine, symhomospermidine. We demonstrate that homospermidine synthase has evolved vertically, primarily in the alpha- Proteobacteria but enzymatically active, diverse HSS orthologues have spread by horizontal gene transfer to other bacteria, bacteriophage, archaea, eukaryotes and viruses. By expressing diverse HSS orthologues in Escherichia coli, we demonstrate in vivo the production of coproducts diaminopropane and N1- aminobutylcadaverine, in addition to symhomospermidine. We show that symhomospermidine is required for normal growth of the alpha-proteobacterium Rhizobium leguminosarum. However, symhomospermidine can be replaced, for growth restoration, by the structural analogues spermidine and symnorspermidine, suggesting that the symmetrical or unsymmetrical form, and carbon backbone length are not critical for polyamine function in growth. We found that the HSS enzyme evolved from the alternative spermidine biosynthetic pathway enzyme carboxyspermidine dehydrogenase. The structure of HSS is related to lysine metabolic enzymes, and HSS and carboxyspermidine dehydrogenase evolved from the aspartate family of pathways. Finally, we show that other bacterial phyla such as Cyanobacteria and some alpha-Proteobacteria synthesize sym-homospermidine by an HSS-independent pathway, very probably based on deoxyhypusine synthase orthologues, similar to the alternative homospermidine synthase found in some plants. Thus bacteria can contain alternative biosynthetic pathways for both spermidine and sym-norspermidine and distinct alternative pathways for sym-homospermidine.

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TAB2 scaffolds TAK1 and NLK in repressing canonical WNT signaling.

Publication Date: 2010 Mar 1 PMID: 20194509
Authors: Li, M. - Wang, H. - Huang, T. - Wang, J. - Ding, Y. - Li, Z. - Zhang, J. - Li, L.
Journal: J Biol Chem

The TAK1-NLK cascade is a MAP kinase related pathway that plays an inhibitory role in canonical Wnt/beta-catenin signaling through regulating the LEF1/TCF family transcriptional factors. TAB2 (TAK1 Binding protein 2) is a putative TAK1 interacting protein which is involved in the regulation of TAK1. Here, we found that TAB2 could directly interact with NLK and function as a scaffold protein to facilitate the interaction between TAK1 and NLK. Knocking down TAB2 using siRNA abolished the interaction of TAK1 with NLK in mammalian cells. The intermediate region (Residues 292-417) of TAB2 was mapped for its binding to NLK. TAB2-DeltaM, a TAB2 mutant lacking this region, showed a lower affinity for NLK and became defective in its scaffolding function. In addition, TAB2, but not TAB2-DeltaM, mediated TAK1-dependent activation of NLK and LEF1 poly-ubiquitylation, resulting in the inhibition of canonical Wnt signaling. Moreover, Wnt3a stimulation led to an increase in the interaction of TAB2 with NLK and the formation of a TAK1-TAB2-NLK complex, suggesting that this TAK-TAB-NLK pathway may constitute a negative feedback mechanism for canonical Wnt signaling.

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Experimental IUGR induces alterations in DNA methylation and gene expression in pancreatic islets of rats.

Publication Date: 2010 Mar 1 PMID: 20194508
Authors: Thompson, R. F. - Fazzari, M. J. - Niu, H. - Barzilai, N. - Simmons, R. A. - Greally, J. M.
Journal: J Biol Chem

Intrauterine growth restriction (IUGR) increases susceptibility to age-related diseases including type 2 diabetes (T2DM), and is associated with permanent and progressive changes in gene expression. Our study was designed to test whether epigenomic dysregulation mediates the cellular memory of this intrauterine event. To test this hypothesis, we isolated pancreatic islets from control and IUGR (induced by bilateral uterine artery ligation at day 18 of fetal life) animals at 7 weeks of age. Using the HELP assay, we generated the first genome-wide DNA methylation map at almost 1 million unique sites throughout the rat genome in normal pancreatic islet cells, allowing us to identify the changes that occur as a consequence of IUGR. We validated candidate dysregulated loci with quantitative assays of cytosine methylation and gene expression. IUGR changes cytosine methylation at ~1,400 loci (false discovery rate of 4.2%) in male rats at 7 weeks of age, preceding the development of diabetes and thus representing candidate loci for mediating the pathogenesis of metabolic disease that occurs later in life. Epigenetic dysregulation occurred preferentially at conserved intergenic sequences, frequently near genes regulating processes known to be abnormal in IUGR islets, such as vascularization, beta-cell proliferation, insulin secretion, and cell death, associated with concordant changes in mRNA expression at Fgfr1, Gch1, Pcsk5, and Vgf. These results demonstrate that epigenetic dysregulation is a strong candidate for propagating the cellular memory of intrauterine events, causing changes in expression of nearby genes and long-term susceptibility to T2DM.

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Ribosomal protein L11 associates with c-Myc at 5S rRNA and tRNA genes and regulates their expression.

Publication Date: 2010 Mar 1 PMID: 20194507
Authors: Dai, M. S. - Sun, X. X. - Lu, H.
Journal: J Biol Chem

The c-Myc oncoprotein promotes cell growth by enhancing ribosomal biogenesis. Overexpression of c-Myc and aberrant ribosomal biogenesis lead to deregulated cell growth and tumorigenesis. Hence, c-Myc activity and ribosomal biogenesis must be tightly coordinated during normal homeostasis. We previously found that ribosomal protein L11 inhibits c-Myc activity by blocking the recruitment of its co-activator TRRAP to the promoter regions of c-Myc target genes that are transcribed by RNA polymerases I and II. In this study, we extended the role of L11 to the regulation of c-Myc-driven transcription of the 5S rRNA and tRNA genes by RNA Pol III. L11 co-resided with c-Myc at the 5S rRNA and tRNA genes and significantly inhibited the binding of TRRAP to these genes. Knocking down endogenous L11 enhanced c-Myc-dependent transcription of these genes. Interestingly, in response to ribosomal stress induced by the treatment of cells with a low dose of actinomycin D or serum starvation, L11 binding to these genes was increased, and inversely TRRAP binding to these genes was decreased. Consistently, knockdown of L11 rescued the reduction of the expression of these genes by the two treatments. These results demonstrate that L11 suppresses c-Myc-dependent and RNA Pol III-catalyzed transcription of 5S rRNA and tRNA genes in response to ribosomal stress, ensuing a tight coordination between c-Myc activity and ribosomal biogenesis.

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Targeting the active site gate to yield hyperactive variants of 5-aminolevulinate synthase.

Publication Date: 2010 Mar 1 PMID: 20194506
Authors: Lendrihas, T. - Hunter, G. A. - Ferreira, G. C.
Journal: J Biol Chem

The rate of porphyrin biosynthesis in mammals is controlled by the activity of the pyridoxal 5'-phosphate-dependent enzyme 5-aminolevulinate synthase (EC 2.3.1.37). Based on the postulate that turnover in this enzyme is controlled by conformational dynamics associated with a highly conserved active site loop, we constructed a variant library by targeting imperfectly conserved non-catalytic loop residues and examined the effects on product and porphyrin production. Functional loop variants of the enzyme were isolated via genetic complementation in Escherichia coli strain HU227. Colony porphyrin fluorescence varied widely when bacterial cells harboring the loop variants were grown on inductive media; this facilitated identification of clones encoding unusually active enzyme variants. Nine loop variants leading to high in vivo porphyrin production were purified and characterized kinetically. Steady-state catalytic efficiencies for the two substrates were increased by up to one hundred-fold. Pre-steady state single turnover reaction data indicated that the second step of quinonoid intermediate decay, previously assigned as reaction rate-limiting, was specifically accelerated such that in three of the variants this step was no longer kinetically significant. Overall, our data support the postulate that the active site loop controls the rate of product and porphyrin production in vivo, and suggest the possibility of an as yet undiscovered means of allosteric regulation.

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Formation of a cyclopropyl epoxide via a leukotriene a synthase-related pathway in an anaerobic reaction of soybean lipoxygenase-1 with 15s-hydroperoxyeicosatetraenoic acid. Evidence that oxygen access is a determinant of secondary reactions with fatty acid hydroperoxides.

Publication Date: 2010 Mar 1 PMID: 20194505
Authors: Zheng, Y. - Brash, A. R.
Journal: J Biol Chem

The further conversion of an arachidonic acid hydroperoxide to a leukotriene A-type (LTA) epoxide by specific lipoxygenase (LOX) enzymes constitutes a key step in inflammatory mediator biosynthesis. Whereas mammalian 5-LOX transforms its primary product (5S-HPETE) almost exclusively to LTA4, the model enzyme, soybean LOX-1, normally produces no detectable leukotrienes and instead further oxygenates its primary product 15S-HPETE to 5,15- and 8,15-dihydroperoxides. Mammalian 15-LOX-1 displays both types of activity. We reasoned that availability of molecular oxygen within the LOX active site favors oxygenation whereas lack of O2 promotes LTA epoxide synthesis. To test this, we reacted 15S-HPETE with soybean LOX-1 under anaerobic conditions and identified the products by HPLC, UV, mass spectrometry and NMR. Among the products we identified a pair of 8,15-dihydroxy diastereomers with all-trans conjugated trienes that incorporated 18O from H218O at C-8, indicative of the formation of 14,15-LTA4. A pair of 5,15-dihydroxy diastereomers containing two trans-trans conjugated dienes (6E,8E,11E,13E) and that incorporated 18O from H218O at C-5 was deduced to arise from hydrolysis of a novel epoxide containing a cyclopropyl ring, 14,15-epoxy-[9,10,11-cyclopropyl]-eicosa-5Z,7E,13E-trienoic acid. Also identified was the delta-lactone of the 5,15-diol, a derivative that exhibited no 18O incorporation due to its formation by intramolecular reaction of the carboxyl anion with the proposed epoxide intermediate. Our results support a model in which access to molecular oxygen within the active site directs the outcome from competing pathways in the secondary reactions of lipoxygenases.

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Origin of the Allyl group in FK506 biosynthesis.

Publication Date: 2010 Mar 1 PMID: 20194504
Authors: Goranovic, D. - Kosec, G. - Mrak, P. - Fujs, S. - Horvat, J. - Kuscer, E. - Kopitar, G. - Petkovic, H.
Journal: J Biol Chem

FK506 (tacrolimus) is a secondary metabolite with a potent immunosuppressive activity, currently registered for use as immunosuppressant after organ transplantation. FK506 and FK520 are biogenetically related natural products which are synthesized by combined polyketide synthase / non-ribosomal peptide synthetase systems. The entire gene cluster for biosynthesis of FK520 from Streptomyces hygroscopicus var. ascomyceticus has been cloned and sequenced. On the other hand, the FK506 gene cluster from Streptomyces sp. MA6548 (ATCC55098) was sequenced only partially and it was reasonable to expect that additional genes would be required for the provision of substrate supply. Here we report the identification of a previously unknown region of the FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 containing genes encoding the provision of unusual building blocks for FK506 biosynthesis as well as a regulatory gene. Among others, we identified a group of genes encoding biosynthesis of the extender unit which forms the allyl group at carbon 21 of FK506. Interestingly, we have identified a small independent diketide synthase system involved in the biosynthesis of the allyl-group. Inactivation of one of these genes, encoding an unusual ketosynthase domain, resulted in an FK506 non-producing strain and the production was restored when a synthetic analog of the allylmalonyl-CoA extender unit was added to the cultivation medium. Based on our results, we propose a biosynthetic pathway for the provision of an unusual five-carbon extender unit which is carried out by a novel diketide synthase complex.

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Mechanisms of activity and inhibition of the hepatitis C virus RNA-dependent RNA-polymerase.

Publication Date: 2010 Mar 1 PMID: 20194503
Authors: Reich, S. - Golbik, R. P. - Geissler, R. - Lilie, H. - Behrens, S. E.
Journal: J Biol Chem

The RNA-dependent RNA-polymerase NS5B is a key enzyme of the replication of hepatitis C virus (HCV) and a major therapeutic target. Applying a novel continuous assay with highly purified protein and a fluorescent RNA-template we provide for the first time a comprehensive mechanistic description of the enzymatic reaction. Using fluorescence spectroscopy, the kinetics of NS5B were confirmed to consist of two half-reactions, namely substrate binding and turnover. Determining the binding constants of the substrates and the rate constants of individual reaction steps, NS5B was shown to bind the template single-stranded (ss) RNA with high affinity (nanomolar range) and in a stepwise process that reflect the substrate positioning. As demonstrated by circular dichroism, NTP(s) binding caused a tertiary structural change of the enzyme into an active conformation. The second half-reaction was dissected into a sequential polymerization and a subsequent, rate-limiting product release reaction. Taking advantage of these tools, we analyzed the mechanism of action of the NS5B inhibitor HCV-796, which was shown to interfere with the formation of double-stranded RNA by blocking the second half-reaction.

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Determinants for stop-transfer and post-import pathways for protein targeting to the chloroplast inner envelope membrane.

Publication Date: 2010 Mar 1 PMID: 20194502
Authors: Viana, A. A. - Li, M. - Schnell, D. J.
Journal: J Biol Chem

The inner envelope membrane (IEM) of the chloroplast plays key roles in controlling metabolite transport between the organelle and cytoplasm, and is a major site of lipid and membrane synthesis within the organelle. IEM biogenesis requires the import and integration of nucleus-encoded membrane proteins. Previous reports have led to the conclusion that membrane proteins are inserted into the IEM during protein import from the cytoplasm via a stop-transfer mechanism or are completely imported into the stroma and then inserted into the IEM in a post-import mechanism. In this study, we examined the determinants for each pathway by comparing the targeting of Albino or Pale Green Mutant 1 (APG1), an example of a stop-transfer substrate, and atTic40, an example of a post-import substrate. We show that the APG1 transmembrane domain (TMD) is sufficient to direct stop-transfer insertion. The APG1 TMD also functions as a topology determinant. We also show that the ability of the post-import signals within atTic40 to target proteins to the IEM is dependent upon their context within the full protein sequence. In the incorrect context, the atTic40 signals can behave as stop-transfer signals or fail to target fusion proteins to the IEM. These data suggest that the post-import pathway are complex and have evolved to avoid stop-transfer insertion.

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Human GLUD2 glutamate dehydrogenase is expressed in neural and testicular supporting cells.

Publication Date: 2010 Mar 1 PMID: 20194501
Authors: Spanaki, C. - Zaganas, I. - Kleopa, K. A. - Plaitakis, A.
Journal: J Biol Chem

Mammalian glutamate dehydrogenase is an allosterically regulated enzyme that is expressed widely. Its activity is powerfully inhibited by GTP and thought to be controlled by the need of the cell in ATP. In addition to this housekeeping hGDH1, humans have acquired (via a duplication event) a highly homologous isoenzyme (hGDH2) that is resistant to GTP. While transcripts of GLUD2, the gene encoding hGDH2, have been detected in human neural and testicular tissues, data on the endogenous protein are lacking. Here we developed an antibody specific for hGDH2 and used it to study human tissues. Western blot analyses revealed to our surprise that endogenous hGDH2 is more densely expressed in testis than in brain. At the subcellular level, hGDH2 localized to mitochondria. Study of testicular tissue using immuno-cytochemical and immuno-fluorescence methods revealed that the Sertoli cells were strongly labeled by our anti-hGDH2 antibody. In human cerebral cortex, a robust labeling of astrocytes was detected, with neurons showing faint hGDH2 immuno-reactivity. Astrocytes and Sertoli cells are known to support neurons and germ cells, respectively, providing them with lactate that largely derives from the TCA cycle via conversion of glutamate to alpha-ketoglutarate (GDH reaction). As hGDH2 is not subject to GTP control, the enzyme is able to metabolize glutamate even when the TCA cycle generates GTP amounts sufficient to inactivate the housekeeping hGDH1. Hence the selective expression of hGDH2 by astrocytes and Sertoli cells may provide a significant biological advantage by facilitating metabolic recycling processes essential to the supportive role of these cells.

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Functional identification of two non-redundant Arabidopsis {alpha}(1,2)fucosyltransferases specific to arabinogalactan-proteins.

Publication Date: 2010 Mar 1 PMID: 20194500
Authors: Wu, Y. - Williams, M. - Bernard, S. - Driouich, A. - Showalter, A. M. - Faik, A.
Journal: J Biol Chem

Virtually nothing is known about the mechanisms and enzymes responsible for the glycosylation of arabinogalactan-proteins (AGPs). Glycosyltransferase (GT)37 family contains plant-specific enzymes, which suggests involvement in plant-specific organs such as the cell wall. Our working hypothesis is that AtFUT4 and AtFUT6 genes encode alpha(1,2)fucosyltransferases (FUTs) for AGPs. Multiple lines of evidence support this hypothesis. First, over-expression of the two genes in tobacco BY2 cells, known to contain non-fucosylated AGPs, resulted in a staining of transgenic cells with eel lectin, which specifically binds to terminal alpha-linked fucose (Fuc). Second, monosaccharide analysis by high pH anion exchange chromatography and electrospray ionization mass spectrometry indicated the presence of Fuc in AGPs from transgenic cell lines but not in AGPs from wild type cells. Third, detergent extracts from microsomal membranes prepared from transgenic lines were able to fucosylate, in-vitro, purified AGPs from BY2 wild type cells. Susceptibility of [14C]fucosylated-AGPs to alpha(1,2)fucosidase, and not to alpha(1,3/4)fucosidase, indicated that an alpha(1,2) linkage is formed. Furthermore, dearabinosylated AGPs were not substrate acceptors for these enzymes indicating that arabinosyl residues represent the fucosylation sites on these molecules. Testing of several polysaccharides, oligosaccharides, and glycoproteins as potential substrate acceptors in the fucosyl transfer reactions indicated that the two enzymes are specific for AGPs, but are not functionally redundant as they differentially fucosylate certain AGPs. AtFUT4 and AtFUT6 are the first enzymes to be characterized for AGP glycosylation and further our understanding of cell wall biosynthesis.

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G{alpha}q-mediated activation of GRK2 by mechanical stretch in cardiac myocytes; The role of protein kinase C.

Publication Date: 2010 Mar 1 PMID: 20194499
Authors: Malhotra, R. - D'Souza, K. M. - Staron, M. L. - Birukov, K. G. - Bodi, I. - Akhter, S. A.
Journal: J Biol Chem

G protein-coupled receptor kinase-2 (GRK2) is a critical regulator of beta-adrenergic receptor (beta-AR) signaling and cardiac function. We studied the effects of mechanical stretch, a potent stimulus for cardiac myocyte hypertrophy, on GRK2 activity and beta-AR signaling. To eliminate neurohormonal influences, neonatal rat ventricular myocytes were subjected to cyclical equi-biaxial stretch. A hypertrophic response was confirmed by fetal gene upregulation. GRK2 activity in cardiac myocytes was increased 4.2-fold at 48 hours of stretch versus unstretched controls. Adenylyl cyclase (AC) activity was blunted in sarcolemmal membranes after stretch demonstrating beta-AR desensitization. The hypertrophic response to mechanical stretch is mediated, primarily, through the Galphaq-coupled Angiotensin II AT1 receptor leading to activation of protein kinase C (PKC). PKC is known to phosphorylate GRK2 at the N-terminal serine29 residue, leading to kinase activation. Overexpression of a mini-gene that inhibits receptor-Galphaq coupling blunted stretch-induced hypertrophy and GRK2 activation. shRNA-mediated knockdown of PKCalpha also significantly attenuated stretch-induced GRK2 activation. Overexpression of a GRK2 mutant (S29A) in cardiac myocytes inhibited phosphorylation of GRK2 by PKC, abolished stretch-induced GRK2 activation, and restored AC activity. Cardiac-specific activation of PKCalpha in transgenic mice led to impaired beta-agonist-stimulated ventricular function, blunted cyclase activity, and increased GRK2 phosphorylation and activity. Phosphorylation of GRK2 by PKC appears to be the primary mechanism of increased GRK2 activity and impaired beta-AR signaling following mechanical stretch. Cross-talk between hypertrophic signaling at the level of PKC and beta-AR signaling regulated by GRK2 may be an important mechanism in the transition from compensatory ventricular hypertrophy to heart failure.

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T cell signal regulation by the actin cytoskeleton.

Publication Date: 2010 Mar 1 PMID: 20194498
Authors: Chichili, G. R. - Westmuckett, A. - Rodgers, W.
Journal: J Biol Chem

T cells form an immunological synapse (IS) that sustains and regulates signals for cell stimulation. Enriched in the IS is the Src family kinase (SFK) Lck. Conversely, the membrane phosphatase CD45, which activates SFKs, is excluded, and this is necessary to avoid quenching of TCR phosphosignals. Data suggest this arrangement occurs by an enrichment of cholesterol-dependent rafts in the IS. However, the role of rafts in structuring the IS remains unclear. To address this question, we used fluorescence resonance energy transfer (FRET) to interrogate the nanoscopic structure of the IS. The FRET probes consisted of membrane-anchored fluorescent proteins (FPs) with distinct affinities for rafts. Both the raft and nonraft probes exhibited clustering in the IS. However, co-clustering of raft donor-acceptor pairs was 10-fold greater than co-clustering of raft-nonraft pairs. We measured the effect of disrupting rafts in the IS on CD45 localization and Lck regulation by treating stimulated T cells with filipin. The filipin specifically disrupted co-clustering of the raft FRET pairs in the IS, and allowed targeting of CD45 to the IS and dephosphorylation of the regulatory tyrosine of Lck. Clustering of the raft probes was also sensitive to latrunctulin B (Lat B), which disrupts actin filaments. Strikingly, enriching the cortical cytoskeleton using jasplakinolide maintained raft probe co-clustering, CD45 exclusion, and Lck regulation in the IS following addition of filipin. These data show the actin cytoskeleton maintains a membrane raft environment in the IS that promotes Lck regulation by excluding CD45.

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Tead-1 over-expression in the mouse heart promotes an age-dependent heart dysfunction.

Publication Date: 2010 Mar 1 PMID: 20194497
Authors: Tsika, R. W. - Ma, L. - Kehat, I. - Schramm, C. - Simmer, G. - Morgan, B. - Fine, D. M. - Hanft, L. M. - McDonald, K. S. - Molkentin, J. D. - Krenz, M. - Yang, S. - Ji, J.
Journal: J Biol Chem

TEA-domain transcription factor-1 (TEAD-1) is essential for proper heart development, is implicated in cardiac-specific gene expression and the hypertrophic response of primary cardiomyocytes to hormonal and mechanical stimuli, and its activity increases in the pressure overloaded hypertrophied rat heart. To investigate whether TEAD-1 is an in vivo modulator of cardiac-specific gene expression and hypertrophy, we developed transgenic mice expressing HA-tagged TEAD-1 under the control of the muscle creatine kinase (MCK) promoter. We show that a sustained increase in TEAD-1 protein lead to an age-dependent dysfunction. Magnetic resonance imaging (MRI) revealed decreases in cardiac output, stroke volume, ejection fraction, and fractional shortening. Isolated TEAD-1 hearts revealed decreased left ventricular power output that correlated with increased betaMyHC protein. Histological analysis showed altered alignment of cardio-myocytes, septal wall thickening, and fibrosis while ECG displayed a left axis shift of mean electrical axis. Transcripts representing most members of the fetal heart gene program remained elevated from fetal- through adult-life. Western blot analyses revealed decreases in p-phospholamban, SERCA2a, p-CX43, p-GSK-3alpha/beta, nuclear beta-catenin, GATA4, NFATc3/c4, and increased NCX1, nuclear DYKR1A and Puralpha/beta protein. TEAD-1 mice did not display cardiac hypertrophy. TEAD-1 mice do not tolerate stress as they die 6-hours after surgical induction of pressure overload. These data provide the first in vivo evidence that increased TEAD-1 can induce characteristics of cardiac remodeling associated with cardiomyopathy and heart failure.

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Peroxynitrite is the major species formed from different flux ratios of co-generated nitric oxide and superoxide: direct reaction with boronate-based fluorescent probe.

Publication Date: 2010 Mar 1 PMID: 20194496
Authors: Zielonka, J. - Sikora, A. - Joseph, J. - Kalyanaraman, B.
Journal: J Biol Chem

There is much interest in the nitration and oxidation reaction mechanisms initiated by superoxide radical anion (O(2)(.-)) and nitric oxide ((.)NO). It is well known that O(2)(.-) and (.)NO rapidly react to form a potent oxidant, peroxynitrite anion (ONOO(-)). However, indirect measurements with the existing probes (e.g., dihydrorhodamine) previously revealed a bell-shaped response to co-generated (.)NO and O(2)(.-) fluxes, with the maximal yield of the oxidation or nitration product occurring at a 1:1 ratio. These results raised doubts on the formation of ONOO(-) per se at various fluxes of (.)NO and O(2)(.-). Using a novel fluorogenic probe, coumarin-7-boronic acid, that reacts stoichiometrically and rapidly with ONOO(-) (k = 1.1 x 10(6) M(-1)s(-1)), we report that ONOO(-) formation increased linearly and began to plateau after reaching a 1:1 ratio of co-generated ( .)NO and O(2)(.-) fluxes. We conclude that ONOO(-) is formed as the primary intermediate during the reaction between ( .)NO and O(2)(.-) co-generated at different fluxes.

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PRP conformational transitions alter species-preference of a PrP specific antibody.

Publication Date: 2010 Mar 1 PMID: 20194495
Authors: Zou, W. Q. - Langeveld, J. - Xiao, X. - Chen, S. - McGeer, P. L. - Yuan, J. - Payne, M. C. - Kang, H. E. - McGeehan, J. - Sy, M. S. - Greenspan, N. S. - Kaplan, D. - Wang, G. X. - Parchi, P. - Hoover, E. - Kneale, G. - Telling, G. - Surewicz, W. K. - Kong, Q. - Guo, J. P.
Journal: J Biol Chem

The epitope of the 3F4 antibody most commonly used in human prion disease diagnosis is believed to consist of residues Met-Lys-His-Met (MKHM) corresponding to human PrP109-112. This assumption is based mainly on the observation that 3F4 reacts with human and hamster PrP but not with PrP from mouse, sheep and cervids, in which Met at residue 112 is replaced by Val (V). Here we report that, by brain histoblotting, 3F4 did not react with PrP of uninfected transgenic (Tg) mice expressing elk PrP; however, it did show distinct immunoreactivity in Tg mice infected with chronic wasting disease (CWD). Moreover, conventional Western blotting revealed that although 3F4 did not react with PrP of uninfected elk, it detected elk PrPSc after enrichment. Compared to human PrP, the 3F4 reactivity with the recombinant elk PrP was two orders of magnitude weaker, as indicated by both Western blotting and surface plasmon resonance. To investigate the molecular basis of these species- and conformer-dependent preferences of 3F4, the epitope was probed by peptide membrane array and antigen competition experiments. Remarkably, the 3F4 antibody did not react with MKHM but reacted strongly with KTNMK (corresponding to human PrP106-110), a sequence that is also present in cervids, sheep and cattle. 3F4 reacted also with elk PrP peptides containing KTNMKHV. We concluded that the minimal sequence for the 3F4 epitope consists of residues KTNMK, and the species- and conformer-dependent preferences of 3F4 arise largely from the interactions between Met112 (human PrP) or Val115 (cervid PrP) and adjacent residues.

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A proteome-wide analysis of kinase-substrate network in the DNA damage response.

Publication Date: 2010 Feb 27 PMID: 20190278
Authors: Chen, S. H. - Albuquerque, C. P. - Liang, J. - Suhandynata, R. T. - Zhou, H.
Journal: J Biol Chem

The DNA damage checkpoint, consisting of an evolutionarily conserved protein kinase cascade, controls the DNA damage response in eukaryotes. Knowledge of the in vivo substrates of the checkpoint kinases is essential towards understanding their functions. Here we used quantitative mass spectrometry to identify 53 new and 34 previously known targets of Mec1/Tel1, Rad53 and Dun1 in Saccharomyces cerevisiae. Analysis of Replication Protein-A (RPA) associated proteins reveals extensive physical interactions between RPA-associated proteins and Mec1/Tel1 specific substrates. Among them, multiple subunits of the chromatin remodeling complexes including ISW1, ISW2, INO80, SWR1, RSC and SWI/SNF are identified and they undergo DNA damage induced phosphorylation by Mec1 and Tel1. Taken together, this study greatly expands the existing knowledge of the targets of DNA damage checkpoint kinases and provides insights into the role of RPA-associated chromatins in mediating Mec1 and Tel1 substrate phosphorylation in vivo.

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Drf1-dependent kinase (DDK) interacts with Claspin through a conserved protein motif.

Publication Date: 2010 Mar 2 PMID: 20190277
Authors: Gold, D. A. - Dunphy, W. G.
Journal: J Biol Chem

The Dbf4/Drf1-dependent kinase (DDK) is required for the initiation of DNA replication in eukaryotes. Another protein, Claspin, mediates the activation of a cellular checkpoint response to stalled replication forks and is also a regulator of replication. In this study, we found that DDK phosphorylates Claspin in vitro and forms a nuclear complex containing Cdc7, Drf1, and Claspin in Xenopus egg extracts. In addition, purified Claspin and DDK are capable of a direct in vitro interaction. We identified a conserved binding site on Claspin required for its interaction with DDK. This site corresponds to the first of two sequence repeats in the Chk1-binding domain (CKBD) of Claspin. Furthermore, we have established that two amino acids in this motif, D861 and Q866, are essential for the interaction between Claspin and DDK. We found that mutant forms of Claspin incapable of interacting with DDK are still able to associate with and activate Chk1 in response to DNA replication blockages. However, Claspin-depleted egg extracts that have been reconstituted with these mutants of Claspin undergo DNA replication more slowly. These findings suggest that the interaction of DDK with Claspin mediates a checkpoint-independent function of Claspin related to DNA replication.

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Increased E4 activity in mice leads to Ubiquitin-containing aggregates and degeneration of hypothalamic neurons resulting in obesity.

Publication Date: 2010 Feb 26 PMID: 20190229
Authors: Susaki, E. - Kaneko-Oshikawa, C. - Miyata, K. - Tabata, M. - Yamada, T. - Oike, Y. - Katagiri, H. - Nakayama, K. I.
Journal: J Biol Chem

Obesity has become a serious public health problem worldwide. Although neural degeneration in specific brain regions has been suggested to contribute to the obesity phenotype in humans, a causal relation between these two conditions has not been demonstrated experimentally. We now show that E4B (also known as UFD2a), a mammalian ubiquitin chain elongation factor (E4), induces the formation of intracellular aggregates positive for ubiquitin and the adaptor protein p62 when overexpressed in cultured cells or the brain. Mice transgenic for E4B manifested neural degeneration in association with aggregate formation, and they exhibited functional impairment specifically in a subset of hypothalamic neurons that regulate food intake and energy expenditure, resulting in the development of hyperphagic obesity and related metabolic abnormalities. The neural pathology of E4B transgenic mice was similar to that of human neurodegenerative diseases associated with the formation of intracellular ubiquitin-positive deposits, indicating the existence of a link between such diseases and obesity and related metabolic disorders. Our findings thus provide experimental evidence for a role of hypothalamic neurodegeneration in obesity, and the E4B transgenic mouse should prove to be a useful animal model for studies of the relation between neurodegenerative diseases and obesity.

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Redundant control of adipogenesis by histone deacetylases 1 and 2.

Publication Date: 2010 Feb 26 PMID: 20190228
Authors: Haberland, M. - Carrer, M. - Mokalled, M. H. - Montgomery, R. L. - Olson, E. N.
Journal: J Biol Chem

Adipocyte differentiation is a well-defined process that is under the control of transcriptional activators and repressors. We show that histone deacetylase (HDAC) inhibitors efficiently block adipocyte differentiation in vitro. This effect is specific to adipogenesis, as another mesenchymal differentiation process, osteoblastogenesis, is enhanced upon HDAC inhibition. Through the systematic genetic deletion of HDAC genes in cultured mesenchymal precursor cells, we show that deletion of HDAC1 and HDAC2 leads to reduced lipid accumulation, revealing redundant and requisite roles of these class I HDACs in adipogenesis. These findings unveil a previously unrecognized role for HDACs in the control of adipogenesis.

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Improved insulin sensitivity in high-fat high-cholesterol fed LDLR-/- mice with macrophage-specific transgenic expression of cholesteryl ester hydrolase: Role of macrophage inflammation and infiltration into adipose tissue.

Publication Date: 2010 Feb 26 PMID: 20189995
Authors: Bie, J. - Zhao, B. - Song, J. - Ghosh, S.
Journal: J Biol Chem

Cellular cholesterol balance induces changes in the inflammatory status of macrophage and low grade chronic inflammation is increasingly being recognized as one of the key steps in the development of atherosclerosis as well as insulin resistance. Cholesteryl ester hydrolase (CEH) catalyzes the hydrolysis of intracellular stored cholesteryl esters (CE) and thereby enhance free cholesterol efflux and reduces cellular CE content. We have earlier reported reduced atherosclerosis and lesion necrosis in macrophage-specific CEH transgenic mice in Ldlr-/- background. In the present study we tested the hypothesis that reduced intracellular accumulation of CE in macrophages from CEH transgenic mice will attenuate expression of pro-inflammatory mediators thereby reducing infiltration into adipose tissue, alleviating inflammation and resulting in improved insulin sensitivity. Western diet fed Ldlr-/-CEH transgenic mice showed improved insulin sensitivity as assessed by glucose and insulin tolerance tests. Macrophages from CEH transgenic mice expressed significantly lower levels of pro-inflammatory cytokines (IL-1b, IL-6) and chemokine (MCP-1). Attenuation of NF-kB and AP-1 driven gene expression was determined to be the underlying mechanism. Infiltration of macrophages into the adipose tissue that increases inflammation and impairs insulin signaling was also significantly reduced in Ldlr-/-CEH transgenic mice. In OP-9 adipocyte peritoneal macrophage co-culture system, macrophages from CEH transgenic mice had significantly reduced effect on insulin signaling as measured by Akt phosphorylation compared to non-transgenic macrophages. Taken together, these studies demonstrate that macrophage-specific over-expression of CEH decreases expression of pro-inflammatory mediators and attenuates macrophage infiltration into the adipose tissue resulting in decreased circulating cytokines and improved insulin sensitivity.

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Morphine-like opiates antagonize selectively receptor-arrestin interactions.

Publication Date: 2010 Feb 26 PMID: 20189994
Authors: Molinari, P. - Vezzi, V. - Sbraccia, M. - Gro, C. - Riitano, D. - Ambrosio, C. - Casella, I. - Costa, T.
Journal: J Biol Chem

The addictive potential of opioids may be related to their differential ability to induce G protein signaling and endocytosis. We compared the ability of 20 ligands (sampled from the main chemical classes of opioids) to promote the association of mu and delta receptors with G protein or beta-arrestin 2. Receptor-arrestin binding was monitored by bioluminescence resonance energy transfer (BRET) in intact cells, where pertussis toxin experiments indicated that the interaction was minimally affected by G protein signaling. To assess receptor-G protein coupling without competition from arrestins, we employed a cell-free BRET assay, using membranes isolated from cells expressing luminescent receptors and fluorescent Gbeta1. In this system, the agonist-induced enhancement of BRET (indicating shortening of distance between the two proteins) was Galpha-mediated (as shown by sensitivity to pertussis toxin and guanine nucleotides) and yielded data consistent with the known pharmacology of the ligands. We found marked differences of efficacy for G protein and arrestin, with a pattern suggesting more restrictive structural requirements for arrestin efficacy. The analysis of such differences identified a subset of structures showing a marked discrepancy between efficacies for G protein and arrestin. Addictive opiates like morphine and oxymorphone exhibited large differences both at delta and mu receptors. Thus, they were effective agonists for G protein coupling, but acted as competitive enkephalin antagonists (delta) or partial agonists (mu) for arrestin. This arrestin-selective antagonism resulted in inhibition of short and long term events mediated by arrestin, such as rapid receptor internalization and down-regulation.

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GATA3 inhibits breast cancer metastasis through the reversal of epithelial-mesenchymal transition.

Publication Date: 2010 Feb 26 PMID: 20189993
Authors: Yan, W. - Cao, Q. J. - Arenas, R. B. - Bentley, B. - Shao, R.
Journal: J Biol Chem

GATA3, a transcription factor which regulates T lymphocyte differentiation and maturation, is exclusively expressed in early stage well-differentiated breast cancers, but not in advanced invasive cancers. Yet, little is understood regarding its activity and mechanisms underlying this differential expression in cancers. Here, we employed GATA3-positive, non-invasive (MCF-7) and GATA3-negative, invasive (MDA-MB-231) breast cancer cells to define its role in the transformation between these two distinct phenotypes. Ectopic expression of GATA3 in MDA-MB-231 cells led to a cuboidal like epithelial phenotype and reduced cell invasive activity. These cells also increased E-cadherin expression; but decreased levels of vimentin, N-cadherin, and MMP-9. Further, MDA-MB-231 cells expressing GATA3 grew smaller primary tumors without metastasis compared with larger metastatic tumors derived from control MDA-MB-231 cells in xenografted mice. GATA3 was found to induce E-cadherin expression through binding GATA-like motifs located in E-cadherin promoter. Blockade of GATA3 using siRNA gene knockdown in MCF-7 cells triggered fibroblastic transformation and cell invasion, resulting in distant metastasis. Studies of human breast cancer showed that GATA3 expression correlated with elevated E-cadherin levels, ER expression, and long disease-free survival. These data suggest that GATA3 drives invasive breast cancer cells to undergo the reversal of epithelial-mesenchymal transition (EMT), leading to the suppression of cancer metastasis.

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An important role of the SRC family kinase LYN in stimulating platelet granule secretion.

Publication Date: 2010 Feb 26 PMID: 20189992
Authors: Li, Z. - Zhang, G. - Liu, J. - Stojanovic, A. - Ruan, C. - Lowell, C. A. - Du, X.
Journal: J Biol Chem

The Src family kinases (SFK) have been proposed to play stimulatory and inhibitory roles in platelet activation. The mechanisms for these apparently contradictory roles are unclear. Here we show that SFK, mainly Lyn, is important in stimulating a common signaling pathway leading to secretion of platelet granules. Lyn knockout or an isoform-nonselective SFK inhibitor, PP2, inhibited platelet secretion of both dense and alpha granules and the secretion-dependent platelet aggregation induced by thrombin, collagen and thromboxane A2 (TXA2). The inhibitory effect of Lyn knockout on platelet aggregation was reversed by supplementing granule content ADP, indicating that the primary role of Lyn is to stimulate granule secretion. Inhibitory effect of PP2 on platelet aggregation induced by thrombin and TXA2 were also reversed by supplementing ADP. Furthermore, PP2-treatment or Lyn knockout diminished agonist-induced Akt activation and cGMP production. The inhibitory effect of PP2 or Lyn knockout on platelet response can be corrected by supplementing cGMP. These data indicate that Lyn stimulates platelet secretion by activating the phosphoinositide 3-kinase-Akt-nitric oxide (NO)-cGMP pathway, and also provide an explanation why Lyn can both stimulate and inhibit platelet activation.

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Use of venom peptides to probe ion channel structure and function.

Publication Date: 2010 Feb 26 PMID: 20189991
Authors: Dutertre, S. - Lewis, R. J.
Journal: J Biol Chem

Venoms of snakes, scorpions, spiders, insects, sea anemones and cone snails are complex mixtures comprising mostly peptides and small proteins, which have evolved for prey capture and/or defence. These deadly animals have long fascinated scientists and the general public alike. Early studies isolated lethal components in the search for cures and an understanding of their mechanisms of action. Interestingly, ion channels emerged as the targets for many of the venom peptides, providing researchers with highly selective and potent molecular probes useful in unravelling ion channel structure and function. This review describes the remarkable features of these unique pharmacological tools, highlighting the molecular details of their receptor-toxin interactions and opportunities for the development of new peptide therapeutics.

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Dependence of phospholipase D1 multi-monoubiquitination on its enzymatic activity and palmitoylation.

Publication Date: 2010 Feb 26 PMID: 20189990
Authors: Yin, H. - Gui, Y. - Du, G. - Frohman, M. A. - Zheng, X. L.
Journal: J Biol Chem

Phospholipase D (PLD) is an important lipase in many cellular processes including vesicular trafficking, cell survival and cell migration. In the present study, we show that PLD1, but not PLD2, is posttranslationally modified by multi-monoubiquitination. Intriguingly, suppression of lipase activity either by mutation of the HKD motif (PLD1H896R, K898R, D903A) or the phosphatidylinositol 4,5-bisphosphate binding motif (PLD1R691, 695G) or through use of PLD-selective inhibitors impaired the ubiquitination of PLD1, although stimulation of lipase activity by phorbol 12-myristate 13-acetate (PMA) did not enhance its ubiquitination. A palmitoylation-deficient mutant PLD1 allele, which exhibits altered patterns of vesicular trafficking, had significantly lower levels of monoubiquitination. In addition, the expression of ubiquitin-fused PLD1 induced aberrant enlarged vesicles partially co-localized with the Golgi complex, but not with early endosomes. The altered localization was reduced by K898R mutation, suggesting a role of multi-monoubiquitination in PLD1 subcellular localization. Surprisingly, the degradation of PLD1, but not PLD1K898R nor PLD2, was blocked by inhibitors of proteasomes, but not by inhibitors of lysosomes or other proteases, suggesting a role of the ubiquitination in proteasomal degradation of PLD1. In summary, our studies show that PLD1, but not PLD2, is multi-monoubiquitinated. The ubiquitination modification might represent a novel regulatory mechanism in PLD1 functioning, particularly in the context of subcellular trafficking in between different membrane compartments.

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CDK5 nuclear localization is p27-dependent in nerve cells: Implications for cell cycle suppression and caspase-3 activation.

Publication Date: 2010 Feb 26 PMID: 20189989
Authors: Zhang, J. - Li, H. - Herrup, K.
Journal: J Biol Chem

Initiation of a cell cycle in an adult neuron leads to cell death, placing great importance on the mechanisms that normally suppress the neuronal cell cycle. We have previously shown that the cyclin dependent kinase, Cdk5, is an important part of this process, but only when it is present in the nucleus. We report here that Cdk5 nuclear localization relies on its binding to the cyclin dependent kinase inhibitor, p27. Cdk5 has no intrinsic nuclear localization signal; in the absence of p27, two weak nuclear export signals that bind CRM1 cause it to shuttle to the cytoplasm. When a neuron is subjected to stress, such as exposure to beta-amyloid, the Cdk5-p27 interaction is lost, reducing Cdk5 levels in the nucleus, and depriving the neuron of a major cell cycle suppression mechanism. Caspase-3 is activated within hours, but death is not immediate; elevated levels of cytoplasmic Cdk5 appear to retard neuronal death by a mechanism that may involve Bcl2. These data suggest a model in which Cdk5 exerts a double protective function in neurons: chronically suppressing the cell cycle when located in the nucleus and transiently delaying cell death in the cytoplasm.

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Isoform specific regulation of Akt signaling by the endosomal protein WDFY2.

Publication Date: 2010 Feb 26 PMID: 20189988
Authors: Walz, H. A. - Shi, X. - Chouinard, M. - Bue, C. A. - Navaroli, D. M. - Hayakawa, A. - Zhou, Q. L. - Nadler, J. - Leonard, D. M. - Corvera, S.
Journal: J Biol Chem

Recent work has led to the identification of novel endocytic compartments with functional roles in both protein trafficking and growth-factor signal transduction. The PI3P-binding, FYVE domain-containing protein WDFY2 is localized to a distinct subset of early endosomes, which are localized close to the plasma membrane. Here we find that the serine/threonine kinase Akt interacts with these endosomes in an isoform-specific manner. Using quantitative fluorescence microscopy we demonstrate specific co-localization of WDFY2 with endogenous Akt2, but not Akt1. Moreover, depletion of WDFY2 leads to impaired phosphorylation of Akt in response to insulin due to isoform specific reduction of Akt2, but not Akt1, protein levels, and to a marked reduction in the insulin-stimulated phosphorylation of numerous Akt substrates. This is accompanied by an impairment in insulin-stimulated glucose transport and, after prolonged silencing, a reduction in the level of expression of adipogenic genes. We propose that WDFY2-enriched endosomes serve as a scaffold that enables specificity of insulin signaling through Akt2.

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Direct transactivator-transcription factor IID (TFIID) contacts drive yeast ribosomal protein gene transcription.

Publication Date: 2010 Feb 26 PMID: 20189987
Authors: Layer, J. H. - Miller, S. G. - Weil, P. A.
Journal: J Biol Chem

Transcription factor IID (TFIID) plays a key role in regulating eukaryotic gene expression by directly binding promoters and enhancer-bound transactivator proteins. However, the precise mechanisms and outcomes of transactivator-TFIID interaction remain unclear. Transcription of yeast ribosomal protein genes (RPGs) requires TFIID and the DNA-binding transactivator Rap1. We have previously shown that Rap1 directly binds to the TFIID complex through interaction with its TBP Associated Factor (Taf) subunits Taf4, 5 and 12. Here we identify and characterize the Rap1 Binding Domains (RBDs) of Taf4 and Taf5. These RBDs are essential for viability but dispensable for Taf-Taf interactions and TFIID stability. Cells expressing altered Rap1 Binding Domains exhibit conditional growth, synthetic phenotypes when expressed in combination or with altered Rap1, and are selectively defective in RPG transcription. Taf4 and Taf5 proteins with altered RBDs bind Rap1 with reduced affinity. We propose that collectively the Taf4, Taf5 and Taf12 subunits of TFIID represent the physical and functional targets for Rap1 interaction, and further, that these interactions drive ribosomal protein gene transcription.

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Regulation of FGF1 gene promoter through transcription factor RFX1.

Publication Date: 2010 Feb 26 PMID: 20189986
Authors: Hsu, Y. C. - Liao, W. C. - Kao, C. Y. - Chiu, I. M.
Journal: J Biol Chem

Fibroblast growth factor 1 (FGF1) has been suggested to have an important role in cell growth, proliferation, and neurogenesis. Human FGF1 gene 1B promoter (-540 to +31)-driven green fluorescence (F1BGFP) has been shown to monitor endogenous FGF1 expression. F1BGFP could also be used to isolate neural stem/progenitor cells (NSPCs) from embryonic, neonatal and adult mouse brains or to isolate glioblastoma stem cells (GBM-SCs) from human glioblastoma tissues. Here, we present evidence that transcription factor RFX1 could bind the 18-bp cis-elements (-484 to -467) of the F1B promoter, modulate F1BGFP expression, endogenous FGF1 expression, and further regulate the maintenance of GBM-SCs. These observations were substantiated by using yeast-one hybrid assay, electrophoretic mobility shift assay, chromatin immunoprecipitation assay, gain- and loss-of-function assays, and neurosphere assays. Overexpression of RFX1 was shown to down-regulate FGF-1B mRNA expression and neurosphere formation in human glioblastoma cells, whereas RNAi knockdown of RFX1 demonstrated the opposite effects. Our findings provide insight into FGF1 gene regulation and suggest that the roles of FGF1 and RFX1 in the maintenance of GBM-SCs. RFX1 may negatively regulate the self-renewal of GBM-SCs through modulating FGF-1B and FGF1 expression levels by binding the 18-bp cis-elements of F1B promoter.

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Regulation of inositol 1,4,5-trisphosphate (IP3) receptors by cyclic AMP independent of cyclic AMP-dependent protein kinase.

Publication Date: 2010 Feb 26 PMID: 20189985
Authors: Tovey, S. C. - Dedos, S. G. - Rahman, T. - Taylor, E. J. - Pantazaka, E. - Taylor, C. W.
Journal: J Biol Chem

In HEK cells stably expressing type 1 receptors for parathyroid hormone (PTH), PTH causes a sensitization of IP(3) receptors (IP(3)R) to IP(3) that is entirely mediated by cAMP and requires cAMP to pass directly from type 6 adenylyl cyclase (AC6) to IP(3)R2. Using DT40 cells expressing single subtypes of mammalian IP(3)R, we demonstrate that high concentrations of cAMP similarly sensitize all IP(3)R isoforms to IP(3) by a mechanism that does not require cAMP-dependent protein kinase (PKA). IP(3) binding to IP(3)R2 is unaffected by cAMP, and sensitization is not mediated by the site through which ATP potentiates responses to IP(3). In single channel recordings from excised nuclear patches of cells expressing IP(3)R2, cAMP alone had no effect, but it increased the open probability of IP(3)R2 activated by a submaximal concentration of IP(3) alone or in combination with a maximally effective concentration of ATP. These results establish that cAMP itself increases the sensitivity of all IP(3)R subtypes to IP(3). For IP(3)R2, this sensitization results from cAMP binding to a novel site that increases the efficacy of IP(3). Using stably expressed short-hairpin RNA to reduce expression of the G-protein, Galphas, we demonstrate that attenuation of AC activity by loss of Galphas more substantially reduces sensitization of IP(3)R by PTH than does comparable direct inhibition of AC. This suggests that Galphas may also specifically associate with each AC-IP(3)R complex. We conclude that all three subtypes of IP(3)R are regulated by cAMP independent of PKA. In HEK cells, where IP(3)R2 selectively associates with AC6, Galphas also associates with the AC-IP(3)R signaling junction.

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SOD1 mutations targeting surface hydrogen bonds promote ALS without reducing apo-state stability.

Publication Date: 2010 Feb 26 PMID: 20189984
Authors: Bystrom, R. - Andersen, P. M. - Grobner, G. - Oliveberg, M.
Journal: J Biol Chem

IIn good accord with the protein-aggregation hypothesis for neurodegenerative diseases, ALS-associated SOD1 mutations are found to reduce structural stability or net repulsive charge. Moreover there are weak indications that the ALS disease progression rate is correlated with the degree of mutational impact on the apo-SOD1 structure. A bottleneck for obtaining more conclusive information about these structure-disease relationships, however, is the large intrinsic variability in patient survival times and insufficient disease statistics for the majority of ALS-provoking mutations. As an alternative test of the structure-disease relationship we focus here on the SOD1 mutations that appear to be outliers in the data set. The results identify several ALS-provoking mutations whose only effect on apo SOD1 is the elimination or introduction of a single charge, i.e., D76V/Y, D101N and N139D/K. The thermodynamic stability and folding behaviour of these mutants are indistinguishable from the wildtype control. Moreover, D101N is an outlier in the plot of stability loss vs. patient survival time by having rapid disease progression. Common to the identified mutations is that they truncate conserved salt-links and/or H-bond networks in the functional loops IV or VII. The results show that the local impact of ALS-associated mutations on the SOD1 molecule can sometimes overrun their global effects on apo-state stability and net repulsive charge, and point at the analysis of property outliers as an efficient strategy for mapping out new ALS-provoking features.

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Apoptosis protection by MCL-1 and BCL-2 modulation of inositol 1,4,5-trisphosphate receptor dependent Ca2+ signaling.

Publication Date: 2010 Feb 26 PMID: 20189983
Authors: Eckenrode, E. F. - Yang, J. - Velmurugan, G. V. - Foskett, J. K. - White, C.
Journal: J Biol Chem

Members of the Bcl-2 protein family play a central role in the regulation of apoptosis. An interaction between anti-apoptotic Bcl-x(L) and the endoplasmic reticulum (ER) localized inositol trisphosphate receptor Ca(2+) release channel (InsP(3)R) enables Bcl-x(L) to be fully efficacious as a anti-apoptotic mediator [White, C. et al. 2005. The endoplasmic reticulum gateway to apoptosis by Bcl-x(L) modulation of the InsP(3)R. Nature Cell Biology 7:1021-1028]. Physiologically, Bcl-x(L) binds to the InsP(3)R to enhance its gating and Ca(2+) signaling. Here we have discovered that structurally related proteins Bcl-2 and Mcl-1 function similarly. Bcl-2, Mcl-1 and Bcl-x(L) bind with comparable affinity to the carboxyl-termini of all three mammalian InsP(3)R isoforms with important functional consequences. Stable expression of Bcl-2 or Mcl-1 lowered ER Ca(2+) content and enhanced the rate of InsP(3)R -mediated Ca(2+) release in response sub-maximal InsP(3)R stimulation in permeabilized wild type DT40 cells but not in cells lacking InsP(3)R. In addition, expression of either Bcl-2 or Mcl-1 enhanced spontaneous InsP(3)R-dependent Ca(2+) oscillations and spiking in intact cells in the absence of agonist stimulation. Bcl-2 and Mcl-1 mediated protection from apoptosis induced by staurosporine or etoposide was enhanced in cells expressing InsP(3)R, demonstrating that their interactions with InsP(3)R enable Bcl-2 and Mcl-1 to be fully efficacious anti-apoptotic mediators. Our data suggest a molecular mechanism that is shared by several anti-apoptotic Bcl-2 proteins that provides apoptosis resistance by direct interactions at the ER with the InsP(3)R that impinges on cellular Ca(2+) homeostasis.

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Hearts of hypoxia-inducible factor prolyl 4-hydroxylase-2 hypomorphic mice show protection against acute ischemia-reperfusion injury.

Publication Date: 2010 Feb 25 PMID: 20185832
Authors: Hyvarinen, J. - Hassinen, I. E. - Sormunen, R. - Maki, J. M. - Kivirikko, K. I. - Koivunen, P. - Myllyharju, J.
Journal: J Biol Chem

Hypoxia-inducible factor (HIF) has a pivotal role in oxygen homeostasis and cardioprotection mediated by ischemic preconditioning. Its stability is regulated by HIF prolyl 4-hydroxylases (HIF-P4Hs), the inhibition of which is regarded as a promising strategy for treating diseases such as anemia and ischemia. We generated a viable Hif-p4h-2 hypomorph mouse line (Hif-p4h-2(gt/gt)) that expresses decreased amounts of wild-type Hif-p4h-2 mRNA, 8% in the heart, 15% in the skeletal muscle, 34-47% in the kidney, spleen, lung and bladder, 60% in the brain and 85% in the liver. These mice have no polycythemia and show no signs of the dilated cardiomyopathy or hyperactive angiogenesis observed in mice with broad-spectrum conditional Hif-p4h-2 inactivation. We focused here on the effects of chronic Hif-p4h-2 deficiency in the heart. Hif-1 and Hif-2 were stabilized and the mRNA levels of glucose transporter-1, several enzymes of glycolysis, pyruvate dehydrogenase kinase 1, angiopoietin-2 and adrenomedullin were increased in the Hif-p4h-2(gt/gt) hearts. When isolated Hif-p4h-2(gt/gt) hearts were subjected to ischemia-reperfusion, the recovery of mechanical function and coronary flow rate was significantly better than in wild type, while cumulative release of lactate dehydrogenase reflecting the infarct size was reduced. The pre-ischemic amount of lactate was increased and the ischemic vs. pre-ischemic [CrP]/[Cr] and [ATP] remained at higher levels in Hif-p4h-2(gt/gt) hearts indicating enhanced glycolysis and improved cellular energy state. Our data suggest that chronic stabilization of Hif-1alpha and Hif-2alpha by genetic knock-down of Hif-p4h-2 promotes cardioprotection by induction of many genes involved in glucose metabolism, cardiac function and blood pressure.

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Natural angiogenesis inhibitor signals through Erk5 activation of peroxisome proliferator activated receptor gamma (PPAR{gamma}).

Publication Date: 2010 Feb 25 PMID: 20185831
Authors: Biyashev, D. - Veliceasa, D. - Kwiatek, A. - Sutanto, M. M. - Cohen, R. N. - Volpert, O. V.
Journal: J Biol Chem

Erk-5, a member of the MAPK superfamily, has the catalytic domain similar to Erk1/2 and a unique C-terminal domain enabling binding with transcription factors. Aberrant vascularization in the Erk5 null mice suggested a link to angiogenesis. Ectopic expression of constitutively active Erk5 blocks endothelial cell morphogenesis and causes HIF1-alpha destabilization/degradation. However the mechanisms, by which endogenous Erk5 regulates angiogenesis, remained unknown. We show that Erk5 and its activating kinase MEK5 are the upstream mediators of the anti-angiogenic signal by the natural angiogenesis inhibitor, pigment epithelial-derived factor (PEDF). We demonstrate that Erk5 phosphorylation allows activation of PPARgamma transcription factor by displacement of SMRT co-repressor. PPARgamma, in turn is critical for NFkappaB activation, PEDF-dependent apoptosis and anti-angiogenesis. The dominant negative MEK5 mutant and Erk5 shRNA diminished PEDF-dependent apoptosis, inhibition of the endothelial cell chemotaxis and angiogenesis. This is the first evidence of Erk5 dependent transduction of the signals by endogenous angiogenesis inhibitor.

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Slc4a11 prevents osmotic imbalance leading to corneal endothelial dystrophy, deafness, and polyuria.

Publication Date: 2010 Feb 25 PMID: 20185830
Authors: Groeger, N. - Froehlich, H. - Maier, H. - Olbrich, A. - Kostin, S. - Braun, T. - Boettger, T.
Journal: J Biol Chem

Maintenance of ion-concentration gradients is essential for the function of many organs including the kidney, the cornea, and the inner ear. Ion concentrations and fluid content in the cornea are regulated by endothelial cells that separate the collagenous avascular corneal stroma from the anterior eye-chamber. Failure to maintain correct ion concentrations leads to swelling and destruction of the cornea. In the inner ear, the Stria vascularis is responsible to generate proper ion concentrations in the endolymphe, which is essential for hearing. Mutations of SLC4A11 in humans lead to syndromes associated with corneal dystrophy and perceptive deafness. The molecular mechanisms underlying these symptoms are poorly understood, impeding therapeutic interventions. The ion-transporter SLC4A11 mediates sodium dependent transport of borate as well as flux of sodium and hydroxyl ions in vitro. Here, we show that SLC4A11 is expressed in the endothelial cells of the cornea where it prevents severe morphological changes of the cornea caused by increased sodium chloride concentrations in the stroma. In the inner ear, SLC4A11 is located in fibrocytes underlying the Stria vascularis. Loss of SLC4A11 leads to morphological changes in the fibrocytes and deafness. We demonstrate that SLC4A11 is essential for the generation of the endocochlear potential but not for regulation of potassium concentrations in the endolymph. In the kidney, SLC4A11 is expressed in the thin descending limb of Henle's loop. SLC4A11 is essential for urinary concentration, suggesting that SLC4A11 participates in the countercurrent multiplication that concentrates urine in the kidney medulla.

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Identification of Ror2 as a hypoxia inducible factor (HIF) target in von Hippel-Lindau (VHL)-associated renal cell carcinoma.

Publication Date: 2010 Feb 25 PMID: 20185829
Authors: Wright, T. M. - Rathmell, W. K.
Journal: J Biol Chem

Ror2 is an orphan receptor tyrosine kinase with expression normally restricted to early stages of development. However, emerging evidence has placed aberrantly expressed Ror2, leading to an invasive phenotype, in several cancers including renal cell carinoma (RCC). Though Ror2 is currently identified as upregulated in an assortment of cancers, neither the regulatory role or mechanism of action have been delineated. We sought to place Ror2 in RCC's most commonly mutated pathway, the loss of the tumor suppressor von Hippel-Lindau, VHL, which causes hypoxia induced factor (HIF)-1alpha and 2alpha stabilization and the transcriptional activation of a broad repertoire of response genes. We found that Ror2 was indeed associated with pVHL loss in RCC as well as with VHL somatic mutations tightly coordinated with the induction of RCC. Additionally, knockdown and rescue analysis of HIF expression suggests that Ror2 is dependent on the pathologic stabilization of either HIF-1alpha or HIF-2alpha. Subsequent evaluation of the Ror2 promoter suggests that HIF-2alpha and its dimerization partner, aryl hydrocarbon nuclear transferase, ARNT, localize to the Ror2 promoter via a cryptic transcriptional element. This data substantiates a unique regulation pattern for Ror2 in the VHL-HIF axis that has the potential to be applied to other cancer etiologies.

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Activation of acid sensing ion channel 1a (ASIC1a) by surface trafficking.

Publication Date: 2010 Feb 25 PMID: 20185828
Authors: Chai, S. - Li, M. - Branigan, D. - Xiong, Z. G. - Simon, R. P.
Journal: J Biol Chem

Acid-sensing ion channels (ASICs) are voltage-independent Na+ channels activated by extracellular protons. ASIC1a is expressed in neurons in mammalian brain and is implicated in long-term potentiation of synaptic transmission that contributes to learning and memory. In ischemic brain injury, however, activation of this Ca2+-permeable channel plays a critical role in acidosis-mediated, glutamate-independent, Ca2+-toxicity. We report here the identification of insulin as a regulator of ASIC1a surface expression. In modeled ischemia using CHO cells, serum depletion caused a significant increase in ASIC1a surface expression which resulted in the potentiation of ASIC1a activity. Among the components of serum, insulin was identified as the key factor that maintains a low level of ASIC1a on the plasma membrane. Neurons subjected to insulin-depletion increased surface expression of ASIC1a with resultant potentiation of ASIC1a currents. Intracellularly, ASIC1a is predominantly localized to the ER in CHO cells and this intracellular localization is also observed in neurons. Under conditions of serum or insulin depletion, the intracellular ASIC1a is translocated to the cell surface increasing the surface expression level. These results reveal an important trafficking mechanism of ASIC1a that is relevant to both the normal physiology and pathological activity of this channel.

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Transcriptional regulation of the base excision repair pathway by BRCA1.

Publication Date: 2010 Feb 25 PMID: 20185827
Authors: Saha, T. - Rih, J. K. - Roy, R. - Ballal, R. - Rosen, E. M.
Journal: J Biol Chem

Inactivation of the breast cancer susceptibility gene BRCA1 plays a significant role in the development of a subset of breast cancers, although the major tumor suppressor function of this gene remains unclear. Previously, we showed that BRCA1 induces antioxidant response gene expression and protects cells against oxidative stress. We now report that BRCA1 stimulates the base excision repair pathway, a major mechanism for the repair of oxidized DNA, by stimulating the activity of key BER enzymes, including 8-oxoguanine DNA glycosylase (OGG1), the DNA glycosylase NTH1, and the apurinic endonuclease REF1/APE1, in human breast carcinoma cells. The increase in BER enzyme activity appears to be due, primarily, to an increase in enzyme expression. The ability of BRCA1 to stimulate the expression of the three BER enzymes and to enhance NTH1 promoter activity was dependent upon the octamer-binding transcription factor OCT1. Finally, we found that OGG1, NTH1, and REF1/APE1 each contribute to the BRCA1 protection against oxidative stress due to hydrogen peroxide and that hydrogen peroxide stimulates the expression of BRCA1 and the three BER enzymes. These findings identify a novel mechanism through which BRCA1 may regulate the repair of oxidative DNA damage.

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A role for Huntington's disease protein in dendritic RNA granules.

Publication Date: 2010 Feb 25 PMID: 20185826
Authors: Savas, J. N. - Ma, B. - Deinhardt, K. - Culver, B. P. - Restituito, S. - Wu, L. - Belasco, J. G. - Chao, M. V. - Tanese, N.
Journal: J Biol Chem

Regulated transport and local translation of mRNA in neurons are critical for modulating synaptic strength, maintaining proper neural circuitry and the establishment of long-term memory. Neuronal RNA granules are ribonucleoprotein particles that serve to transport mRNA along microtubules and control local protein synthesis in response to synaptic activity. Studies suggest that neuronal RNA granules share similar structures and functions with somatic P-bodies. We recently reported that the Huntington's disease protein huntingtin (Htt) associates with Argonaute (Ago) and localizes to cytoplasmic P-bodies, which serve as sites of mRNA storage, degradation and small RNA-mediated gene silencing. Here we report that wild-type Htt associates with Ago2 and components of neuronal granules and co-traffics with mRNA in dendrites. Htt was found to co-localize with RNA containing the 3'UTR sequence of known dendritically targeted mRNAs. Knockdown of Htt in neurons caused altered localization of mRNA. When tethered to a reporter construct, Htt down-regulated reporter gene expression in a manner dependent on Ago2, suggesting that Htt may function to repress translation of mRNAs during transport in neuronal granules.

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Glycosylation of wall teichoic acid in Staphylococcus aureus by TarM.

Publication Date: 2010 Feb 25 PMID: 20185825
Authors: Xia, G. - Maier, L. - Sanchez-Carballo, P. - Li, M. - Otto, M. - Holst, O. - Peschel, A.
Journal: J Biol Chem

Wall teichoic acid (WTA) glycopolymers are major constituents of cell envelopes in Staphylococcus aureus and related Gram-positive bacteria with important roles in cell wall maintenance, susceptibility to antimicrobial molecules, biofilm formation, and host interaction. Most S. aureus strains express poly-ribitolphosphate (Rbo-P) WTA substituted with D-alanine and N-acetylglucosamine (GlcNAc). WTA sugar modifications are highly variable and have been implicated in bacterial phage susceptibility and immunogenicity but the pathway and enzymes of staphylococcal WTA glycosylation have remained unknown. Revisiting the structure of S. aureus RN4220 WTA by NMR analysis revealed the presence of canonical Rbo-P WTA bearing only alpha-linked GlcNAc substituents. A RN4220 transposon mutant resistant to WTA-dependent phages was identified and shown to produce altered WTA, which exhibited faster electrophoretic migration and lacked completely the WTA alpha-GlcNAc residues. Disruption of a gene of unknown function, renamed tarM, was responsible for this phenotype. Recombinant TarM was capable of glycosylating WTA in vitro in a UDP-GlcNAc dependent manner thereby confirming its WTA GlcNAc transferase activity. Deletion of the last seven amino acids from the C-terminus abolished the activity of TarM. tarM-related genes were found in the genomes of several WTA-producing bacteria suggesting that TarM-mediated WTA glycosylation is a general pathway in Gram-positive bacteria. Our study represents a basis for dissecting the biosynthesis and function of glycosylated WTA in S. aureus and other bacteria.

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AIRAP: a new human heat shock gene regulated by heat shock factor 1.

Publication Date: 2010 Feb 25 PMID: 20185824
Authors: Rossi, A. - Trotta, E. - Brandi, R. - Arisi, I. - Coccia, M. - Santoro, M. G.
Journal: J Biol Chem

Heat shock factor-1 (HSF1) is the central regulator of heat-induced transcriptional responses leading to rapid expression of molecular chaperones which protect mammalian cells against proteotoxic stress. The main targets for HSF1 are specific promoter elements (HSE) located upstream of heat shock genes encoding a variety of heat shock proteins, including HSP70, HSP90, HSP27 and other proteins of the network. Herein we report that the zinc-finger AN1-type domain-2a gene, also known as AIRAP, behaves as a canonical heat shock gene, whose expression is temperature-dependent and strictly controlled by HSF1. Transcription is triggered at temperatures above 40 degrees C in different types of human cancer and primary cells, including peripheral blood monocytes. As shown by ChIP analysis, HSF1 is recruited to the AIRAP promoter rapidly after heat treatment, with a kinetics that parallels HSP70 promoter HSF1-recruitment. In transfection experiments HSF1-silencing abolished heat-induced AIRAP promoter-driven transcription, which could be rescued by exogenous Flag-HSF1 expression. The HSF1-binding HSE-sequence in the AIRAP promoter critical for heat-induced transcription was identified. Since its expression is induced at febrile temperatures in human cells, AIRAP may represent a new potential component of the protective response during fever in humans.

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Epigenetic repression of PDLIM2: implications for the biology and treatment of breast cancer.

Publication Date: 2010 Feb 25 PMID: 20185823
Authors: Qu, Z. - Fu, J. - Yan, P. - Hu, J. - Cheng, S. - Xiao, G.
Journal: J Biol Chem

The NF-kappaB transcription factor plays a pivotal role in breast cancer progression and therapy resistance. However, the mechanisms by which the tightly regulated NF-kappaB becomes constitutively activated during breast cancer pathogenesis remain obscure. Here, we report that PDLIM2, an essential terminator of NF-kappaB activation, is repressed in both estrogen receptor (ER)-positive and ER-negative breast cancer cells, suggesting one important mechanism for the constitutive activation of NF-kappaB. Indeed, PDLIM2 re-expression inhibited constitutive NF-kappaB activation and expression of NF-kappaB-targeted genes in those breast cancer cells. Importantly, the PDLIM2, but not its mutants defective in NF-kappaB termination, could suppress in vitro anchorage-independent growth and in vivo tumor formation of those malignant breast cells. In addition, we have shown that the PDLIM2 repression involves promoter methylation. Accordingly, treatment of the breast cancer cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) reverses the methylation of the PDLIM2 promoter, restored PDLIM2 expression and suppressed tumorigenicities of human breast cancer cells both in vitro and in vivo. These studies thus provide important mechanistic insights into breast cancer pathogenesis. These studies also suggest a tumor suppression function of PDLIM2 and a therapeutic strategy for breast cancer.

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DAXX is a new AIRE interacting protein.

Publication Date: 2010 Feb 25 PMID: 20185822
Authors: Meloni, A. - Fiorillo, E. - Corda, D. - Incani, F. - Serra, M. L. - Contini, A. - Cao, A. - Rosatelli, M. C.
Journal: J Biol Chem

The AIRE protein plays a remarkable role as a regulator of the central tolerance by controlling the promiscuous expression of tissue-specific Ags (TSAs) in thymic medullary epithelial cells (mTECs). Defects in the AIRE gene cause the Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a rare disease frequent in Iranian Jews, Finns and Sardinian population. To this day the precise function of AIRE protein in regulating transcription as well as its interacting proteins are still yet to be entirely clarified. The knowledge of novel AIRE interactors could lead to gain a more comprehensive functional picture of its biological role and serve to better address some of foremost autoimmunity-related questions. In this study we have used yeast two hybrid system to identify AIRE interacting proteins. The Yeast Two Hybrid approach led us to the discovery of a new AIRE interacting protein called DAXX. The protein is known to be a multifunctional adaptor with functions both in apoptosis and in transcription regulation pathways. The interaction between AIRE and DAXX has been validated by in vivo coimmunoprecipitation analysis and colocalization study in mammalian cells. Moreover it has been investigated through transactivation tests which highlighted a strong identifiable repressive role of DAXX on AIRE transcriptional activity.

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A pathway involving FXR and shp positively regulates hepatic SIRT1 levels via MIR-34A inhibition.

Publication Date: 2010 Feb 25 PMID: 20185821
Authors: Lee, J. - Padhye, A. - Sharma, A. - Song, G. - Miao, J. - Mo, Y. Y. - Wang, L. - Kemper, J. K.
Journal: J Biol Chem

SIRT1 is a NAD-dependent deacetylase that is critically involved in diverse cellular processes including metabolic disease, cancer, and possibly aging. Despite extensive studies on SIRT1 function, how SIRT1 levels are regulated remains relatively unknown. Here we report that the nuclear bile acid receptor FXR inhibits microRNA-34a (miR-34a) in the liver, which results in a positive regulation of SIRT1 levels. Activation of FXR by the synthetic agonist GW4064 decreases hepatic miR-34a levels in normal mice and consistently, hepatic miR-34a levels are elevated in FXR null mice. FXR induces expression of SHP, an orphan nuclear receptor and transcriptional corepressor, which in turn results in repression of p53, a key activator of the miR-34a gene, by inhibiting p53 occupancy at the promoter. MiR-34a decreased SIRT1 levels by binding to the 3' untranslated region of SIRT1 mRNA and adenoviral-mediated overexpression of miR-34a substantially decreased SIRT1 protein levels in mouse liver. Remarkably, miR-34a levels were elevated and SIRT1 protein levels were reduced in diet-induced obese mice, and FXR activation in these mice reversed the miR-34a and SIRT1 levels, indicating an intriguing link between FXR activation, decreased miR-34a, and subsequently, increased SIRT1 levels. Our study demonstrates an unexpected role of the FXR/SHP pathway in controlling SIRT1 levels via miR-34a inhibition and that elevated miR-34a levels in obese mice contribute to decreased SIRT1 levels. Manipulation of this regulatory network may be useful for treating diseases of aging, such as metabolic disease and cancer.

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Foxp1/2/4-NuRD interactions regulate gene expression and epithelial injury response in the lung via regulation of IL-6.

Publication Date: 2010 Feb 25 PMID: 20185820
Authors: Chokas, A. L. - Trivedi, C. M. - Lu, M. M. - Tucker, P. W. - Li, S. - Epstein, J. A. - Morrisey, E. E.
Journal: J Biol Chem

To determine the underlying mechanism of Foxp1/2/4 mediated transcriptional repression, a yeast two-hybrid screen was performed which identified p66beta, a transcriptional repressor and component of the NuRD chromatin remodeling complex. We show that direct interactions between Foxp1/4 and p66beta are mediated by the CR2 domain within p66beta and the zinc finger/leucine zipper repression domain found in Foxp1/2/4. These direct interactions are functionally relevant as over-expression of p66beta in combination with Foxp factors cooperatively represses Foxp target gene expression, while loss of p66 and Foxp factors results in de-repression of endogenous Foxp target genes in lung epithelial cells. Moreover, the NuRD components HDAC1/2 associate in a macromolecular complex with Foxp proteins and loss of expression or inhibition of HDAC1/2 activity leads to de-repression of Foxp target gene expression. Importantly, we show in vivo that Foxp1 and HDAC2 act cooperatively to regulate expression of the cytoprotective cytokine IL-6, which results in increased resistance to hyperoxic lung injury in Foxp1/HDAC2 compound mutant animals. These data reveal an important interaction between the Foxp transcription factors and the NuRD chromatin remodeling complex that modulates transcriptional repression critical for the lung epithelial injury response.

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TRAF3 controls activation of the canonical and alternative NF{kappa}B by the lymphotoxin beta receptor.

Publication Date: 2010 Feb 25 PMID: 20185819
Authors: Bista, P. - Zeng, W. - Ryan, S. - Bailly, V. - Browning, J. - Lukashev, M. E.
Journal: J Biol Chem

Components of lymphotoxin beta receptor (LTBR)-associated signaling complexes, including TRAF2, TRAF3, NIK, IKK1, and IKK2 have been shown to participate in the coupling of LTBR to NFkappaB. Here, we report that TRAF3 functions as a negative regulator of LTBR signaling via both canonical and non-canonical NFkappaB pathways by two distinct mechanisms. Analysis of NFkappaB signaling in cell lines with functionally intact NFkappaB pathway but lacking LTBR-mediated induction of NFkappaB target genes revealed an inverse association of cellular TRAF3 levels with LTBR-specific defect in canonical NFkappaB activation. Increased expression of TRAF3 correlated with its increased recruitment to LTBR-induced signaling complexes, decreased recruitment of TRAF2, and attenuated phosphorylation of IkappaBalpha and RelA. In contrast, activation of NFkappaB by TNF did not depend on TRAF3 levels. siRNA-mediated depletion of TRAF3 promoted recruitment of TRAF2 and IKK1 to activated LTBR, enabling LTBR-inducible canonical NFkappaB signaling and NFkappaB target gene expression. TRAF3 knockdown also increased mRNA and protein expression of several non-canonical NFkappaB components, including NFkappaB2/p100, RelB, and NIK, accompanied by processing of NFkappaB2/p100 into p52. These effects of TRAF3 depletion did not require LTBR signaling and were consistent with autonomous activation of the non-canonical NFkappaB pathway. Our data illustrate the function of TRAF3 as a dual-mode repressor of LTBR signaling that controls activation of canonical NFkappaB, and de-repression of the intrinsic activity of non-canonical NFkappaB. Modulation of cellular TRAF3 levels may thus contribute to regulation of NFkappaB-dependent gene expression by LTBR by affecting the balance of LTBR-dependent activation of canonical and non-canonical NFkappaB pathways.

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Direct ubiquitination of {beta}-catenin by siah-1 and regulation by the exchange factor TBL1.

Publication Date: 2010 Feb 24 PMID: 20181957
Authors: Dimitrova, Y. N. - Li, J. - Lee, Y. T. - Rios-Esteves, J. - Friedman, D. B. - Choi, H. J. - Weis, W. I. - Wang, C. Y. - Chazin, W. J.
Journal: J Biol Chem

beta-Catenin is a key component of the Wnt signaling pathway that functions as a transcriptional co-activator of Wnt-target genes. Upon UV-induced DNA damage, beta-catenin is recruited for poly-ubiquitination and subsequent proteasomal degradation by a unique, p53-induced SCF-like complex (SCF(TBL1)), comprised of Siah-1, Siah-1-interacting protein (SIP), Skp1, Transducin beta-like 1 (TBL1) and Adenomatous Polyposis Coli (APC). Given the complexity of the various factors involved and the novelty of ubiquitination of the non-phosphorylated beta-catenin substrate, we have investigated Siah-1 mediated ubiquitination of beta-catenin in vitro and in cells. Over-expression and purification protocols were developed for each of the SCF(TBL1) proteins, enabling a systematic analysis of beta-catenin ubiquitination using an in vitro ubiquitination assay. This study revealed that Siah-1 alone was able to poly-ubiquitinate beta-catenin. In addition, TBL1 was shown to play a role in protecting beta-catenin from Siah-1 ubiquitination in vitro and from Siah-1-targeted proteasomal degradation in cells. Siah-1 and TBL1 were found to bind to the same armadillo repeat domain of beta-catenin, suggesting that poly-ubiquitination of beta-catenin is regulated by competition between Siah-1 and TBL1 during Wnt signaling.

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Structural basis of binding of P-body associated proteins GW182 and Ataxin-2 by the Mlle domain of poly(A)-binding protein.

Publication Date: 2010 Feb 24 PMID: 20181956
Authors: Kozlov, G. - Safaee, N. - Rosenauer, A. - Gehring, K.
Journal: J Biol Chem

Poly(A) binding protein (PABPC1) is involved in multiple aspects of mRNA processing and translation. It is a component of RNA stress granules and binds the RNA-induced silencing complex (RISC) to promote degradation of silenced mRNAs. Here, we report the crystal structures of the C-terminal Mlle (or PABC) domain in complex with peptides from GW182 (TNRC6C) and Ataxin-2. The structures reveal overlapping binding sites but with unexpected diversity in the peptide conformation and residues involved in binding. The mutagenesis and binding studies show low to sub-micromolar binding affinity with overlapping but distinct specificity determinants. These results rationalize the role of the Mlle domain of PABPC1 in miRNA-mediated mRNA deadenylation and suggest a more general function in the assembly of cytoplasmic RNA granules.

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Arabidopsis AtSerpin1: crystal structure and in vivo interaction with its target protease responsive to desiccation-21 (RD21).

Publication Date: 2010 Feb 24 PMID: 20181955
Authors: Lampl, N. - Budai-Hadrian, O. - Davydov, O. - Joss, T. V. - Harrop, S. J. - Curmi, P. M. - Roberts, T. H. - Fluhr, R.
Journal: J Biol Chem

In animals, protease inhibitors of the serpin family are associated with many physiological processes including blood coagulation and innate immunity. Serpins feature a reactive center loop (RCL), which displays a protease target sequence as a bait. RCL cleavage results in an irreversible, covalent serpin-protease complex. AtSerpin1 is an Arabidopsis protease inhibitor that is expressed ubiquitously throughout the plant. The X-ray crystal structure of recombinant AtSerpin1 in its native stressed conformation was determined at 2.2 A. The electrostatic surface potential below the RCL was found to be highly positive, while the breach region critical for RCL insertion is an unusually open structure. AtSerpin1 accumulates in plants as a full-length and a cleaved form. Fractionation of seedling extracts by non-reducing SDS-PAGE revealed the presence of an additional slower-migrating complex that was absent when leaves were treated with the specific cysteine protease inhibitor E-64. Significantly, RESPONSIVE TO DESICCATION-21 (RD21) was the major protease labeled with the E-64 derivative DCG-04 in wild type extracts but not in extracts of mutant plants constitutively over expressing AtSerpin1, indicating competition. Fractionation by non-reducing SDS-PAGE followed by immunoblotting with RD21-specific antibody revealed that the protease accumulated both as a free enzyme and in a complex with AtSerpin1. Importantly, both RD21 and AtSerpin1 knock-out mutants lacked the serpin protease complex. The results establish that the major Arabidopsis plant serpin interacts with RD21. This is the first report of the structure and in vivo interaction of a plant serpin with its target protease.

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SENP3-mediated de-conjugation of SUMO2/3 from PML is correlated with accelerated cell proliferation under mild oxidative stress.

Publication Date: 2010 Feb 25 PMID: 20181954
Authors: Han, Y. - Huang, C. - Sun, X. - Xiang, B. - Wang, M. - Yeh, E. T. - Chen, Y. - Li, H. - Shi, G. - Cang, H. - Sun, Y. - Wang, J. - Wang, W. - Gao, F. - Yi, J.
Journal: J Biol Chem

Small ubiquitin-like modifier (SUMO) 2/3 is known to conjugate to substrates in response to a variety of cellular stresses. However, whether and how SUMO2/3-specific proteases are involved in de-conjugation under cell stress is unclear. Here, we show that low doses of hydrogen peroxide (H2O2) induce an increase of the SENP3 protein, which removes SUMO2/3 from PML. Low-dose H2O2 causes SENP3 to co-localize with PML bodies and reduces the number of PML bodies in a SENP3-dependent manner. Furthermore, de-conjugation of SUMO2/3 from PML is responsible for the accelerated cell proliferation caused by low-dose H2O2. Knocking down PML promotes basal cell proliferation as expected. This can be reversed by reconstitution with wild type PML, but not its mutant lacking SUMOylation, indicating that only the SUMOylated PML can play an inhibitory role for cell proliferation. Thus, SENP3 appears to be a key mediator in mild oxidative stress-induced cell proliferation via regulation of the SUMOylation status of PML. Furthermore, SENP3 is over-expressed in a variety of primary human cancers including colon adenocarcinoma in which PML is hypo-SUMOylated. These results reveal an important role of SENP3 and the SUMOylation status of PML in the regulation of cell proliferation under oxidative stress.

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Structural basis of dimerization-dependent ubiquitination by the SCFFBX4 ubiquitin ligase.

Publication Date: 2010 Feb 24 PMID: 20181953
Authors: Li, Y. - Hao, B.
Journal: J Biol Chem

The F-box proteins are the substrate-recognition subunits of the SCF (Skp1-Cul1-Rbx1-F box protein) ubiquitin ligase complexes that control the stability of numerous regulators in eukaryotic cells. Here we show that dimerization of the F-box protein Fbx4 is essential for SCFFbx4 ubiquitination activity toward the telomere regulator Pin2. The crystal structure of Fbx4 in complex with an adaptor protein Skp1 reveals an antiparallel dimer configuration in which the linker domain of Fbx4 interacts with the C-terminal substrate-binding domain of the other protomer, while the C-terminal domain of the protein adopts a compact alpha/beta fold distinct from those of known F-box proteins. Biochemical studies indicate that both the N-terminal domain and a loop connecting the linker and C-terminal domain of Fbx4 are critical for the dimerization and activation of the protein. Our findings provide a framework for understanding the role of F-box dimerization in the SCF-mediated ubiquitination reaction.

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Supramolecular protein engineering. Design of zinc-stapled insulin hexamers as a long-acting depot.

Publication Date: 2010 Feb 24 PMID: 20181952
Authors: Phillips, N. B. - Wan, Z. L. - Whittaker, L. - Hu, S. Q. - Huang, K. - Hua, Q. X. - Whittaker, J. - Ismail-Beigi, F. - Weiss, M. A.
Journal: J Biol Chem

Bottom-up control of supramolecular protein assembly can provide a therapeutic nanobiotechnology. We demonstrate that insulin's pharmacological properties can be enhanced by design of "zinc staples" between hexamers. Paired (i, i+4) His substitutions were introduced at an alpha-helical surface. The crystal structure contains both classical axial zinc ions and novel zinc ions at hexamer-hexamer interfaces. Although soluble at pH 4, the combined electrostatic effects of the substitutions and bridging zinc ions cause isoelectric precipitation at neutral pH. Following subcutaneous injection in a diabetic rat, the analog effected glycemic control with time course similar to that of long-acting formulation Lantus(R) Relative to Lantus, however, the analog discriminates at least 30-fold more stringently between the insulin receptor and mitogenic IGF receptor. Because aberrant mitogenic signaling may be associated with elevated cancer risk, such enhanced specificity may improve safety. Zinc stapling provides a general strategy to modify the pharmacokinetic and biological properties of a subcutaneous protein depot.

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Probing DNA and ATP-mediated conformational changes in the muts family of mispair recognition proteins using deuterium exchange mass spectrometry.

Publication Date: 2010 Feb 24 PMID: 20181951
Authors: Mendillo, M. L. - Putnam, C. D. - Mo, A. O. - Jamison, J. W. - Li, S. - Woods, V. L. Jr - Kolodner, R. D.
Journal: J Biol Chem

We have performed Deuterium Exchange Mass Spectrometry (DXMS) to probe the conformational changes that the bacterial MutS homodimer and the homologous eukaryotic heterodimer Msh2-Msh6 undergo when binding to ATP or DNA. The DXMS data support the view that high affinity binding to mispair-containing DNA and low affinity binding to fully base paired DNA both involve forming rings by MutS protein family dimers around the DNA; however, mispair binding protects additional regions from deuterium exchange. DXMS also reveals two distinct conformations upon binding one or two ATP molecules and that binding of two ATP molecules propagates conformational changes to other regions of the protein complexes. The regions showing major changes in deuterium exchange upon ATP binding tend to occur in regions distinct from those involved in DNA binding, suggesting that although communication occurs between DNA and nucleotide binding, sliding clamps formed by binding both ATP and mispairs could result from the simultaneous action of two independent conformational changes.

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Rho kinase inhibition rescues the endothelial cell cerebral cavernous malformation phenotype.

Publication Date: 2010 Feb 24 PMID: 20181950
Authors: Borikova, A. L. - Dibble, C. F. - Sciaky, N. - Welch, C. M. - Abell, A. N. - Bencharit, S. - Johnson, G. L.
Journal: J Biol Chem

Cerebral cavernous malformations (CCM) are vascular lesions causing seizures and stroke. Mutations causing inactivation of one of three genes, ccm1, 2 or 3 are sufficient to induce vascular endothelial cell defects resulting in CCM. Herein, we show that loss of expression of the CCM1, 2 or 3 proteins cause a marked increase in expression of the GTPase RhoA. Live cell imaging with a RhoA-specific biosensor demonstrates increased RhoA activity with loss of CCM1, 2 or 3, with an especially pronounced RhoA activation in both the cytosol and nucleus with the loss of CCM1 expression. Increased RhoA activation was associated with Rho kinase-dependent phosphorylation of myosin-light chain 2. Functionally, loss of CCM1, 2, or 3 inhibited endothelial cell vessel-like tube formation and extracellular matrix invasion, each of which is rescued by chemical inhibition or shRNA knockdown of Rho kinase. The findings, for the first time, define a signaling network for CCM1, 2 and 3 in CCM pathology, whereby loss of CCM1, 2 or 3 protein expression results in increased RhoA activity, with the activation of Rho kinase responsible for endothelial cell dysregulation. The results define Rho kinase as a therapeutic target to rescue endothelial cells from the loss of CCM protein function.

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Interaction between periostin and BMP-1 promotes proteolytic activation of lysyl oxidase.

Publication Date: 2010 Feb 24 PMID: 20181949
Authors: Maruhashi, T. - Kii, I. - Saito, M. - Kudo, A.
Journal: J Biol Chem

Intra- and intermolecular covalent cross-linking between collagen fibrils, catalyzed by lysyl oxidase (LOX), determines the mechanical properties of connective tissues; however, mechanisms that regulate the collagen cross-linking according to tissue specificity are not well understood. Here we show that periostin, a secretory protein in the dense connective tissues, promotes the activation of LOX. Previous studies showed that periostin null mice exhibit reduced collagen cross-linking in their femurs, periosteum, infarcted myocardium, and tendons. Presently, we showed that active LOX protein, formed by cleavage of its propeptide by bone morphogenetic protein-1 (BMP-1), was decreased in calvarial osteoblast cells derived from periostin null mice. Overexpression of periostin promoted the proteolytic cleavage of the propeptide, which promotion increased the amount of active LOX protein. The results of a co-immunoprecipitation and solid phase binding assays revealed that periostin interacted with BMP-1. Furthermore, this interaction probably resulted in enhanced deposition of BMP-1 on the extracellular matrix, suggesting that this enhanced deposition would lead to cleavage of the propeptide of LOX. Thus, we demonstrated that periostin supported BMP-1-mediated proteolytic activation of LOX on the extracellular matrix, which promoted collagen cross-linking.

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Unraveling the specificity of heparanase utilizing synthetic substrates.

Publication Date: 2010 Feb 24 PMID: 20181948
Authors: Peterson, S. B. - Liu, J.
Journal: J Biol Chem

Heparanase is a promising anticancer target because of its involvement in cancer invasion and metastasis. Heparanase cleaves heparan sulfate (HS), a sulfated polysaccharide, and activates a series of HS-mediated cell proliferation and angiogenesis processes. Understanding the substrate specificity of heparanase will aid the discovery of heparanase inhibitors. Here, we sought to determine the specificity of heparanase using synthetic polysaccharide substrates. The substrates were prepared using purified HS biosynthetic enzymes. Using these substrates, we were able to dissect the structural moieties required for heparanase. Our data suggest that heparanase cleaves the linkage between a GlcUA unit and a N-sulfo glucosamine unit carrying either a 3-O-sulfo or a 6-O-sulfo group. In addition, heparanase cleaves the linkage of a GlcUA unit and N-sulfo glucosamine unit with a 2-O-sulfated GlcUA residue, not a 2-O-sulfated IdoUA residue, in proximity. We also discovered that the polysaccharide with repeating disaccharide units of IdoUA2S-GlcNS inhibits the activity of heparanase. Our findings advance the understanding of the substrate specificity of heparanase and identify a lead compound for developing polysaccharide-based heparanase inhibitors.

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Interaction of syndecan and alpha6beta4 integrin cytoplasmic domains: Regulation of ErbB2-mediated integrin activation.

Publication Date: 2010 Feb 24 PMID: 20181947
Authors: Wang, H. - Leavitt, L. - Ramaswamy, R. - Rapraeger, A. C.
Journal: J Biol Chem

The alpha6beta4 integrin is a laminin332 (LN332) receptor central to the formation of hemidesmosomes in epithelial layers. However, the integrin becomes phosphorylated by keratinocytes responding to EGF in skin wounds, or by squamous cell carcinomas that overexpress/hyperactivate the tyrosine kinases ErbB2, EGFR or c-Met. We show here that the beta4-dependent signaling in A431 human squamous carcinoma cells is dependent on the syndecan family of matrix receptors. Yeast two-hybrid analysis identifies an interaction within the distal third (amino acids 1473-1752) of the beta4 cytoplasm domain and the conserved C2 region of the syndecan cytoplasmic domain. Via its C2 region, Sdc1 forms a complex with the alpha6beta4 integrin along with the receptor tyrosine kinase ErbB2 and the cytoplasmic kinase Fyn in A431 cells. Engagement of LN332, or clustering the alpha6beta4 integrin with integrin-specific antibodies, causes phosphorylation of ErbB2, Fyn, and the beta4 subunit, as well as activation of PI-3K and Akt and their assimilation into this complex. This leads to PI-3K-dependent cell spreading and Akt-dependent protection from apoptosis. This is disrupted by RNAi silencing of Sdc1, but can be rescued by mouse Sdc1 or Sdc4, but not by syndecan mutants lacking their C-terminal C2 region. This disruption does not prevent the phosphorylation of ErbB2 or Fyn, but blocks the Fyn-mediated phosphorylation of the beta4 tail. We propose that syndecans engage the distal region of the beta4 cytoplasmic domain and bring it to the plasma membrane where it can be acted upon by src-family kinases.

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Vinculin facilitates cell invasion into 3D collagen matrices.

Publication Date: 2010 Feb 24 PMID: 20181946
Authors: Mierke, C. T. - Kollmannsberger, P. - Zitterbart, D. P. - Diez, G. - Koch, T. M. - Marg, S. - Ziegler, W. H. - Goldmann, W. H. - Fabry, B.
Journal: J Biol Chem

The cytoskeletal protein vinculin contributes to the mechanical link of the contractile actomyosin cytoskeleton to the extracellular matrix (ECM) through integrin receptors. In addition, vinculin modulates the dynamics of cell adhesions and is associated with decreased cell motility on two-dimensional (2D) ECM substrates. The effect of vinculin on cell invasion through dense three-dimensional (3D) ECM gels is unknown. Here, we report how vinculin expression affects cell invasion into 3D collagen matrices. Cell motility was investigated in vinculin knock-out and vinculin expressing wild-type mouse embryonic fibroblasts. Vinculin knock-out cells were 2-fold more motile on 2D collagen-coated substrates compared to wild-type cells, but 3-fold less invasive in 2.4 mg/ml 3D collagen matrices. Vinculin knock-out cells were softer and remodeled their cytoskeleton more dynamically, which is consistent with their enhanced 2D motility but does not explain their reduced 3D invasiveness. Importantly, vinculin-expressing cells adhered stronger to collagen and generated 3-fold higher traction forces compared to vinculin knock-out cells. Moreover, vinculin expressing cells were able to migrate into dense (5.8 mg/ml) 3D collagen matrices that were impenetrable for vinculin knock-out cells. These findings suggest that vinculin facilitates 3D matrix invasion through upregulation or enhanced transmission of traction forces that are needed to overcome the steric hindrance of ECMs.

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Photox: A novel actin-targeting mono-ADP-ribosyltransferase from Photorhabdus luminescens.

Publication Date: 2010 Feb 24 PMID: 20181945
Authors: Visschedyk, D. D. - Perieteanu, A. A. - Turgeon, Z. J. - Fieldhouse, R. J. - Dawson, J. F. - Merrill, A. R.
Journal: J Biol Chem

Photorhabdus luminescens is a pathogenic bacterium that produces many toxic proteins. The mono-ADP- ribosyltransferases (mARTs) are an enzyme class produced by numerous pathogenic bacteria and participate in disease in plants and animals, including humans. Herein we report a novel mART from P. luminescens called Photox. This 46 kDa toxin shows high homology to other actin-targeting mARTs in hallmark catalytic regions and a similar core catalytic fold. Furthermore, Photox shows in vivo cytotoxic activity against yeast, with protection occurring when catalytic residues are substituted with alanine. In vitro, enzymatic activity (kcat, 1680 +/- 75 min-1) is higher than that of the related iota toxin, and diminishes by nearly 14,000-fold following substitution of the catalytic Glu (E355A). This toxin specifically ADP-ribosylates monomeric alpha skeletal actin and nonmuscle beta and gamma actin at Arg-177, inhibiting regular polymerization of actin filaments. These results indicate that Photox is indeed an ADP-ribosyltransferase, making it the newest member of the actin-targeting mART family.

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Trimeric Structure of Langerin.

Publication Date: 2010 Feb 24 PMID: 20181944
Authors: Feinberg, H. - Powlesland, A. S. - Taylor, M. E. - Weis, W. I.
Journal: J Biol Chem

Langerin, an endocytic receptor of Langerhans cells, binds pathogens such as HIV by recognition of surface glycoconjugates and mediates their internalization into Birbeck granules. Langerin has an extracellular region consisting of a C-type carbohydrate-recognition domain (CRD) and a neck region that stabilizes formation of trimers. As in many other C-type lectins, oligomerisation is required for high affinity binding to glycan ligands and is also likely to be important for determining specificity. To facilitate structural analysis of the human langerin trimer, a truncated form of the extracellular region, consisting of part of the neck and the CRD, has been characterized. Like the full-length protein, truncated langerin exists as a stable trimer in solution. Glycan array screening with the trimeric fragment shows that high mannose oligosaccharides are the best ligands for langerin. Structural analysis of the trimeric fragment of langerin confirms that the neck region forms a coiled-coil of alpha-helices. Multiple interactions between the neck region and the CRDs make the trimer a rigid unit with the three CRDs in fixed positions and the primary sugar binding sites separated by a distance of 42 A. The fixed orientation of the sugar-binding sites in the trimer is likely to place constraints on the ligands that can be bound by langerin.

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Structural and kinetic analysis of Schwanniomyces occidentalis invertase reveals a new oligomerization pattern and the role of its supplementary domain in substrate binding.

Publication Date: 2010 Feb 24 PMID: 20181943
Authors: Alvaro-Benito, M. - Polo, A. - Gonzalez, B. - Fernandez-Lobato, M. - Sanz-Aparicio, J.
Journal: J Biol Chem

Schwanniomyces occidentalis invertase is an extracellular enzyme that hydrolizes sucrose and releases beta-fructose from various oligosaccharides, and essential storage fructan-polymers such as inulin. We report here the three-dimensional structure of Sw. occidentalis invertase at 2.9 A resolution, and its complex with fructose at 1.9 A resolution. The monomer presents a bi-modular arrangement common to other GH32 enzymes, with an N-terminal fivefold beta-propeller catalytic domain and a C-terminal beta-sandwich domain with up to now unknown function. However, the dimeric nature of Sw. occidentalis invertase reveals a unique active-site cleft shaped by both subunits that may be representative of other yeast enzymes reported to be multimeric. Binding of the tetrasaccharide nystose and the polymer inulin has been investigated by docking analysis, which suggests that medium and long-size substrates are recognized by residues from both subunits. The identified residues have been mutated and the enzymatic activity of the mutants against sucrose, nystose and inulin has been investigated by kinetic analysis. The replacements that showed the largest effect on catalytic efficiency are Q228V, a residue involved in direct binding of nystose and inulin, and Ser281I, involved in a hydrogen bond linking the catalytic domains of both subunits. Moreover, a three-fold decrease of catalytic efficiency against inulin is observed in the mutants Q435A and Y462A, both located in the beta-sandwich domain of the other subunit, highlighting the essential function that oligomerization plays in substrate specificity and assigning, for the first time, a direct catalytic role to the supplementary domain of a GH32 enzyme.

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p35 is required for CDK5 activation in cellular senescence.

Publication Date: 2010 Feb 24 PMID: 20181942
Authors: Mao, D. - Hinds, P. W.
Journal: J Biol Chem

The retinoblastoma tumor suppressor gene (RB-1) is a key regulator of cellular senescence. Expression of the retinoblastoma protein (pRB) in human tumor cells that lack it results in senescence-like changes. The induction of the senescent phenotype by pRB requires the postmitotic kinase CDK5, the best known function of which is in neuronal development and postmitotic neuronal activities. Activation of CDK5 in neurons depends on its activators p35 and p39; however, little is known about how CDK5 is activated in non-neuronal senescent cells. Here we report that p35 is required for the activation of CDK5 in the process of cellular senescence. We demonstrate (i) p35 is expressed in osteosarcoma cells, (ii) p35 is required for CDK5 activation induced by pRB during senescence, (iii) p35 is required for the senescent morphological changes in which CDK5 is known to be involved as well as for expression of the senescence secretome, and (iv) p35 is upregulated in senescing cells. Taken together, these results suggest that p35 is at least one of the activators of CDK5 that is mobilized in the process of cellular senescence, which may provide insight into cancer cell proliferation and future cancer therapeutics.

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The role of polyamines in supporting growth of mammalian cells is mediated through their requirement for translation initiation and elongation.

Publication Date: 2010 Feb 24 PMID: 20181941
Authors: Landau, G. - Bercovich, Z. - Park, M. H. - Kahana, C.
Journal: J Biol Chem

Polyamines are essential cell constituents whose depletion results in growth cessation. Here we have investigated potential mechanisms of action of polyamines in supporting mammalian cell proliferation. We demonstrate that polyamines regulate translation both at the initiation and at the elongation steps. DFMO treatment resulting in polyamine depletion reduces protein synthesis via inhibition of translation initiation. N1-guanyl-diaminoheptane (GC7), a spermidine analogue that inhibits eIF5A hypusination, also caused inhibition of translation initiation. In contrast, depletion of eIF5A by shRNA inhibits translation elongation as was recently demonstrated in yeast and drosophila. These results suggest that in addition to competing with spermidine in the hypusination reaction, GC7 also competes with spermidine at yet undefined sites required for translation initiation. Finally, we show that either polyamine depletion or GC7 treatment induced eIF2alpha phosphorylation and reduced phosphorylation of 4E-BP, thus setting the molecular basis for the observed inhibition of translation initiation.

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Crystal structure of the putidaredoxin reductase-putidaredoxin electron transfer complex.

Publication Date: 2010 Feb 23 PMID: 20179327
Authors: Sevrioukova, I. F. - Poulos, T. L. - Churbanova, I. Y.
Journal: J Biol Chem

In the camphor monooxygenase system from Pseudomonas putida, the [2Fe-2S]-containing putidaredoxin (Pdx) shuttles electrons between the NADH-dependent putidaredoxin reductase (Pdr) and cytochrome P450cam. The mechanism of the Pdr-Pdx redox couple has been investigated by a variety of techniques. One of the exceptions is x-ray crystallography as the native partners associate weakly and resist co-crystallization. Here we present the 2.6 angstrom x-ray structure of a catalytically active complex between Pdr and Pdx Cys73Ser/Cys85Ser chemically crosslinked via the Lys409Pdr-Glu72Pdx pair. The 365 square angstrom Pdr-Pdx interface is predominantly hydrophobic with one central Arg310Pdr-Asp38Pdx salt bridge, likely assisting docking and orienting the partners optimally for electron transfer, and a few peripheral hydrogen bonds. A predicted 12 angstrom-long electron transfer route between FAD and [2Fe-2S] includes flavin flanking Trp330Pdr and the iron ligand Cys39Pdx. The x-ray model agrees well with the experimental and theoretical results and suggests that the linked Pdx must undergo complex movements during turnover to accommodate P450cam, which could limit the Pdx-to-P450cam electron transfer reaction.

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Selection of a novel and highly specific TNF{alpha} antagonist: insight from the crystal structure of the antagonist-TNF{alpha} complex.

Publication Date: 2010 Feb 23 PMID: 20179326
Authors: Byla, P. - Andersen, M. H. - Holtet, T. L. - Jacobsen, H. - Munch, M. - Gad, H. H. - Thogersen, H. C. - Hartmann, R.
Journal: J Biol Chem

Inhibition of TNFalpha is a favourable way of treating several important diseases such as rheumatoid arthritis, Crohns disease and psoriasis. Therefore an extensive range of TNFalpha inhibitory proteins, most of them based upon an antibody scaffold, has been developed and used with variable success as therapeutic. We have developed a novel technology platform to using C-type lectins as vehicle for the creation of novel trimeric therapeutic proteins with increased avidity and unique properties compared to current protein therapeutics. We chose TNFalpha as a test target to validate this new technology because of the extensive experience available in protein based TNFalpha antagonist. Here we present a novel and highly specific TNFalpha antagonist developed using this technology. Furthermore, we have solved the 3 dimensional structure of the antagonist-TNFalpha complex by X-ray crystallography and this structure is presented here. The structure has given us a unique insight in how the selection procedure works at a molecular level. Surprisingly little change is seen in the CTLD structure outside of the randomized regions, where as substantial change is seen within the randomized loops. Thus the overall integrity of the CTLD is maintained where as the specificity and binding affinity is change by the introduction of a range of specific contact with TNFalpha.

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Transcription factor SMAD3 is required for the inhibition of adipogenesis by retinoic acid.

Publication Date: 2010 Feb 23 PMID: 20179325
Authors: Marchildon, F. - St-Louis, C. - Akter, R. - Roodman, V. - Wiper-Bergeron, N. L.
Journal: J Biol Chem

The process of adipocyte differentiation is driven by a highly coordinated cascade of transcriptional events that results in the development of the mature adipocyte and in lipid accumulation. One of the early events of differentiation is the upregulation of CCAAT/Enhancer Binding Protein beta (C/EBPbeta) expression. C/EBPbeta then acts to upregulate the expression of adipogenic factors such as C/EBPalpha, which control the late stage of adipogenesis. Retinoic acid (RA) is a potent inhibitor of adipogenesis, and its action appears to block C/EBPbeta transcriptional potential early during differentiation. Using preadipocytes and mesenchymal stem cell models, we show that RA specifically blocks the occupancy of C/EBPbeta of the C/EBPalpha promoter, thereby abrogating the differentiation process. RA does not act directly on C/EBPbeta but rather stimulates the expression of the Transforming Growth Factor beta-effector protein Smad3, which can interact with C/EBPbeta via its Mad Homology 1 (MH1) domain and can interfere with C/EBPalpha DNA binding. The RA-induced increase in Smad3 expression results in increased cytoplasmic and nuclear Smad3, an important event as ectopic expression of Smad3 in preadipocytes in the absence of RA treatment only modestly inhibits adipogenesis and C/EBPbeta DNA binding, suggesting that Smad3 alone is not sufficient to completely recapitulate the effects of retinoic acid treatment during differentiation. However, in the absence of Smad3, RA is not able to inhibit adipocyte differentiation or to elicit a decrease in C/EBPbeta DNA occupancy suggesting that Smad3 is necessary to convey the inhibitory effects of retinoic acid during adipogenesis.

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Activation of canonical Wingless-type MMTV integration site family (WNT) signaling in mature adipocytes increases &-{beta}catenin levels and leads to cell dedifferentiation and insulin resistance.

Publication Date: 2010 Feb 23 PMID: 20179324
Authors: Gustafson, B. - Smith, U.
Journal: J Biol Chem

Canonical Wnt ligands are secreted by several cell types in the adipose tissue. We examined if also mature adipocytes can be target cells and found that canonical Wnt activation by Wnt3a induced a marked dedifferentiation of both 3T3-L1 and human adipocytes. Typical adipogenic markers were reduced while undifferentiated cell markers like Pref-1, Wnt10b and GATA2 were increased. The cells also became insulin resistant with impaired upstream insulin signaling and reduced glucose uptake. Wnt3a stabilised beta-catenin in the absence of the LRP6 receptor and with maintained axin and Dickkopf-1 protein expression. PPARgamma was repressed and PPARgamma ligands could not restore the adipogenic markers or reduce the gamma-catenin levels. The dedifferentiated adipocytes expressed the myofibroblast marker alpha-SMA and were also susceptible to osteogenic transdifferentiation. These results identify a novel pathway in mature adipose cells which is critical for maintaining the normal adipocyte phenotype and insulin sensitivity.

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Replication fork arrest and rDNA silencing are two independent and separable functions of the replication terminator protein Fob1 of saccharomyces cerevisiae.

Publication Date: 2010 Feb 23 PMID: 20179323
Authors: Bairwa, N. K. - Zzaman, S. - Mohanty, B. K. - Bastia, D.
Journal: J Biol Chem

The replication terminator protein Fob1 of Saccharomyces cerevisiae (Sc Fob1) is multifunctional and it not only promotes polar replication fork arrest at the tandem Ter sites located in the intergenic spacer region (IGS) of rDNA but also loads the NAD-dependent histone deacetylase, Sir2, at the Ter sites via a protein complex called RENT (Regulator of Nucleolar Silencing and Telophase exit). Sir2 is a component of the RENT complex and its loading not only silences intra-chromatid recombination in rDNA but also RNA polymerase II-catalyzed transcription. Here, we present three lines of evidence showing that the two aforementioned activities of Fob1 are independent of each other and functionally separable. First, a Fob1 ortholog of S. baynus (Sb Fob1) expressed in a fob1 deleted strain of S. cerevisiae restored polar fork arrest at Ter but not rDNA silencing. Second, a mutant form (I407T) of Sc Fob1 retained normal fork arresting activity but was severely defective in rDNA silencing. We show further that the silencing defect of Sb Fob1 and the I407T mutant of Sc Fob1 were caused by the failure of the proteins to interact with two members of the Sc RENT complex namely Sc Sir2 and Sc Net1. Third, deletions of the intra-S phase checkpoint proteins Tof1 and Csm3 abolished fork arrest by Fob1 at Ter without causing loss of silencing. Taken together, the data support the conclusion that unlike some other functions of Fob1, rDNA silencing at Ter is independent of fork arrest.

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DHHC5 interacts with PDZ domain 3 of post-synaptic density-95 (PSD-95) protein and plays a role in learning and memory.

Publication Date: 2010 Feb 22 PMID: 20178993
Authors: Li, Y. - Hu, J. - Hofer, K. - Wong, A. M. - Cooper, J. D. - Birnbaum, S. G. - Hammer, R. E. - Hofmann, S. L.
Journal: J Biol Chem

A family of integral membrane proteins containing a signature DHHC motif have been shown to display protein S-acyltransferase activity, modifying cysteine residues in proteins with fatty acids. The physiological roles of these proteins have largely been unexplored. Here we report that mice homozygous for a hypomorphic allele of a previously uncharacterized member, DHHC5, are born at half the expected rate and survivors show a marked deficit in contextual fear conditioning, an indicator of defective hippocampal-dependent learning. DHHC5 is highly enriched in a post-synaptic density preparation and co-immunoprecipates with post-synaptic density protein-95 (PSD-95), an interaction that is mediated through binding of the carboxyl terminus of DHHC5 and the PDZ3 domain of PSD-95. Immunohistochemistry demonstrated that DHHC5 is expressed in the CA3 and dentate gyrus in the hippocampus. These findings point to a previously unsuspected role for DHHC5 in post-synaptic function affecting learning and memory.

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Overlapping roles for Yen1 and Mus81 in cellular Holliday junction processing.

Publication Date: 2010 Feb 22 PMID: 20178992
Authors: Tay, Y. D. - Wu, L.
Journal: J Biol Chem

Eukaryotic Holliday junction (HJ) resolvases have attracted much attention recently with the identification of at least three distinct proteins that, in vitro, can cleave model HJs. However, the specific DNA structure(s) these proteins act upon in the cell is unknown. Here we describe a system in budding yeast to directly and quantitatively monitor in vivo HJ resolution. We find that Yen1 acts redundantly with Mus81, but not Slx1, to resolve a model HJ in vivo. This functional overlap specifically extends to the repair/bypass of lesions that impede the progression of replication forks but not to the repair of DSBs induced by ionizing radiation. Together, these results suggest a direct role for Yen1 in the response to DNA damage and, moreover, implicate overlapping HJ resolution functions of Yen1 with Mus81 during replication fork repair.

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Electrostatic interaction between oxysterol binding protein and VAMP-associated protein-A revealed by NMR and mutagenesis studies.

Publication Date: 2010 Feb 23 PMID: 20178991
Authors: Furuita, K. - Jee, J. - Fukada, H. - Mishima, M. - Kojima, C.
Journal: J Biol Chem

Oxysterol binding protein (OSBP), a cytosolic receptor of cholesterol and oxysterols, is recruited to endoplasmic reticulum by binding to the cytoplasmic major sperm protein (MSP) domain of integral endoplasmic reticulum protein VAMP-associated protein-A (VAP-A), a process essential for the stimulation of sphingomyelin synthesis by 25-hydroxycholesterol. To delineate the interaction mechanism between VAP-A and OSBP, we determined the complex structure between the VAP-A MSP domain (VAP-A(MSP)) and OSBP fragment containing a VAP-A binding motif FFAT (OSBP(F)) by NMR. This solution structure explained that 5 out of 6 conserved residues in the FFAT motif are required for the stable complex formation, and 3 of 5, including three critical intermolecular electrostatic interactions, were not explained before. By combining NMR relaxation and titration, isothermal titration calorimetry and mutagenesis experiments with structural information, we further elucidated the detailed roles of the FFAT motif and underlying motions of VAP-A(MSP), OSBP(F) and the complex. Our results show that OSBP(F) is disordered in the free state and VAP-A(MSP) and OSBP(F) form a final complex by means of intermediates, where electrostatic interactions through acidic residues including an acid patch preceding the FFAT motif probably play a collective role. Additionally, we report that the mutation which causes the familial motor neuron disease decreases the stability of the MSP domain.

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Calcium-dependent conformational flexibility of a CUB domain controls activation of the complement serine protease C1r.

Publication Date: 2010 Feb 23 PMID: 20178990
Authors: Major, B. - Kardos, J. - Kekesi, K. A. - Lorincz, Z. - Zavodszky, P. - Gal, P.
Journal: J Biol Chem

C1, the first component of the complement system is a Ca2+-dependent heteropentamer complex of C1q and two modular serine proteases, C1r and C1s. Current functional models assume significant flexibility of the subcomponents. Non-catalytic modules in C1r have been proposed to provide the flexibility required for function. Using recombinant CUB2-CCP1 domain-pair and the individual CCP1 module, we showed that binding of Ca2+ induces the folding of CUB2 domain and stabilizes its structure. In the presence of Ca2+, CUB2 shows a compact, folded structure, while in the absence of Ca2+ it has a flexible, disordered conformation. CCP1 module is Ca2+ insensitive. ITC titration revealed that CUB2 binds one single Ca2+ with a relatively high KD (430 microM). In blood, the CUB2 domain of C1r is only partially (74%) saturated by Ca2+, therefore the disordered, Ca2+-free form could provide the flexibility required for C1 activation. In accordance with this assumption, the effect of Ca2+ on the autoactivation of native, isolated C1r zymogen was proved. In the case of infection-inflammation when the local Ca2+ concentration decreases this property of CUB2 domain could serve as subtle means to trigger the activation of the classical pathway of complement. The CUB2 domain of C1r is a novel example for globular protein domains with marginal stability, high conformational flexibility, and proteolytic sensitivity. The physical nature of the behavior of this domain is similar to that of intrinsically unstructured proteins, providing a further example of functionally relevant ligand-induced reorganization of a polypeptide chain.

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The lycopene cyclase CrtY from pantoea ananatis (ex Erwinia Uredovora) catalyzes an FADred -dependent non-redox reaction.

Publication Date: 2010 Feb 23 PMID: 20178989
Authors: Yu, Q. - Schaub, P. - Ghisla, S. - Al-Babili, S. - Krieger-Liszkay, A. - Beyer, P.
Journal: J Biol Chem

The cyclization of lycopene generates provitamin A carotenoids, such as beta-carotene and paves the way towards the formation of cyclic xanthophylls playing distinct roles in photosynthesis and as precursors for regulatory molecules in plants and animals. The biochemistry of lycopene cyclization has been enigmatic since the previously proposed acid-base catalysis conflicted with the possibility of redox-catalysis as predicted by the presence of a dinucleotide binding site. We show that reduced FAD is the essential cofactor in CrtY and CrtL-type lycopene cyclases. Using flavin analogs, mass spectrometry and mutagenesis evidence is obtained based on which a catalytic mechanism relying on cryptic (net) electron transfer can be refuted. The role of reduced FAD is proposed to reside in the stabilization of a transition state carrying a (partial) positive charge or of a positively charged intermediate via a charge transfer interaction, acid-base catalysis serving as the underlying catalytic principle. Lycopene cyclase thus ranks among the novel class non-redox flavoproteins, such as isopentenyl diphosphate:dimethyallyl diphosphate isomerase type 2 (IDI-2) that requires the reduced form of the cofactor.

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Two immunoregulatory peptides with antioxidant activity from tick salivary glands.

Publication Date: 2010 Feb 24 PMID: 20178988
Authors: Wu, J. - Wang, Y. - Liu, H. - Yang, H. - Ma, D. - Li, J. - Li, D. - Lai, R. - Yu, H.
Journal: J Biol Chem

Ticks are blood feeding arthropods that may secrete immunosuppressant molecules, which inhibit host inflammatory and immune responses and provide survival advantages to pathogens at tick bleeding sites in hosts. In current work, two families of immunoregulatory peptides, hyalomin A and B were firstly identified from salivary glands of hard tick Hyalomma asiaticum asiaticum. Three copies of them are encoded by identical gene and released from the same protein precursor. Both hyalomin A and B can exert significant antiinflammatory functions, either by directly inhibiting the host secretion of inflammatory factors such as TNF alpha, MCP1 and IFN gamma, or by indirectly increasing the secretion of immunosuppressant cytokine of IL10. Hyalomin A and B were both found to potently scavenge free radical in vitro in a rapid manner, and inhibited adjuvant induced inflammation in mouse models in vivo. The JNK SAPK subgroup of MAPKs signaling pathway was involved in such immunoregulatory functions of hyalomin A and B. These results showed that immunoregulatory peptides of tick salivary glands suppress host inflammatory response by modulating cytokine secretion and detoxifying reactive oxygen species.

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FAS apoptosis inhibitory molecule regulates T cell receptor-mediated apoptosis of thymocytes by modulating AKT activation and NUR77 expression.

Publication Date: 2010 Feb 23 PMID: 20178987
Authors: Huo, J. - Xu, S. - Lam, K. P.
Journal: J Biol Chem

Fas apoptosis inhibitory molecule (FAIM) had been demonstrated to confer resistance to Fas-induced apoptosis of lymphocytes and hepatocytes in vitro and in vivo. Here, we show that FAIM is up-regulated in thymocytes upon T cell receptor (TCR) engagement and that faim-/- thymocytes are highly susceptible to TCR-mediated apoptosis with increased activation of caspase-8 and -9. Furthermore, injection of anti-CD3 antibodies leads to augmented depletion of CD4+CD8+ T cells in the thymus of faim-/- mice compared with wild-type control, suggesting that FAIM plays a role in thymocyte apoptosis. Cross-linking of the TCR on faim-/- thymocytes leads to an elevated protein level of the orphan nuclear receptor Nur77, which plays a role in thymocyte apoptosis. Interestingly, in the absence of FAIM, there are reduced ubiquitination and degradation of Nur77 protein. Faim-/- thymocytes also exhibit a defective TCR-induced activation of Akt whose activity we now show is required for Nur77 ubiquitination. Further analyses utilizing FAIM-deficient primary thymocytes and FAIM-overexpressing DO-11.10 T-cells indicate that FAIM acts upstream of Akt during TCR signaling and influences the localization of Akt to lipid rafts and hence, affecting its activation. Taken together, our study defined a TCR-induced FAIM/Akt/Nur77 signaling axis that is critical for modulating the apoptosis of developing thymocytes.

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Phosphorylation of the Mycobacterium tuberculosis {beta}-ketoacyl-ACP reductase MabA regulates mycolic acid biosynthesis.

Publication Date: 2010 Feb 23 PMID: 20178986
Authors: Veyron-Churlet, R. - Zanella-Cleon, I. - Cohen-Gonsaud, M. - Molle, V. - Kremer, L.
Journal: J Biol Chem

Mycolic acids are key cell wall components for the survival, pathogenicity and antibiotic resistance of the human tubercle bacillus. Although it was thought that Mycobacterium tuberculosis tightly regulates their production to adapt to prevailing environmental conditions, the molecular mechanisms governing mycolic acid biosynthesis remained extremely obscure. Meromycolic acids, the direct precursors of mycolic acids, are synthesized by a type II fatty acid synthase (FAS-II) from acyl carrier protein (ACP)-bound substrates that are extended iteratively, with a reductive cycle in each round of extention, the second step of which is catalyzed by the essential b-ketoacyl-ACP reductase, MabA. In this study, we investigated whether post-translational modifications of MabA might represent a strategy employed by M. tuberculosis to regulate mycolic acid biosynthesis. Indeed, we show here that MabA was efficiently phosphorylated in vitro by several M. tuberculosis Ser/Thr protein kinases (STPK). Mass spectrometric analyses identified three phosphothreonines, with Thr191 being the primary phosphor-acceptor. A MabA_T191D mutant, designed to mimic constitutive phosphorylation, exhibited markedly decreased ketoacyl reductase activity compared to the wild-type protein, as well as impaired binding of the NADPH cofactor, as demonstrated by fluorescence spectroscopy. The hypothesis that phosphorylation of T191 alters the enzymatic activity of MabA, and subsequently mycolic acid biosynthesis, was further supported by the fact that constitutive over-expression of the mabA(T191D) allele in Mycobacterium bovis BCG strongly impaired mycobacterial growth. Importantly, conditional expression of the phosphomimetic MabA_T191D led to a significant inhibition of de novo biosynthesis of mycolic acids. This study provides the first information on the molecular mechanism(s) involved in mycolic acid regulation through STPK-dependent phosphorylation of a FAS-II enzyme.

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Targeted deletion of hepatocyte ABCA1 leads to VLDL triglyceride over-production and LDL hypercatabolism.

Publication Date: 2010 Feb 23 PMID: 20178985
Authors: Chung, S. - Timmins, J. M. - Duong, M. - Degirolamo, C. - Rong, S. - Sawyer, J. K. - Singaraja, R. R. - Hayden, M. R. - Maeda, N. - Rudel, L. L. - Shelness, G. S. - Parks, J. S.
Journal: J Biol Chem

Loss of ABCA1 activity in Tangier disease (TD) is associated with abnormal apoB-lipoprotein (Lp) metabolism in addition to the complete absence of HDL. We used hepatocyte-specific ABCA1 knock out (HSKO) mice to test the hypothesis that hepatic ABCA1 plays dual roles in regulating apoB-lipoprotein (Lp) metabolism and nascent HDL formation. HSKO mice reca-pitulated the TD lipid phenotype with post-prandial hypertriglyceridemia, markedly de-creased LDL, and near absence of HDL. Triglyceride (TG) secretion was two-fold higher in HSKO compared to wild type (WT) mice, primarily due to secretion of larger TG-enriched VLDL secondary to reduced hepatic PI3 kinase signaling. HSKO mice also displayed delayed clearance of postprandial TG and reduced post-heparin plasma lipolytic activity. In addition, hepatic LDLr expression and plasma LDL catabolism were increased two-fold in HSKO compared to WT mice. Lastly, adenoviral repletion of hepatic ABCA1 in HSKO mice normalized plasma VLDL TG and hepatic PI3 kinase signaling, with a partial recovery of HDL-C levels, providing evidence that hepatic ABCA1 is involved in the reciprocal regulation of apoB-Lp production and HDL formation. These findings suggest that altered apoB Lp metabolism in TD subjects may result from hepatic VLDL TG overproduction and increased hepatic LDLr expression and highlight hepatic ABCA1 as an important regulatory factor for apoB-containing Lp metabolism.

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Impaired glucose tolerance and predisposition to the fasted state in liver glycogen synthase knockout mice.

Publication Date: 2010 Feb 23 PMID: 20178984
Authors: Irimia, J. M. - Meyer, C. M. - Peper, C. L. - Zhai, L. - Bock, C. B. - Previs, S. F. - McGuinness, O. P. - Depaoli-Roach, A. A. - Roach, P. J.
Journal: J Biol Chem

Conversion to glycogen is a major fate of ingested glucose in the body. A rate-limiting enzyme in the synthesis of glycogen is glycogen synthase (GS) encoded by two genes, GYS1, expressed in muscle and other tissues, and GYS2, primarily expressed in liver (LGS). Defects in GYS2 cause the inherited monogenic disease Glycogen Storage Disease 0 (GSD0). We have generated mice with a liver specific disruption of the GYS2 gene (LGSKO mice), using Lox-P/Cre technology. Conditional mice, carrying floxed GYS2, were crossed with mice expressing Cre recombinase under the albumin promoter. The resulting LGSKO mice are viable, develop liver GS deficiency, and have a 95% reduction in fed liver glycogen content. They have mild hypoglycemia but dispose glucose less well in a glucose tolerance test. Fed, LGSKO mice also have reduced capacity for exhaustive exercise compared to mice carrying floxed alleles but the difference disappears after an overnight fast. Upon fasting, LGSKO mice reach within 4 hours decreased blood glucose levels attained by control floxed mice only after 24 hours of food deprivation. The LGSKO mice maintain this low blood glucose for at least 24 hours. Basal gluconeogenesis is increased in LGSKO mice and insulin suppression of endogenous glucose production is impaired as assessed by euglycemic-hyperinsulinemic clamp. This observation correlates with an increase in the liver gluconeogenic enzyme phosphoenolpyruvate carboxykinase expression and activity. This mouse model mimics the pathophysiology of GSD0 patients and highlights the importance of liver glycogen stores in whole body glucose homeostasis.

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Molecular interplay between mTOR, A{beta} and tau: Effects on cognitive impairments.

Publication Date: 2010 Feb 23 PMID: 20178983
Authors: Caccamo, A. - Majumder, S. - Richardson, A. - Strong, R. - Oddo, S.
Journal: J Biol Chem

Accumulation of amyloid-[beta] (A[beta]) and tau is an invariant feature of Alzheimer disease (AD). The upstream role of A[beta] accumulation in disease pathogenesis is widely accepted, and there is strong evidence showing that A[beta] accumulation causes cognitive impairments. However, the molecular mechanisms linking A[beta] to cognitive decline remain to be elucidated. Here we show that the buildup of A[beta] increases the mammalian target of rapamycin (mTOR) signaling, while decreasing mTOR signaling reduces A[beta] levels, thereby highlighting an interrelation between mTOR signaling and A[beta]. The mTOR pathway plays a central role in controlling protein homeostasis and hence neuronal functions; indeed mTOR signaling regulates different forms of learning and memory. Using an animal model of AD, we show that pharmacologically restoring mTOR signaling with rapamycin rescues cognitive deficits and ameliorates A[beta] and tau pathology by increasing autophagy. Indeed, we further show that autophagy induction is necessary for the rapamycin-mediated reduction in A[beta] levels. The results presented here provide a molecular basis for the A[beta]-induced cognitive deficits and, moreover, show that rapamycin, an FDA approved drug, improves learning and memory and reduces A[beta] and tau pathology.

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O-Acetylation of peptidoglycan in Gram-negative bacteria: Identification and characterization of peptidoglycan O-acetyltransferase in Neisseria gonorrhoeae.

Publication Date: 2010 Feb 23 PMID: 20178982
Authors: Moynihan, P. - Clarke, A. J.
Journal: J Biol Chem

The ape2 gene encoding a hypothetical O-acetylpeptidoglycan esterase was amplified from genomic DNA of N. gonorrhoeae FA1090 and cloned to encode either the full-length protein or a truncated version lacking its hypothetical signal sequence. Expression trials revealed that production of the full length version possessing either an N-terminal or C-terminal hexa-His tag was toxic to Escherichia coli transformants and that the host rapidly degraded the small amount of protein that was produced. An N-terminally truncated protein could be produced in sufficient yields for purification only if it possessed an N-terminal hexa-His tag. This form of the protein was isolated and purified to apparent homogeneity and its enzymatic properties were characterized. Whereas the protein could bind to insoluble peptidoglycan, it did not function as an esterase. Phenotypic characterization of E. coli transformants producing various forms of the protein revealed that it functions instead to O-acetylate peptidoglycan within the periplasm, and it was thus renamed peptidoglycan O-acetyltransferase B. This activity was found to be dependent upon a second protein which functions to translocate the source of acetate from the cytoplasm to the periplasm, demonstrating that the O-acetylation of peptidoglycan in N. gonorrhoeae, and other gram-negative bacteria, requires a two component system.

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The crystal structure of SmcR, a quorum-sensing master regulator of Vibrio vulnificus, provides insight into its regulation of transcription.

Publication Date: 2010 Feb 23 PMID: 20178981
Authors: Kim, Y. - Kim, B. S. - Park, Y. J. - Choi, W. C. - Hwang, J. - Kang, B. S. - Oh, T. K. - Choi, S. H. - Kim, M. H.
Journal: J Biol Chem

Quorum sensing has been implicated as an important global regulatory system controlling the expression of numerous virulence factors in bacterial pathogens. SmcR, a homologue of Vibrio harveyi LuxR, has been proposed as a quorum-sensing master regulator of V. vulnificus, an opportunistic human pathogen. Previous studies demonstrated that SmcR is essential for the survival and pathogenesis of V. vulnificus, indicating that inhibiting SmcR is an attractive approach to combat infections by the bacteria. In this study, we therefore determined the crystal structure of SmcR at 2.1 Angstron resolution. The protein structure reveals a typical TetR superfamily fold consisting of an N-terminal DNA binding domain and a C-terminal dimerization domain. In vivo and in vitro functional analysis of the dimerization domain suggested that dimerization of SmcR is vital for its biological regulatory function. The N-terminal DNA recognition and binding residues were assigned based on the protein structure, and the results of in vivo and in vitro mutagenesis experiments. Furthermore, protein-DNA interaction experiments suggested that SmcR may have a sophisticated mechanism that enables the protein to recognize each of its many target operators with different affinities.

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cAMP/CREB-mediated transcriptional regulation of ectonucleoside triphosphate diphosphohydrolase 1 (CD39) expression.

Publication Date: 2010 Feb 23 PMID: 20178980
Authors: Liao, H. - Hyman, M. C. - Baek, A. E. - Fukase, K. - Pinsky, D. J.
Journal: J Biol Chem

CD39 is a transmembrane enzyme that inhibits platelet reactivity and inflammation by phosphohydrolyzing ATP and ADP to AMP. Cyclic AMP (cAMP) is important in regulating genes controlling vascular homeostasis. We tested the hypothesis that cAMP might positively regulate the expression of CD39. Cd39 mRNA was induced in RAW cells treated with a membrane-permeant cAMP analogue (8-Br-cAMP). Flow cytometry, immunofluorescence, and thin layer chromatographic assays demonstrated that both CD39 protein expression and enzymatic activity were increased in cells treated with 8-Bromo-cAMP, but not in cells transfected with shRNA against CD39. 8-Br-cAMP significantly increased transcriptional activity at the CD39 promoter, though not when its CRE sites were mutated. Pretreatment with PKA, Pi3K, or ERK inhibitors nearly obliterated the cAMP-driven increase in Cd39 mRNA, protein expression, and promoter activity. 8-Br-cAMP greatly increased the phosphorylation of CREB1 (Ser133) and ATF2 (Thr71) in a PKA-, Pi3K-, and ERK-dependent fashion. ChIP assays demonstrated that binding of phosphorylated CREB1 and ATF2 to CRE-like sites was significantly increased with 8-Br-cAMP treatment and that binding was reduced with PKA, Pi3K, and ERK inhibition, where as transfection of CREB1 and ATF2 overexpression constructs enhanced cAMP-driven Cd39 mRNA expression. Transfection of RAW cells with mutated CREB1 (S133A) reduced cAMP-driven Cd39 mRNA expression. Furthermore, the cAMP-mediated induction of CD39 mRNA, protein, and phosphohydrolytic activity was replicated in primary peritoneal macrophages. These data identify cAMP as a crucial regulator of macrophage CD39 expression, and demonstrate that cAMP acts through the PKA/CREB, PKA/PI3K/ATF2, and PKA/ERK/ATF2 pathways to control a key vascular homeostatic mediator.

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The anti-tumorigenic properties of the peroxisomal proliferator activated receptor (alpha) are arachidonic acid epoxygenase-mediated.

Publication Date: 2010 Feb 23 PMID: 20178979
Authors: Pozzi, A. - Popescu, V. - Yang, S. - Mei, S. - Shi, M. - Puolitaival, S. M. - Caprioli, R. M. - Capdevila, J. H.
Journal: J Biol Chem

Prevalence and mortality make cancer a health challenge in need of effective and better tolerated therapeutical approaches, with tumor angiogenesis identified as a promising target for drug development. The epoxygenase products, the epoxyeicosatrienoic acids, are pro-angiogenic, and down-regulation of their biosynthesis by peroxisomal proliferator activated receptor alpha (PPARa) ligands reduces tumor angiogenesis and growth. Endothelial cells lacking a Cyp2c44 epoxygenase, a PPARa target, show reduced proliferative and tubulogenic activities that are reversed by the enzyme metabolites. In a mouse xenograft model of tumorigenesis, disruption of the host Cyp2c44 gene causes marked reductions in tumor volume, mass, and vascularization. The relevance of these studies to human cancer is indicated by the demonstration that: a) activation of human PPARa down-regulates endothelial cell CYP2C9 epoxygenase expression, and blunts proliferation and tubulogenesis, b) in a PPARa-humanized mouse model, activation of the receptor inhibits tumor angiogenesis and growth, and c) the CYP2C9 epoxygenase is expressed in the vasculature of human tumors. The identification of anti-angiogenic/anti-tumorigenic properties of PPARa points to a role for the receptor and its epoxygenase regulatory target in the pathophysiology of cancer, and for its ligands as candidates for the development of a new generation of safer and better tolerated anti-cancer drugs.

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Kinetic and spectroscopic studies of the molybdenum-copper CO dehydrogenase from Oligotropha carboxidovorans.

Publication Date: 2010 Feb 23 PMID: 20178978
Authors: Zhang, B. - Hemann, C. F. - Hille, R.
Journal: J Biol Chem

Carbon monoxide dehydrogenase from the aerobic bacterium Oligotropha carboxidovorans catalyzes the oxidation of CO to CO2, yielding two electrons and two H+. The steady-state kinetics of the enzyme exhibit a pH optimum of 7.2, with kcat of 93.3 s-1 and Km of 10.7 uM at 25 oC. kred for the reductive half-reaction agrees well with kcat and exhibits a similar pH optimum, indicating that the rate-limiting step of overall turnover is likely in the reductive half-reaction. No dependence on CO concentration was observed in the rapid reaction kinetics, however, suggesting that CO initially binds rapidly to the enzyme, possibly at the Cu(I) of the active site, prior to undergoing oxidation. A Mo(V) species that exhibits strong coupling to the copper of the active center (I = 3/2) has been characterized by EPR. The signal is further split when [13C]-CO is used to generate it, demonstrating that substrate (or product) is a component of the signal-giving species. Finally, resonance Raman spectra of CODH reveal the presence of FAD, Fe/S clusters, and a [CuSMoO2] coordination in the active site, consistent with earlier X-ray absorption and crystallographic results.

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Interaction of VPX and APOBEC3A correlates with efficient lentivirus infection of monocytes.

Publication Date: 2010 Feb 26 PMID: 20178977
Authors: Berger, A. - Munk, C. - Schweizer, M. - Cichutek, K. - Schule, S. - Flory, E.
Journal: J Biol Chem

The accessory protein Vpx is encoded by lentiviruses of the human immunodeficiency virus type 2 (HIV-2) and the simian immunodeficiency SIVsm/SIVmac lineage. It is packaged into virions and is indispensable in early steps of monocyte infection. HIV-1, which does not encode Vpx, is not able to infect human monocytes, but Vpx enables infection with HIV-1. The underlying mechanism is not completely understood. In this work, we focus on Vpx-mediated intracellular post-entry events as counteraction of host cell proteins. We found that Vpx binds to APOBEC3A (A3A), a member of the family of cytidine deaminases, present in monocytes. This interaction led to a reduction of the steady-state protein level of A3A. A single point mutation in Vpx (H82A) abrogated binding to A3A and single-round infection of monocytes by HIV-1. Taken together, our data indicate that lentiviral Vpx counteracts A3A in human monocytes.

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Modulating native-like residual structure in the fully denatured state of photoactive yellow protein affects its refolding.

Publication Date: 2010 Feb 23 PMID: 20178976
Authors: Lee, B. C. - Kumauchi, M. - Hoff, W. D.
Journal: J Biol Chem

Residual structure in the fully unfolded state is a key element for understanding protein folding. We show that residual structure in fully denatured photoactive yellow protein (PYP) is affected by isomerization of its p-coumaric acid (pCA) chromophore. The exposure of total surface area and hydrophobic surface area upon unfolding were quantified by denaturant m values and heat capacity changes DeltaCp, respectively. The exposure of buried surface area upon the unfolding of the acid denatured state of PYP containing trans-pCA is ~20% smaller than that of the native state. In contrast, for the partially unfolded pB photocycle intermediate containing cis-pCA unfolding-induced exposure of surface area is not decreased. These results show that pCA photoisomerization reduces residual structure in the fully unfolded state. Thus, residual structure in the fully unfolded state of PYP is under direct experimental control by photoexcitation. The sensitivity of the unfolded state to pCA isomerization provides a novel criterion that residual structure in the unfolded state of PYP is native-like, involving native-like protein-chromophore interactions. A largely untested prediction is that native-like residual structure facilitates the conformational search during folding. In the case of PYP refolding from the less disordered fully unfolded state containing trans-pCA indeed is substantially accelerated. The burial of significant hydrophobic surface area in the fully unfolded state suggests that a significant part of the hydrophobic collapse process already has occurred in the denatured state.

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Increased monomerization of mutant HSPB1 leads to protein hyperactivity in Charcot-Marie-Tooth neuropathy.

Publication Date: 2010 Feb 23 PMID: 20178975
Authors: Almeida-Souza, L. - Goethals, S. - de Winter, V. - Dierick, I. - Gallardo, R. - Van Durme, J. - Irobi, J. - Gettemans, J. - Rousseau, F. - Schymkowitz, J. - Timmerman, V. - Janssens, S.
Journal: J Biol Chem

Small heat shock proteins are molecular chaperones capable of maintaining denatured proteins in a folding-competent state. We have previously shown that missense mutations in the small heat shock protein HSPB1 (HSP27) cause distal hereditary motor neuropathy and axonal Charcot-Marie-Tooth (CMT) disease. Here we investigated the biochemical consequences of HSPB1 mutations that are known to cause peripheral neuropathy. In contrast to other chaperonopathies, our results revealed that particular HSPB1 mutations presented higher chaperone activity compared to wild-type. Hyperactivation of HSPB1 was accompanied by a change from its wild-type dimeric state to a monomer without dissociation of the 24-meric state. Purification of protein complexes from wild-type and HSPB1 mutants showed that the hyperactive isoforms also presented enhanced binding to client proteins. Furthermore, we show that the wild-type HSPB1 protein undergoes monomerization during heat-shock activation, strongly suggesting that the monomer is the active form of the HSPB1 protein.

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Heat shock protein 70 interacts with nucleolin and inhibits its cleavage, down-regulation and apoptosis induced by hydrogen peroxide in myocytes.

Publication Date: 2010 Feb 22 PMID: 20177076
Authors: Wang, K. - Jiang, L. - Deng, G. - Chen, G. - Tan, S. - Tu, Z. - Jiang, B. - Xiao, X.
Journal: J Biol Chem

Increasing evidence proved that apoptosis plays a key role in the pathogenesis in a variety of cardiovascular diseases due to the loss of terminally differentiated cardiac myocytes. Nucleolin (C23) is a nucleolar anti-apoptotic protein and can be cleaved and down-regulated during the apoptotic process. It has been demonstrated that heat shock protein 70 (Hsp70) inhibits apoptosis in myocytes. However, the molecular mechanisms of Hsp70 inhibiting apoptosis are, at the present time, not well understood. Here, we report for the first time that oxidative stress induces an interaction between Hsp70 and C23, as a consequence of their interaction, Hsp70 inhibited the cleavage, down-regulation of C23, and apoptosis in myocytes. Hydrogen peroxide (H2O2, 0.5 mmol/L) induced the cleavage and down-regulation of C23, whereas over-expression of Hsp70 inhibited cleavage, down-regulation of C23 and apoptosis in C2C12 myogenic cells. Moreover, C23 regulated apoptosis by affecting the Bcl-2 expression, Cytochrome C (Cyt C) release and activity of Caspase-3. We further observed that C23 over-expression reversed the blockage of Hsp70 antisense oligonucleotides on the protection of Hsp70; and that Hsp70 redistributed into nucleus and interacted with C23 N-terminal via the peptide-binding domain (PBD) after H2O2 exposure. Finally, our results showed that the deletion of Hsp70 PBD blocked the interaction and abolished the protection of Hsp70 against the cleavage and down-regulation of C23 and apoptosis. In a word, we revealed that C23 is a novel binding partner of Hsp70 in the nucleus and their interaction is a novel mechanism by which Hsp70 inhibits apoptosis in myocytes.

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Potentiation of TNF-{alpha}-induced tumor cell apoptosis by a small-molecule inhibitor for anti-apoptotic protein hPEBP4.

Publication Date: 2010 Feb 22 PMID: 20177075
Authors: Qiu, J. - Xiao, J. - Han, C. - Li, N. - Shen, X. - Jiang, H. - Cao, X.
Journal: J Biol Chem

hPEBP4 (human phosphatidylethanolamine-binding protein 4) has been identified by us to be able to potentiate the resistance of breast, prostate and ovarian cancers, with the preferential expression of hPEBP4, to tumor necrosis factor alpha (TNF-alpha) or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, suggesting that inhibitors targeting the anti-apoptotic protein hPEBP4 may be useful to increase the sensitivity of hPEBP4-expressing cancer cells to TNF-alpha or TRAIL-induced apoptosis. By structure-based virtual screening and following surface plasmon resonance-based binding assay, 7 small compounds were found to potently bind with hPEBP4. The hit compounds were further functionally screened for their ability to inhibit cancer cell growth, and one small compound, IOI-42, was identified to be able to promote TNF-alpha-mediated growth inhibition of MCF-7 breast cancer cells. IOI-42 could potentiate TNF-alpha-induced apoptosis of MCF-7 cells by inhibiting hPEBP4, and suppress anchorage-independent cell growth of MCF-7 cells. We further demonstrated that IOI-42 could reduce the endogenous association of hPEBP4 with Raf-1/MEK1, enhance the activation of ERK1/2 and JNK, while inhibit Akt activation. Furthermore, IOI-42 also promoted TRAIL-induced cell apoptosis of prostate cancer cells. Taken together, our data suggest that IOI-42, as the first chemical inhibitor of anti-apoptotic protein hPEBP4, may serve as a potential antitumor drug by sensitizing tumor cells to apoptotic inducers.

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Identification of MYB-binding protein 1A (MYBBP1A) as a novel substrate for aurora B kinase.

Publication Date: 2010 Feb 22 PMID: 20177074
Authors: Perrera, C. - Colombo, R. - Valsasina, B. - Carpinelli, P. - Troiani, S. - Modugno, M. - Gianellini, L. - Cappella, P. - Isacchi, A. - Moll, J. - Rusconi, L.
Journal: J Biol Chem

Aurora kinases are mitotic enzymes involved in centrosome maturation and separation, spindle assembly and stability, chromosome condensation, segregation and cytokinesis and represent well known targets for cancer therapy as their deregulation has been linked to tumorigenesis. The availability of suitable markers is of crucial importance to investigate Auroras functions and monitor kinase inhibition in in-vivo models and in clinical trials. Extending the knowledge on Aurora substrates could help to better understand their biology and could be a source for clinical biomarkers. Using biochemical, mass spectrometry and cellular approaches we identified MYBBP1A as a novel Aurora B substrate and Serine 1303 as the major phosphorylation site. MYBBP1A is phosphorylated in nocodazole arrested cells and is dephosphorylated upon Aurora B silencing or by treatment with Danusertib, a small molecule inhibitor of Aurora kinases. Furthermore, we show that MYBBP1A depletion by RNAi causes mitotic progression delay and spindle assembly defects. MYBBP1A has until now been described as a nucleolar protein, mainly involved in transcriptional regulation. The results presented herein show MYBBP1A as a novel Aurora B kinase substrate and reveal a not yet recognised link of this nucleolar protein to mitosis.

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Phosphorylation of TRPC6 channels at Thr69 is required for anti-hypertrophic effects of phosphodiesterase 5 inhibition.

Publication Date: 2010 Feb 22 PMID: 20177073
Authors: Nishida, M. - Watanabe, K. - Sato, Y. - Nakaya, M. - Kitajima, N. - Ide, T. - Inoue, R. - Kurose, H.
Journal: J Biol Chem

Activation of Ca2+ signaling induced by receptor stimulation and mechanical stress plays a critical role in the development of cardiac hypertrophy. A canonical transient receptor potential protein subfamily member TRPC6, which is activated by diacylglycerol (DAG) and mechanical stretch, works as an upstream regulator of Ca2+ signaling pathway. Although activation of protein kinase G (PKG) inhibits TRPC6 channel activity and cardiac hypertrophy, respectively, it is unclear whether PKG suppresses cardiac hypertrophy through inhibition of TRPC6. Here, we show that inhibition of cGMP-selective phosphodiesterase 5 (PDE5) suppresses endothelin-1-, DAG analog- and mechanical stretch-induced hypertrophy through inhibition of Ca2+ influx in rat neonatal cardiomyocytes. Inhibition of PDE5 suppressed the increase in frequency of Ca2+ spikes induced by agonists or mechanical stretch. However, PDE5 inhibition did not suppress the hypertrophic responses induced by high KCl or the activation of protein kinase C, suggesting that PDE5 inhibition suppresses Ca2+ influx itself or molecule(s) upstream of Ca2+ influx. PKG activated by PDE5 inhibition phosphorylated TRPC6 proteins at Thr69 and prevented TRPC6-mediated Ca2+ influx. Substitution of Ala for Thr69 in TRPC6 abolished the anti-hypertrophic effects of PDE5 inhibition. In addition, chronic PDE5 inhibition by oral sildenafil treatment actually induced TRPC6 phosphorylation in mouse hearts. Knockdown of regulator of G protein signaling 2 (RGS2) and RGS4, both of which are activated by PKG to reduce Galphaq-mediated signaling, did not affect the suppression of receptor-activated Ca2+ influx by PDE5 inhibition. These results suggest that phosphorylation and functional suppression of TRPC6 underlies prevention of pathological hypertrophy by PDE5 inhibition.

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Ataxia telangiectasia mutated (ATM) inhibition transforms human mammary gland epithelial cells.

Publication Date: 2010 Feb 22 PMID: 20177072
Authors: Mandriota, S. J. - Buser, R. - Lesne, L. - Stouder, C. - Favaudon, V. - Maechler, P. - Bena, F. - Clement, V. - Ruegg, C. - Montesano, R. - Sappino, A. P.
Journal: J Biol Chem

Carriers of mutations in the cell cycle checkpoint protein kinase ataxia-telangiectasia mutated (ATM), which represent 1-2% of the general population, have an increased risk of breast cancer. However, experimental evidence that ATM deficiency contributes to human breast carcinogenesis is lacking. We report here that in MCF-10A and MCF-12A cells, which are well established normal human mammary gland epithelial cell models, partial or almost complete stable ATM silencing or pharmacological inhibition resulted in cellular transformation, genomic instability and formation of dysplastic lesions in NOD/SCID mice. These effects did not require the activity of exogenous DNA damaging agents and were preceded by an unsuspected and striking increase in cell proliferation also observed in primary human mammary gland epithelial cells. Increased proliferation correlated with a dramatic, transient and proteasome-dependent reduction of p21(WAF1/CIP1) and p27(KIP1) protein levels, whereas little or no effect was observed on p21(WAF1/CIP1) or p27(KIP1) mRNAs. p21(WAF1/CIP1) silencing also increased MCF-10A cell proliferation, thus identifying p21(WAF1/CIP1) downregulation as a mediator of the proliferative effect of ATM inhibition. Our findings provide the first experimental evidence that ATM is a human breast tumor suppressor. In addition, they mirror the sensitivity of ATM tumor suppressor function and unveil a new mechanism by which ATM might prevent human breast tumorigenesis, namely a direct inhibitory effect on the basal proliferation of normal mammary epithelial cells.

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Inflammasome-independent role of apoptosis-associated speck-like protein containing a CARD (ASC) in T cell priming is critical for collagen-induced arthritis.

Publication Date: 2010 Feb 22 PMID: 20177071
Authors: Ippagunta, S. K. - Brand, D. D. - Luo, J. - Boyd, K. L. - Calabrese, C. - Stienstra, R. - Van de Veerdonk, F. L. - Netea, M. G. - Joosten, L. A. - Lamkanfi, M. - Kanneganti, T. D.
Journal: J Biol Chem

Rheumatoid arthritis (RA) is an autoimmune disease with 1% prevalence in the industrialized world. The contributions of the inflammasome components Nalp3, ASC and caspase-1 in the pathogenesis of collagen-induced arthritis (CIA) have not been characterized. Here we show that ASC-/- mice were protected from arthritis, whereas Nalp3-/- and caspase-1-/- mice were susceptible to CIA. Unlike Nalp3-/- and caspase-1-/- mice, the production of collagen-specific antibodies was abolished in ASC-/- mice. This was due to a significantly reduced antigen-specific activation of lymphocytes by ASC-/- dendritic cells. Antigen-induced proliferation of purified ASC-/- T cells was restored upon incubation with wild type dendritic cells, but not when cultured with ASC-/- dendritic cells. Moreover, direct T cell receptor (TCR) ligation with CD3 and CD28 antibodies induced a potent proliferation of ASC-/- T cells, indicating that ASC is specifically required in dendritic cells for antigen-induced T cell activation. Therefore, ASC fulfils a hitherto unrecognized inflammasome-independent role in dendritic cells that is crucial for T cell priming and the induction of antigen-specific cellular and humoral immunity and the onset of collagen-induced arthritis.

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Epithelial myosin light chain kinase activation induces mucosal interleukin-13 expression to alter tight junction ion selectivity.

Publication Date: 2010 Feb 22 PMID: 20177070
Authors: Weber, C. R. - Raleigh, D. R. - Su, L. - Shen, L. - Sullivan, E. A. - Wang, Y. - Turner, J. R.
Journal: J Biol Chem

Intestinal barrier function is reduced in inflammatory bowel disease (IBD). Tumor necrosis factor (TNF) and interleukin- (IL-)13, which are upregulated in IBD, induce barrier defects that are associated with myosin light chain kinase (MLCK) activation and increased claudin-2 expression, respectively, in cultured intestinal epithelial monolayers. Here we report that these independent signaling pathways, which have distinct effects on tight junction barrier properties, interact in vivo. MLCK activation perturbs size selectivity to enhance paracellular flux of uncharged macromolecules without affecting charge selectivity and is rapidly-reversed by MLCK inhibition. In contrast, IL-13-dependent claudin-2 expression increases paracellular cation flux without altering tight junction size selectivity but is unaffected by MLCK inhibition in vitro. In vivo, MLCK activation increases paracellular flux of uncharged macromolecules but also triggers IL-13 expression, claudin-2 synthesis, and increased paracellular cation flux. We conclude that reversible, MLCK-dependent permeability increases cause mucosal immune activation that, in turn, feeds back on the tight junction to establish long-lasting barrier defects. Interactions between these otherwise distinct tight junction regulatory pathways may contribute to IBD pathogenesis.

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Human biliverdin reductase suppresses goodpasture antigen binding protein (GPBP) kinase activity: the reductase regulates TNF-{alpha}- NF-{kappa}B-dependent GPBP expression.

Publication Date: 2010 Feb 22 PMID: 20177069
Authors: Miralem, T. - Gibbs, P. E. - Revert, F. - Saus, J. - Maines, M. D.
Journal: J Biol Chem

The S/T/Y kinase activity of human biliverdin reductase (hBVR) and the expression of Goodpasture antigen-binding protein (GPBP), a nonconventional S/T kinase for the type IV collagen of basement membrane, are regulated by tumor necrosis factor (TNF-alpha). The pro-inflammatory cytokine stimulates kinase activity of hBVR and activates NF-kappaB, a transcriptional regulator of GPBP mRNA. Increased GPBP activity is associated with several autoimmune conditions, including Goodpasture syndrome. Here we show that, in HEK293A cells, hBVR binds to GPBP and down-regulates its TNF-alpha-stimulated kinase activity; this was not due to a decrease in GPBP expression. Findings with si-hBVR and siRNA to the P65 regulatory subunit of NF-kappaB show hBVR's role in the initial stimulation of GPBP expression by TNF-alpha-activated NF-kappaB; hBVR was not a factor in mediating GPBP mRNA stability. The interacting domain was mapped to the C281X10C motif in the C-terminal 24 residues of hBVR. A 7-residue peptide, KKRILHC281, corresponding to the core of the consensus D(delta)-Box motif in the interacting domain, was as effective as the intact 296 residue hBVR polypeptide in inhibiting GPBP kinase activity. GPBP neither regulated hBVR expression nor TNF-alpha dependent NF-kappaB expression. Collectively, our data reveal that hBVR is a regulator of the TNF-alpha-GPBP-collagen type IV signaling cascade and uncover a novel biological interaction that may be of relevance in autoimmune pathogenesis.

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The Hsp90 cochaperone Aha1 is a negative regulator of the Saccharomyces MAL-activator and acts early in the chaperone activation pathway.

Publication Date: 2010 Feb 22 PMID: 20177068
Authors: Ran, F. - Gadura, N. - Michels, C. A.
Journal: J Biol Chem

Aha1 is a ubiquitous cochaperone of the Hsp90/Hsp70 chaperone machine. It binds Hsp90 and stimulates ATPase activity, suggesting a function late in the chaperone pathway. Saccharomyces Mal63 MAL-activator is a DNA-binding transcription factor and Hsp90 client protein. This study utilizes several MAL-activator mutants to investigate Aha1 function in vivo. Deletion of AHA1 enhances induced Mal63-dependent maltase activity levels 2-fold while over-production of Aha1 represses expression. Maltase expression in strains carrying constitutive and super-inducible mutant activators with alterations near the C-terminus (particularly residues 433-463) is unaffected by either aha1Delta or Aha1 over-production. However, another constitutive activator with alterations outside of this C-terminal region is sensitive to Aha1 regulation. Previously, we showed that in the absence of inducer Mal63 forms a stable intermediate complex with Hsp70, Hsp90, and Sti1 while noninducible mutant activators bind only with Hsp70 in an apparent early complex. Here we find that triple Myc-tagged Aha1/Myc3 co-purifies with all noninducible Mal63 mutant activators tested. Aha1/Myc3 association with inducible Mal63 is observed only in a sti1Delta strain, in which Hsp90 binding and intermediate complex formation is defective. Constitutive and super-inducible mutant activators with C-terminal alterations do not bind Aha1 even in a sti1Delta strain. Mal63 binding to Hsp90 and Hsp70 is enhanced 3-fold by loss of Aha1. These results suggest an interaction between Aha1 and residues near the C-terminus of Mal63 thereby regulating Hsp90 association. A novel mechanism for the negative regulation of the MAL-activator by Aha1 cochaperone is proposed.

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Suppression of melanin production by expression of HSP70.

Publication Date: 2010 Feb 22 PMID: 20177067
Authors: Hoshino, T. - Matsuda, M. - Yamashita, Y. - Takehara, M. - Fukuya, M. - Mineda, K. - Maji, D. - Ihn, H. - Adachi, H. - Sobue, G. - Funasaka, Y. - Mizushima, T.
Journal: J Biol Chem

Skin hyperpigmentation disorders due to abnormal melanin production induced by ultraviolet (UV) irradiation are both a clinical and cosmetic problem. UV irradiation stimulates melanin production in melanocytes by increasing intracellular cAMP. Expression of heat shock proteins (HSPs), especially HSP70, is induced by various stressors, including UV irradiation, to provide cellular resistance to such stressors. In this study, we examined the effect of expression of HSP70 on melanin production both in vitro and in vivo. IBMX (3-isobutyl-1-methylxanthine, a cAMP-elevating agent) stimulated melanin production in cultured mouse melanoma cells and this stimulation was suppressed in cells overexpressing HSP70. IBMX-dependent transcriptional activation of the tyrosinase gene was also suppressed in HSP70-overexpressing cells. Expression of microphthalmia-associated transcription factor (MITF), which positively regulates transcription of the tyrosinase gene was up-regulated by IBMX; however, this up-regulation was not suppressed in HSP70-overexpressing cells. On the other hand, immunoprecipitation and immunostaining analyses revealed a physical interaction between and co-localization of MITF and HSP70, respectively. Furthermore, the transcription of tyrosinase gene in nuclear extract was inhibited by HSP70. In vivo, UV irradiation of wild-type mice increased the amount of melanin in the basal layer of the epidermis and this increase was suppressed in transgenic mice expressing HSP70. This study provides the first evidence of an inhibitory effect of HSP70 on melanin production both in vitro and in vivo. This effect seems to be mediated by modulation of MITF activity through a direct interaction between HSP70 and MITF.

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Ubiquitination of PTEN inhibits phosphatase activity and is enhanced by membrane targeting and hyperosmotic stress.

Publication Date: 2010 Feb 22 PMID: 20177066
Authors: Maccario, H. - Perera, N. M. - Gray, A. - Downes, C. P. - Leslie, N. R.
Journal: J Biol Chem

The PTEN tumour suppressor is a phosphatase that inhibits PI3K dependent signalling by metabolising the phosphoinositide lipid, PtdInsP3, at the plasma membrane. PTEN can be mono- or poly-ubiquitinated, and this appears to control its nuclear localisation and stability respectively. Although PTEN phosphorylation at a cluster of C-terminal serine and threonine residues has been shown to stabilise the protein and inhibit polyubiquitination as well as inhibit plasma membrane localisation, details of the regulation of ubiquitination are unclear. Here we show that plasma membrane targeting of PTEN greatly enhances PTEN ubiquitination and that phosphorylation of PTEN in vitro does not affect subsequent ubiquitination. These data suggest that C-terminal phosphorylation indirectly regulates ubiquitination by controlling membrane localisation. We also show that either mono- or poly-ubiquitination in vitro greatly reduces PTEN phosphatase activity. Finally, we show that hyperosmotic stress increases both PTEN ubiquitination and cellular PtdInsP3 levels well before a reduction in PTEN protein levels is observed. Both PTEN ubiquitination and elevated PtdInsP3 levels were reduced within ten minutes after removal of the hyperosmotic stress. Our data indicate that ubiquitination may represent a regulated mechanism of direct reversible control over the PTEN enzyme.

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The Pax6b homeodomain is dispensable for pancreatic endocrine cell differentiation in zebrafish.

Publication Date: 2010 Feb 22 PMID: 20177065
Authors: Verbruggen, V. - Ek, O. - Georlette, D. - Delporte, F. - Von Berg, V. - Detry, N. - Biemar, F. - Coutinho, P. - Martial, J. A. - Voz, M. L. - Manfroid, I. - Peers, B.
Journal: J Biol Chem

Pax6 is a well conserved transcription factor that contains two DNA binding domains, a paired domain and a homeodomain, and plays a key role in the development of eye, brain and pancreas in vertebrates. The recent identification of the zebrafish sunrise mutant, harbouring a mutation in pax6b homeobox and presenting eye abnormalities but no obvious pancreatic defects, raised the question about the role of pax6b in zebrafish pancreas. We show here that pax6b does play an essential role in pancreatic endocrine cell differentiation as revealed by the phenotype of a novel zebrafish pax6b null mutant and of embryos injected with pax6b morpholinos. Pax6b-depleted embryos have almost no beta cells, a strongly reduced number of delta cells, and a significant increase of epsilon cells. Through the use of various morpholinos targetting intron-exon junctions, pax6b RNA splicing was perturbed at several sites leading either to retention of intronic sequences or to deletion of exonic sequences in pax6b transcript. By this strategy, we show that deletion of Pax6b homeodomain in zebrafish embryos does not disturb pancreas development while lens formation is strongly affected. These data thus provide the explanation for the lack of pancreatic defects in the sunrise pax6b mutants. In addition, partial reduction of Pax6b function in zebrafish embryos performed by injection of small amounts of pax6b morpholinos caused a clear rise in alpha cell number and in glucagon expression, emphasizing the importance of the fine tuning of Pax6b level on its biological activity.

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Tracing of conformational transition of abnormal prion proteins during interspecies transmission by using novel antibodies.

Publication Date: 2010 Feb 22 PMID: 20177064
Authors: Ushiki-Kaku, Y. - Endo, R. - Iwamaru, Y. - Shimizu, Y. - Imamura, M. - Masujin, K. - Yamamoto, T. - Hattori, S. - Itohara, S. - Irie, S. - Yokoyama, T.
Journal: J Biol Chem

Conformational differences in abnormal prion proteins (PrPSc) have been postulated to produce different prion phenotypes. During the interspecies transmission of prions, the conformation of PrPSc may change with passage; however, little is known about the mechanism of PrPSc transition. In this study, novel PrPSc-specific monoclonal antibodies (mAbs) were developed that could detect the PrPSc of mouse but not that of sheep. By using these mAbs, we attempted to examine PrPSc accumulated in mice inoculated with sheep scrapie serially up to five passages. The presence of PrPSc in the mice was confirmed at all passages; however, mAb-bound PrPSc conformer was detected only from the third passage onward. The generated mAb enabled tracing of a particular conformer during adaptation in sheep-to-mice transmission of prion, suggesting that conformational transition of PrPSc was caused by propagation of this conformer. Such mAbs capable of discriminating conformational differences may allow us to address questions concerning PrPSc conformation and strain diversity.

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Role of alkaline phosphatase from Manduca sexta in the mechanism of action of Bacillus thuringiensis Cry1Ab toxin.

Publication Date: 2010 Feb 22 PMID: 20177063
Authors: Arenas, I. - Bravo, A. - Soberon, M. - Gomez, I.
Journal: J Biol Chem

Cry toxins produced by Bacillus thuringiensis have been recognized as pore-forming toxins, whose primary action is to lyse midgut epithelial cells in their target insect. In the case of the Cry1A toxins a pre-pore oligomeric intermediated is formed after interaction with cadherin receptor. The Cry1A-oligomer then interacts with glycosylphosphatidyl-inositol (GPI)-anchored receptors. Two M. sexta GPI-anchored proteins, aminopeptidase (APN) and alkaline phosphatase (ALP) have been shown to bind Cry1Ab, although their role in toxicity remains to be determined. Detection of Cry1Ab binding proteins by ligand blot assay revealed that ALP is preferentially expressed earlier during insect development, since it was found in the first larval instars while APN is induced later after the third larval instar. The binding of Cry1Ab-oligomer to pure preparations of APN and ALP showed that this toxin structure interacts with both receptors with high affinity, while the monomer showed weaker binding. Several Cry1Ab non-toxic mutants located in the exposed loop 2 of Domain II or in beta-16 of Domain III were affected in binding to APN and ALP depending on their oligomeric state. In particular monomers of the non-toxic Domain III L511A mutant did not bound ALP but retained APN binding suggesting that initial interaction with ALP is critical for toxicity. The expression pattern of these receptors and the phenotype of L511A mutant suggest that ALP may have a predominant role in toxin action since Cry toxins are highly effective against neonate larvae that is the target for pest control programs.

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Tmem16A encodes the Ca2+-activated Cl- channel in mouse submandibular salivary gland acinar cells.

Publication Date: 2010 Feb 22 PMID: 20177062
Authors: Romanenko, V. G. - Catalan, M. A. - Brown, D. A. - Putzier, I. - Hartzell, H. C. - Marmorstein, A. D. - Gonzalez-Begne, M. - Rock, J. R. - Harfe, B. D. - Melvin, J. E.
Journal: J Biol Chem

Activation of an apical Ca2+-dependent Cl- channel (CaCC) is the rate limiting step for fluid secretion in many exocrine tissues. Here we compared the properties of native CaCC in mouse submandibular salivary gland acinar cells to the Ca2+-gated Cl- currents generated by Tmem16A and Best2, members from two distinct families of Ca2+-activated Cl- channels found in salivary glands. Heterologous expression of Tmem16A and Best2 transcripts in HEK293 cells produced Ca2+-activated Cl- currents with time- and voltage-dependence and inhibitor sensitivity that resembled the Ca2+-activated Cl- current found in native salivary acinar cells. Best2-/- and Tmem16A-/- mice were used to further characterize the role of these channels in the exocrine salivary gland. The amplitude and the biophysical footprint of the Ca2+-activated Cl- current in submandibular gland acinar cells from Best2-deficient mice were the same as in wildtype cells. Consistent with this observation, the fluid secretion rate in Best2 null mice was comparable to that in wildtype mice. In contrast, submandibular gland acinar cells from Tmem16A-/- mice lacked a Ca2+-activated Cl- current and a Ca2+-mobilizing agonist failed to stimulate Cl- efflux, requirements for fluid secretion. Furthermore, saliva secretion was abolished by the CaCC inhibitor niflumic acid in wildtype and Best2-/- mice. Our results demonstrate that both Tmem16A and Best2 generate Ca2+-activated Cl- current in vitro with similar properties to those expressed in native cells, yet only Tmem16A appears to be a critical component of the acinar Ca2+-activated Cl- channel complex that is essential for saliva production by the submandibular gland.

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A cleavage-resistant urokinase plasminogen activator receptor exhibits dysregulated cell-surface clearance.

Publication Date: 2010 Feb 22 PMID: 20177061
Authors: Nieves, E. C. - Manchanda, N.
Journal: J Biol Chem

Urokinase plasminogen activator receptor (u-PAR) binds urokinase plasminogen activator (u-PA) and participates in plasminogen activation in addition to modulating several cellular processes such as adhesion, proliferation, and migration. U-PAR is susceptible to proteolysis by its cognate ligand and several other proteases. To elucidate the biological significance of receptor cleavage by u-PA, we engineered and expressed a two-chain urokinase plasminogen activator (tcu-PA) cleavage resistant u-PAR (cr-uPAR). This mutated receptor was similar to wild-type u-PAR in binding u-PA and initiating plasminogen activation. However, cr-uPAR exhibited accelerated internalization and resurfacing due to direct association with the endocytic receptor alpha2-macroglobulin receptor / low-density lipoprotein receptor related protein (LRP) in the absence of the enzyme-inhibitor complex of tcu-PA and plasminogen activator inhibitor-1 (tcu-PA-PAI-1). Cr-uPAR expressing cells had enhanced migration compared to wt u-PAR expressing cells, and cr-uPAR was less sensitive to chymotrypsin cleavage as compared to wt u-PAR. Our studies suggest that these mutations in the linker region result in a rearrangement within the cr-uPAR structure that makes it resemble its ligand bound form. This constitutively active variant may mimic highly glycosylated cleavage-resistant u-PAR expressed in certain highly malignant cancer-cells.

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Ribosomal protein S6 kinase is a critical downstream effector of the target of rapamycin complex 1 for long-term facilitation in Aplysia.

Publication Date: 2010 Feb 22 PMID: 20177060
Authors: Weatherill, D. B. - Dyer, J. - Sossin, W. S.
Journal: J Biol Chem

Long-term facilitation (LTF) in Aplysia is a leading cellular model for elucidating the biochemical mechanisms of synaptic plasticity underlying learning. In Aplysia, LTF requires translational control downstream of the target of rapamycin (TOR) complex 1 (TORC1). The major known downstream targets of TORC1 are 4E binding protein (4E-BP) and S6 kinase (S6K). By removing the site within these regulators required for their interaction with TORC1, we have generated dominant negative proteins that disrupt specific pathways downstream of TORC1. Expression of dominant negative S6K, but not dominant negative 4E-BP, in Aplysia sensory neurons (SNs) blocked 24-hr LTF. TORC1 is directly activated by the small GTP-binding protein, Rheb. To determine the effects of TORC1 activation on translation in Aplysia neurons, we have examined the effects of expressing a constitutively active form of the Aplysia orthologue of Rheb, ApRheb (ApRheb(Q63L)). Expression of ApRheb(Q63L) increased 4E-BP phosphorylation and the level of general, cap-dependent translation within the SN cell soma in a rapamycin-sensitive manner. This increase in cap-dependent translation was neither blocked by dominant negative 4E-BP nor dominant negative S6K. Thus, we demonstrate that S6K is an important downstream target of TORC1 in Aplysia and that it is necessary for 24-hr LTF, but not for TORC1-mediated increases in somatic cap-dependent translation.

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Human carboxymethylenebutenolidase as a bioactivating hydrolase of olmesartan medoxomil in liver and intestine.

Publication Date: 2010 Feb 19 PMID: 20177059
Authors: Ishizuka, T. - Fujimori, I. - Kato, M. - Noji-Sakikawa, C. - Saito, M. - Yoshigae, Y. - Kubota, K. - Kurihara, A. - Izumi, T. - Ikeda, T. - Okazaki, O.
Journal: J Biol Chem

Olmesartan medoxomil (OM) is a prodrug type angiotensin II type 1 receptor antagonist widely prescribed as an antihypertensive agent. Herein, we describe the identification and characterization of the OM bioactivating enzyme that hydrolyzes the prodrug and converts to its pharmacologically active metabolite olmesartan in human liver and intestine. The protein was purified from human liver cytosol by successive column chromatography and was identified by mass spectrometry to be a carboxymethylenebutenolidase (CMBL) homolog. Human CMBL, whose endogenous function has still not been reported, is a human homolog of Pseudomonas dienelactone hydrolase involved in the bacterial halocatechol degradation pathway. The ubiquitous expression of human CMBL gene transcript in various tissues was observed. The recombinant human CMBL expressed in mammalian cells was clearly shown to activate OM. By comparing the enzyme kinetics and chemical inhibition properties between the recombinant protein and human tissue preparations, CMBL was demonstrated to be the primary OM bioactivating enzyme in liver and intestine. The recombinant CMBL also converted other prodrugs having the same ester structure as OM, faropenem medoxomil and lenampicillin to their active metabolites. CMBL exhibited a unique sensitivity to chemical inhibitors, thus, being distinguishable from other known esterases. Site-directed mutagenesis on the putative active residue Cys132 of the recombinant CMBL caused a drastic reduction of the OM-hydrolyzing activity. We report for the first time that CMBL serves as a key enzyme in the bioactivation of OM, hydrolyzing the ester bond of the prodrug type xenobiotics.

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Myristoylated naked2 antagonizes WNT-{beta}-catenin activity by degrading dishevelled-1 at the plasma membrane.

Publication Date: 2010 Feb 20 PMID: 20177058
Authors: Hu, T. - Li, C. - Cao, Z. - Van Raay, T. J. - Smith, J. G. - Willert, K. - Solnica-Krezel, L. - Coffey, R. J.
Journal: J Biol Chem

In Drosophila, naked cuticle is an inducible antagonist of the Wnt- beta-catenin pathway, likely acting at the level of Dishevelled (Dsh/Dvl), an essential component of this pathway. The mechanism by which naked cuticle and its two vertebrate orthologs, Naked1 (NKD1) and Naked2 (NKD2), inhibit Dvl function is unknown. NKD2 is myristoylated, a co-translational modification that leads to its plasma membrane localization. In contrast, myristoylation-deficient G2A NKD2 is cytoplasmic. Herein we show that the ability of Nkd2/NKD2 to antagonize Wnt- beta-catenin activity during zebrafish embryonic development and in mammalian HEK293 cells is myristoylation-dependent. NKD2 and Dvl-1 interact and co-localize at the lateral membrane of polarized epithelial cells. In reciprocal overexpression and siRNA knockdown experiments, NKD2 and Dvl-1 destabilize each other via enhanced polyubiquitylation; this effect is also dependent upon Naked2 myristoylation. Cell fractionation and ubiquitylation assays indicate that endogenous NKD2 interacts with a slower migrating, ubiquitylated form of Dvl-1 in plasma membrane fractions. These results provide a mechanism by which NKD2 antagonizes Wnt signaling: myristoylated NKD2 interacts with Dvl-1 at the plasma membrane and this interaction leads to their mutual ubiquitin-mediated proteasomal degradation.

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The endoplasmic reticulum Grp170 acts as a nucleotide exchange factor of Hsp70 via a mechanism similar to that of the cytosolic Hsp110.

Publication Date: 2010 Feb 20 PMID: 20177057
Authors: Andreasson, C. - Rampelt, H. - Fiaux, J. - Druffel-Augustin, S. - Bukau, B.
Journal: J Biol Chem

Grp170 and Hsp110 proteins constitute two evolutionary distinct branches of the Hsp70 family that share the ability to function as nucleotide exchange factors (NEFs) for canonical Hsp70s. While the NEF mechanism of the cytoplasmic Hsp110s is well understood, little is known regarding the mechanism used by Grp170s in the endoplasmic reticulum. In this study, we compare the yeast Grp170 Lhs1 with the yeast Hsp110 Sse1. We find that residues important for Sse1 NEF activity are conserved in Lhs1 and that mutations in these residues in Lhs1 compromise NEF activity. Like previously reported for Sse1, Lhs1 requires ATP to trigger nucleotide exchange in its cognate Hsp70 partner Kar2. Using site-specific crosslinking, we show that the nucleotide-binding domain (NBD) of Lhs1 interacts with the NBD of Kar2 face to face, and that Lhs1 contacts the side of the Kar2 NBD via its protruding C-terminal alpha-helical domain. To directly address the mechanism of nucleotide exchange, we have compared the hydrogen-exchange characteristics of a yeast Hsp70 NBD (Ssa1) in complex with either Sse1 or Lhs1. We find that Lhs1 and Sse1 induce very similar changes in the conformational dynamics in the Hsp70. Thus, our findings demonstrate that despite some differences between Hsp110 and Grp170 proteins, they use a similar mechanism to trigger nucleotide exchange.

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Inhibition of Na+-taurocholate co-transporting polypeptide mediated bile acid transport by cholestatic sulphated progesterone metabolites.

Publication Date: 2010 Feb 23 PMID: 20177056
Authors: Abu-Hayyeh, S. - Martinez-Becerra, P. - Sheikh Abdul Kadir, S. H. - Kadir, A. - Selden, C. - Romero, M. R. - Rees, M. - Marschall, H. U. - Marin, J. J. - Williamson, C.
Journal: J Biol Chem

Sulphated progesterone metabolite (P4-S) levels are raised in normal pregnancy and elevated further in intrahepatic cholestasis of pregnancy (ICP), a bile acid-liver disorder of pregnancy. ICP can be complicated by preterm labour and intrauterine death. The impact of P4-S on bile acid uptake was studied using two experimental models of hepatic uptake of bile acids, namely cultured primary human hepatocytes (PHH) and Na(+)-taurocholate co-transporting polypeptide (NTCP)-expressing Xenopus laevis oocytes. Two P4-S compounds, allopregnanolone-sulphate (PM4-S) and epiallopregnanolone-sulphate (PM5-S) reduced (3)H-taurocholate ((3)HTC) uptake in a dose-dependent manner in PHH, with both Na(+)-dependent and -independent bile acid uptake systems significantly inhibited. PM5-S mediated inhibition of TC uptake could be reversed by increasing the TC concentration against a fixed PM5-S dose indicating competitive inhibition. Experiments using NTCP-expressing Xenopus oocytes confirmed that PM4-S/PM5-S are capable of competitively inhibiting NTCP-mediated uptake of (3)HTC, while progesterone and non-sulphated PM4 and PM5 have a minor effect. Total serum PM4-S+PM5-S levels were measured in non-pregnant and third trimester pregnant women using LCMS and were increased in pregnant women, at levels capable of inhibiting TC-uptake. In conclusion, pregnancy levels of P4-S can inhibit Na(+)-dependent and independent influx of taurocholate in PHH and cause competitive inhibition of NTCP-mediated uptake of taurocholate in Xenopus oocytes. Serum levels of P4-S are raised in pregnancy, and more markedly in ICP. Therefore P4-S-mediated competitive inhibition of NTCP uptake of bile acids may contribute to the biochemical phenotype of hypercholanaemia in pregnancy and ICP. P4-S mediated exacerbation of hypercholanaemia may also contribute to ICP-related fetal complications.

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Characterization of the biochemical properties and biological function of the formin homology domains of drosophila DAAM.

Publication Date: 2010 Feb 21 PMID: 20177055
Authors: Barko, S. - Bugyi, B. - Carlier, M. F. - Gombos, R. - Matusek, T. - Mihaly, J. - Nyitrai, M.
Journal: J Biol Chem

We characterised the properties of Drosophila melanogaster DAAM-FH2 and DAAM-FH1-FH2 fragments and their interactions with actin and profilin by using various biophysical methods and in vivo experiments. The results show that while the DAAM-FH2 fragment does not have any conspicuous effect on actin assembly in vivo, in cells expressing the DAAM-FH1-FH2 fragment a profilin-dependent increase in the formation of actin structures is observed. The trachea specific expression of DAAM-FH1-FH2 also induces phenotypic effects leading to the collapse of the tracheal tube and lethality in the larval stages. In vitro, both DAAM fragments catalyze actin nucleation but severely decrease both the elongation and depolymerisation rate of the filaments. Profilin acts as a molecular switch in DAAM function. DAAM-FH1-FH2, remaining bound to barbed ends drives processive assembly of profilin-actin, while DAAM-FH2 forms an abortive complex with barbed ends that does not support profilin-actin assembly. Both DAAM fragments also bind to the sides of the actin filaments and induce actin bundling. These observations show that the Drosophila melanogaster DAAM formin represents an extreme class of barbed end regulators gated by profilin.

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Identification of Functionally Segregated Sarcoplasmic Reticulum Calcium Stores in Pulmonary Arterial Smooth Muscle.

Publication Date: 2010 Feb 21 PMID: 20177054
Authors: Clark, J. H. - Kinnear, N. P. - Kalujnaiab, S. - Cramb, G. - Fleischer, S. - Jeyakumar, L. H. - Wuytack, F. - Evans, A. M.
Journal: J Biol Chem

In pulmonary arterial smooth muscle Ca2+ release from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) may induce constriction and dilation in a manner that is not mutually exclusive. We show here that the targeting of different SR Ca2+ ATPases (SERCA) and RyR subtypes to discrete SR regions explains this paradox. Western blots identified protein bands for SERCA2a and SERCA2b, while immunofluorescence labelling of isolated pulmonary arterial smooth muscle cells revealed striking differences in the spatial distribution of SERCA2a and SERCA2b, and RyR1, RyR2 and RyR3, respectively. Almost all SERCA2a and RyR3 labelling was restricted to a region within 1.5 microns of the nucleus. In marked contrast, SERCA2b labelling was primarily found within 1.5 microns of the plasma membrane, where labelling for RyR1 was maximal. The majority of labelling for RyR2 lay in between these two regions of the cell. Application of the vasoconstrictor endothelin-1 induced global Ca2+ waves in pulmonary arterial smooth muscle cells, which were markedly attenuated upon depletion of SR Ca2+ stores by pre-incubation of cells with the SERCA inhibitor thapsigargin, but remained unaffected after pre-incubation of cells with a second SERCA antagonist, cyclopiazonic acid. We conclude that functionally segregated SR Ca2+ stores exist within pulmonary arterial smooth muscle cells. One sits proximal to the plasma membrane, receives Ca2+ via SERCA2b and likely releases Ca2+ via RyR1 to mediate vasodilation. The other is located centrally, receives Ca2+ via SERCA2a and likely releases Ca2+ via RyR3 and RyR2 to initiate vasoconstriction.

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miR-9 and NFATc3 regulate myocardin in cardiac hypertrophy.

Publication Date: 2010 Feb 21 PMID: 20177053
Authors: Wang, K. - Long, B. - Zhou, J. - Li, P. F.
Journal: J Biol Chem

Myocardial hypertrophy is frequently associated with poor clinical outcomes including the development of cardiac systolic and diastolic dysfunction and ultimately heart failure. In order to prevent cardiac hypertrophy and heart failure, it is necessary to identify and characterize molecules that may regulate hypertrophic program. Our present study reveals that nuclear factor of activated T cells c3 (NFATc3) and myocardin constitute a hypertrophic pathway that can be targeted by miR-9. Our results show that myocardin expression is elevated in response to hypertrophic stimulation with isoproterenol and aldosterone. In exploring the molecular mechanism by which myocardin expression is elevated, we identified that NFATc3 can bind to the promoter region of myocardin and transcriptionally activate its expression. Knockdown of myocardin can attenuate hypertrophic responses triggered by NFATc3, suggesting that myocardin can be a downstream mediator of NFATc3 in the hypertrophic cascades. MicroRNAs (miRNAs) are a class of small non-coding RNAs that mediate post-transcriptional gene silencing. Our data reveals that miR-9 can suppress myocardin expression. However, the hypertrophic stimulation with isoproterenol and aldosterone leads to a decrease in the expression levels of miR-9. Administration of miR-9 could attenuate cardiac hypertrophy and ameliorate cardiac function. Taken together, our data demonstrate that NFATc3 can promote myocardin expression, whereas miR-9 is able to suppress myocardin expression thereby regulating cardiac hypertrophy.

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PARP-1-independent AIF release and cell death are induced by eleostearic acid, and blocked by {alpha}-tocopherol and MEK inhibition.

Publication Date: 2010 Feb 22 PMID: 20177052
Authors: Kondo, K. - Obitsu, S. - Ohta, S. - Matsunami, K. - Otsuka, H. - Teshima, R.
Journal: J Biol Chem

Poly(ADP-ribose)polymerase-1 (PARP-1) is thought to be required for apoptosis-inducing factor (AIF) release from mitochondria in caspase-independent apoptosis. The mechanism by which AIF is released through PARP-1 remains unclear. Here we provide evidence that PARP-1-independent AIF release and cell death is induced by a trienoic fatty acid, alpha-eleostearic acid (alpha-ESA). alpha-ESA induced the caspase-independent and AIF-initiated apoptotic death of neuronal cell lines, independently of PARP-1 activation. The cell death was inhibited by MEK inhibitor U0126 and by knockdown of MEK using siRNA. However, inhibitors for JNK, p38 inhibitors, calpain, PLA2, and PI3K did not block the cell death. AIF was translocated to the nucleus after the induction of apoptosis by alpha-ESA in a differentiated PC12 cells without activating caspase-3 and PARP-1. The alpha-ESA-mediated cell death was not inhibited by PARP inhibitor DPQ and by knockdown of PARP-1 using siRNA. Unlike MNNG treatment, histone H2AX was not phosphorylated by alpha-ESA, which suggests no DNA damage. Overexpression of Bcl-2 did not inhibit the cell death. alpha-ESA caused a small quantity of superoxide production in the mitochondria, resulting in the reduction of mitochondrial membrane potential, both of which were blocked by a trace amount of alpha-Toc localized in the mitochondria. Our results demonstrate that alpha-ESA induces PARP-1-independent AIF release and cell death without activating Bax, cytochome-c, and caspase-3. MEK is also a key molecule, although the link between ERK, AIF release, and cell death remains unknown. Finding molecules that regulates AIF release may be an important therapeutic target for the treatment of neuronal injury.

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Calcium activates Nedd4 E3 ubiquitin ligases by releasing the C2 domain-mediated auto-inhibition.

Publication Date: 2010 Feb 19 PMID: 20172859
Authors: Wang, J. - Peng, Q. - Lin, Q. - Childress, C. - Carey, D. J. - Yang, W.
Journal: J Biol Chem

Nedd4 E3 ligases are members of the HECT E3 ubiquitin ligase family and regulate ubiquitination-mediated protein degradation. In this report, we demonstrate that calcium releases the C2 domain-mediated auto-inhibition in both Nedd4-1 and Nedd4-2. Calcium disrupts binding of the C2 domain to the HECT domain. Consistent with this, calcium activates the E3 ubiquitin ligase activity of Nedd4. Elevation of intracellular calcium by ionomycin treatment, or activation of acetylcholine receptor or epidermal growth factor receptor (EGFR) by carbachol or EGF stimulation induced activation of endogenous Nedd4 in vivo evaluated by assays of either Nedd4 E3 ligase activity or ubiquitination of Nedd4 substrate ENaC-beta. The activation effect of calcium on Nedd4 E3 ligase activity was dramatically enhanced by a membrane-rich fraction, suggesting that calcium-mediated membrane translocation through the C2 domain might be an activation mechanism of Nedd4 in vivo. Our studies have revealed an activation mechanism of Nedd4 E3 ubiquitin ligases and established a connection of intracellular calcium signaling to regulation of protein ubiquitination.

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The BCL-2 protein BAK is required for long-chain ceramide generation during apoptosis.

Publication Date: 2010 Feb 18 PMID: 20172858
Authors: Siskind, L. J. - Mullen, T. D. - Rosales, K. R. - Clarke, C. J. - Hernandez-Corbacho, M. J. - Edinger, A. L. - Obeid, L. M.
Journal: J Biol Chem

The BCL-2 family members BAK and BAX are required for apoptosis and trigger mitochondrial outer membrane permeablization (MOMP). Here we identify a MOMP-independent function of BAK as a required factor for long-chain ceramide production in response to pro-apoptotic stress. UV-C irradiation of wild-type (WT) cells increased long-chain ceramides; blocking ceramide generation prevented caspase activation and cell death, demonstrating that long-chain ceramides play a key role in UV-C-induced apoptosis. In contrast, UV-C irradiation did not increase long-chain ceramides in BAK and BAX double knock-out cells (DKO). Notably, this was not specific to the cell type (BMK, hematopoietic) nor the apoptotic stimulus employed (UV-C, cisplatin, growth factor withdrawal). Importantly, long-chain ceramide generation was dependent on the presence of BAK, but not BAX. However, ceramide generation was independent of the known downstream actions of BAK in apoptosis (MOMP or caspase activation), suggesting a novel role for BAK in apoptosis. Finally, enzymatic assays identified ceramide synthase as the mechanism by which BAK regulates ceramide metabolism. There was no change in CerS expression at the message or protein level, indicating regulation at the post-translational level. Moreover, CerS activity in BAK KO microsomes can be reactivated upon addition of BAK containing microsomes. The data presented indicate that ceramide-induced apoptosis is dependent upon BAK and identify a novel role for BAK during apoptosis. By establishing a unique role for BAK in long-chain ceramide metabolism, these studies further demonstrate that the seemingly redundant proteins BAK and BAX have distinct mechanisms of action during apoptosis induction.

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Identification and characterization of high-molecular-weight complexes formed by m-AAA proteases and prohibitins in mitochondria of Arabidopsis thaliana.

Publication Date: 2010 Feb 19 PMID: 20172857
Authors: Piechota, J. - Kolodziejczak, M. - Juszczak, I. - Sakamoto, W. - Janska, H.
Journal: J Biol Chem

We identify and characterize two m-AAA proteases (AtFtsH3 and AtFtsH10) present in mitochondria of Arabidopsis thaliana. AtFtsH3 is predominant protease in leaves of wild type plants. Both proteases assemble with prohibitins (PHBs) into high-molecular-weight complexes (~2 MDa), similarly to their yeast counterparts. A smaller PHB complex (~1 MDa), without the m-AAA proteases, was also detected. Unlike in yeast, stable prohibitin-independent high-molecular-weight assemblies of m-AAA proteases could not be identified in A. thaliana. AtFtsH3 and AtFtsH10 form at least two types of m-AAA-PHB complexes in wild type plants. The one type contains PHBs and AtFtsH3, the second one is composed of PHBs and both AtFtsH3 and AtFtsH10. Complexes composed of PHBs and AtFtsH10 were found in an Arabidopsis mutant lacking AtFtsH3 (ftsh3). Thus, both AtFtsH3 and AtFtsH10 may form hetero- and homo-oligomeric complexes with prohibitins. The increased level of AtFtsH10 observed in ftsh3 suggests that functions of the homo- and hetero-oligomeric complexes containing AtFtsH3 can be at least partially substituted by AtFtsH10 homo-oligomers. The steady-state level of the AtFtsH10 transcripts did not change in ftsh3 compared to wild-type plants, but we found that almost twice more of the AtFtsH10 transcripts were associated with polysomes in ftsh3. Based on this result we assume that the AtFtsH10 protein is synthesised at a higher rate in the ftsh3 mutant. Our results provide the first data on the composition of m-AAA and PHB complexes in plant mitochondria and suggest that the abundance of m-AAA proteases is regulated not only at the transcriptional, but also at the translational level.

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PB1-F2 influenza a virus protein adopts a beta-sheet conformation and forms amyloid fibers in membrane environments.

Publication Date: 2010 Feb 19 PMID: 20172856
Authors: Chevalier, C. - Al Bazzal, A. - Vidic, J. - Fevrier, V. - Bourdieu, C. - Bouguyon, E. - Le Goffic, R. - Vautherot, J. F. - Bernard, J. - Moudjou, M. - Noinville, S. - Chich, J. F. - Da Costa, B. - Rezaei, H. - Delmas, B.
Journal: J Biol Chem

The influenza A virus PB1-F2 protein, encoded by an alternative reading frame in the PB1 polymerase gene, displays a high sequence polymorphism and is reported to contribute to viral pathogenesis in a sequence-specific manner. To gain insights into the functions of PB1-F2, the molecular structure of several PB1-F2 variants produced in E. Coli was investigated in different environments. Circular dichroism spectroscopy shows that all variants have a random coil secondary structure in aqueous solution. When incubated in TFE polar solvent, all PB1-F2 variants adopt an alpha-helix rich structure, while incubated in acetonitrile, a solvent of medium polarity mimicking membrane environment they display beta-sheet secondary structures. Incubated with asolectin liposomes and SDS micelles, PB1-F2 variants also acquire a beta-sheet structure. Dynamic light scattering revealed that the presence of beta-sheets is correlated with an oligomerisation/aggregation of PB1-F2. Electron microscopy showed that PB1-F2 forms amorphous aggregates in acetonitrile. In contrast, at low concentrations of SDS, PB1-F2 variants exhibited various abilities to form fibers that were evidenced as amyloid fibers in a thioflavine T assay. Using a recombinant virus and its PB1-F2 knocked-out mutant, we show that PB1-F2 also forms amyloid structures in infected cells. Functional membrane permeabilization assays revealed that the PB1-F2 variants can perforate membranes at nanomolar concentrations, but with activities found to be sequence-dependent and not obviously correlated with their differential ability to form amyloid fibers. All these observations suggest that PB1-F2 could be involved in physiological processes through different pathways, permeabilization of cellular membranes and amyloid fiber formation.

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Dimeric arrangement of the parathyroid hormone receptor and a structural mechanism for ligand-induced dissociation.

Publication Date: 2010 Feb 19 PMID: 20172855
Authors: Pioszak, A. A. - Harikumar, K. G. - Parker, N. R. - Miller, L. J. - Xu, H. E.
Journal: J Biol Chem

The parathyroid hormone receptor (PTH1R) is a class B G protein-coupled receptor that is activated by parathyroid hormone (PTH) and PTH-related protein (PTHrP). Little is known about the oligomeric state of the receptor and its regulation by hormone. The crystal structure of the ligand-free PTH1R extracellular domain (ECD) reveals an unexpected dimer in which the C-terminal segment of both ECD protomers forms an alpha-helix that mimics PTH/PTHrP by occupying the peptide-binding groove of the opposing protomer. ECD-mediated oligomerization of intact PTH1R was confirmed in living cells by bioluminescence and fluorescence resonance energy transfer experiments. As predicted by the structure, PTH binding disrupted receptor oligomerization. A receptor rendered monomeric by mutations in the ECD retained wild-type PTH-binding and cAMP signaling ability. Our results are consistent with the hypothesis that PTH1R forms constitutive dimers that are dissociated by ligand binding and that monomeric PTH1R is capable of activating G protein.

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A proprotein convertase subtilisin-like/kexin type 9 (PCSK9) C-terminal domain antibody antigen binding fragment inhibits PCSK9 internalization and restores LDL-uptake.

Publication Date: 2010 Feb 19 PMID: 20172854
Authors: Ni, Y. G. - Condra, J. - Orsatti, L. - Shen, X. - Di Marco, S. - Pandit, S. - Bottomley, M. J. - Ruggeri, L. - Cummings, R. T. - Cubbon, R. M. - Santoro, J. C. - Ehrhardt, A. - Lewis, D. - Fisher, T. S. - Ha, S. - Njimoluh, L. - Wood, D. - Hammond, H. A. - Wisniewski, D. - Volpari, C. - Noto, A. - Lo Surdo, P. - Hubbard, B. - Carfi, A. - Sitlani, A.
Journal: J Biol Chem

PCSK9 binds to the low-density lipoprotein receptor (LDLR) and leads to LDLR degradation and inhibition of plasma LDL cholesterol clearance. Consequently, the role of PCSK9 in modulating circulating LDL makes it a promising therapeutic target for treating hypercholesterolemia and coronary heart disease. Although the C-terminal domain of PCSK9 is not involved in LDLR binding, the location of several naturally-occurring mutations within this region suggests that it has an important role for PCSK9 function. Using a phage display library, we identified an anti-PCSK9 Fab (fragment antigen binding), 1G08, with sub-nanomolar affinity for PCSK9. In an assay measuring LDL uptake in HEK293 and HepG2 cells, 1G08 Fab reduced 50% the PCSK9-dependent inhibitory effects on LDL uptake. Importantly, we found that 1G08 did not affect the PCSK9-LDLR interaction but inhibited the internalization of PCSK9 in these cells. Furthermore, proteolysis and site-directed mutagenesis studies demonstrated that 1G08 Fab binds a region of beta-strands encompassing R549, R580, R582, E607, K609 and E612 in the PCSK9 C-terminal domain. Consistent with these results, 1G08 fails to bind PCSK9DeltaC, a truncated form of PCSK9 lacking the C-terminal domain. Additional studies revealed that lack of the C-terminal domain compromised the ability of PCSK9 to internalize into cells, and to inhibit LDL uptake. Together, the present study demonstrate that the PCSK9 C-terminal domain contribute to its inhibition of LDLR function mainly through its role in the cellular uptake of PCSK9 and LDLR complex. 1G08 Fab represents a useful new tool for delineating the mechanism of PCSK9 uptake and LDLR degradation.

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All-trans retinoic acid promotes trafficking of human concentrative nucleoside transporter-3 (hCNT3) to the plasma membrane by a TGF-{beta}1-mediated mechanism.

Publication Date: 2010 Feb 19 PMID: 20172853
Authors: Fernandez-Calotti, P. - Pastor-Anglada, M.
Journal: J Biol Chem

Human concentrative nucleoside transporter-3 (hCNT3) is a sodium-coupled nucleoside transporter that exhibits high affinity and broad substrate selectivity, making it the most suitable candidate for mediating the uptake and cytotoxic action of most nucleoside-derived drugs. The drug of this class most commonly used in the treatment of chronic lymphocytic leukemia (CLL) is the pro-apoptotic nucleoside analog fludarabine (Flu), which enters CLL cells primarily through human equilibrative nucleoside transporters (hENTs). Although CLL cells lack hCNT3 activity, they do express this transporter protein, which is located mostly in the cytosol. The aim of our study was to identify agents and mechanisms capable of promoting hCNT3 trafficking to the plasma membrane. Here, we report that all-trans retinoic acid (ATRA), currently used in the treatment of acute promyelocytic leukemia (APL), increases hCNT3-related activity through a mechanism that involves trafficking of pre-existing hCNT3 proteins to the plasma membrane. This effect is mediated by the autocrine action of transforming growth factor (TGF)-beta1, which is transcriptionally activated by ATRA in a p38-dependent manner. TGF-beta1 acts through activation of ERK1/2 and the small GTPase RhoA to promote plasma membrane trafficking of the hCNT3 protein.

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Insulin-like growth factor binding protein-5-induced laminin {gamma}1 transcription requires filamin A.

Publication Date: 2010 Feb 18 PMID: 20167606
Authors: Abrass, C. K. - Hansen, K. M.
Journal: J Biol Chem

Insulin-like growth factor binding protein-5 (IGFBP-5) has IGF-1-independent intranuclear effects that are poorly defined. Treatment of cells with IGFBP-5 induces migration, prevents apoptosis, and leads to increased laminin subunit transcription. Similarly, filamin A (FLNa), an actin-binding protein that participates in cell attachment, plays important additional roles in signal transduction and modulation of transcriptional responses. In this report, we show that IGFBP-5 leads to dephosphorylation of FLNa with subsequent FLNa cleavage. Following cleavage, there is enhanced recruitment of Smad3/4 to a C-terminal FLNa fragment with nuclear translocation and subsequent binding to the promoter region of the laminin gamma1 (lamc1) gene. FLNa knockdown prevents IGFBP-5-mediated increases in lamc1 transcription. These data indicate that IGFBP-5 induces formation of a FLNa-based nuclear shuttle that recruits transcription factors and regulates transcription of IGFBP-5 target genes. These studies provide new insights into the mechanisms whereby IGFBP-5 and FLNa exert intranuclear effects.

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Modulation of myocardin function by the ubiquitin E3 ligase, UBR5.

Publication Date: 2010 Feb 18 PMID: 20167605
Authors: Hu, G. - Wang, X. - Saunders, D. N. - Henderson, M. - Russell, A. J. - Herring, B. P. - Zhou, J.
Journal: J Biol Chem

Previous studies have shown myocardin to be a critical transcription factor for stimulating expression of smooth muscle-specific genes, but the mechanisms regulating myocardin activity are still poorly understood. We used a yeast 2-hybrid screen with myocardin as bait to search for factors that may regulate myocardin-induced transcriptional activity. From this screen we identified a HECT domain containing protein UBR5 (ubiquitin protein ligase E3 component n-recognin 5) as a myocardin binding protein. Previous studies have shown that HECT domain containing proteins are ubiquitin E3 ligases that play an important role in protein degradation. UBR5 has however, also been shown to regulate transcription independent of its E3 ligase activity. In the current study we demonstrated that UBR5 localized in the nuclei of smooth muscle cells and forms a complex with myocardin in vivo and in vitro. We also show that UBR5 specifically enhanced trans-activation of smooth muscle-specific promoters by the myocardin family of proteins. In addition, UBR5 significantly augmented myocardin ability to induce expression of endogenous SMC marker genes independent on its E3 ligase function. Conversely, depletion of endogenous UBR5 by siRNA in fibroblast cells attenuated myocardin-induced smooth muscle-specific gene expression, and UBR5 knockdown in smooth muscle cells resulted in down-regulation of smooth muscle-specific genes. Furthermore, we found that UBR5 can attenuate myocardin protein degradation resulting in increased myocardin protein expression without affecting myocardin mRNA expression. The effects of UBR5 on myocardin requires only the HECT and UBR1 domains of UBR5. This study reveals an unexpected role for the ubiquitin E3 ligase UBR5 as an activator of smooth muscle differentiation through its ability to stabilize myocardin protein.

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Interleukin-7 compartmentalizes its receptor signaling complex to initiate CD4 T lymphocyte response.

Publication Date: 2010 Feb 18 PMID: 20167604
Authors: Rose, T. - Pillet, A. H. - Lavergne, V. - Tamarit, B. - Lenormand, P. - Rousselle, J. C. - Namane, A. - Theze, J.
Journal: J Biol Chem

Interleukin-(IL)7 is a central cytokine that controls homeostasis of the CD4 T lymphocyte pool. Here we show on human primary cells that IL-7 binds to preassembled receptor made up of proprietary chain IL-7Ralpha and common chain gammac shared with IL-2, -4, -9, -15 and -21 receptors. Upon IL-7 binding, both chains are driven in cholesterol- and sphingomyelin-rich rafts where associated signaling proteins Jak1, Jak3, STAT1, 3 and 5 are found to be phosphorylated. Meanwhile IL-7/IL-7R complex interacts with the cytoskeleton that halts its diffusion as measured by single-molecule FCS monitored by microimaging. Comparative immunoprecipitations of IL-7Ralpha signaling complex from non-stimulated and IL-7-stimulated cells confirmed recruitment of proteins such as STATs, but many others were also identified by mass spectrometry from 2D-gels. Among recruited proteins, two-thirds are involved in cytoskeleton and raft formation. Thus, early events leading to IL-7 signal transduction involve its receptor compartmentalization into membrane nanodomains and cytoskeleton recruitment.

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DYRK1A and DYRK3 promote cell survival through phosphorylation and activation of SIRT1.

Publication Date: 2010 Feb 18 PMID: 20167603
Authors: Guo, X. - Williams, J. G. - Schug, T. T. - Li, X.
Journal: J Biol Chem

The dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) plays an important role in body growth and brain physiology. Overexpression of this kinase has been associated with development of Down syndrome in both human and animal models, whereas single copy loss-of-function of DYRK1A leads to increased apoptosis and decreased brain size. While more than a dozen of DYRK1A targets have been identified, the molecular basis of its involvement in neuronal development remains unclear. Here we show that DYRK1A and another pro-survival member of the DYRK family, DYRK3, promote cell survival through phosphorylation and activation of SIRT1, an NAD+-dependent protein deacetylase that is essential in a variety of physiological processes including stress response and energy metabolism. DYRK1A and DYRK3 directly phosphorylate SIRT1 at Thr522, promoting deacetylation of p53. A SIRT1 phosphorylation mimetic (SIRT1 T522D) displays elevated deacetylase activity, thus inhibiting cell apoptosis. Conversely, a SIRT1 dephosphorylation mimetic (SIRT1 T522V) fails to mediate DYRK-induced deacetylation of p53 and cell survival. We show that knockdown of endogenous DYRK1A and DYRK3 leads to hypo-phosphorylation of SIRT1, sensitizing cells to DNA damage-induced cell death. We also provide evidence that phosphorylation of Thr522 activates SIRT1 by promoting product release thereby increasing its enzymatic turnover. Taken together, our findings provide a novel mechanism by which two anti-apoptotic DYRK members promote cell survival through direct modification of SIRT1. These findings may have important implications in understanding the molecular mechanism of tumorigenesis, Down syndrome, and aging.

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The YTH domain is a novel RNA binding domain.

Publication Date: 2010 Feb 18 PMID: 20167602
Authors: Zhang, Z. - Theler, D. - Kaminska, K. H. - Hiller, M. - Grange, P. D. - Pudimat, R. - Rafalska, I. - Heinrich, B. - Bujnicki, J. M. - Allain, F. H. - Stamm, S.
Journal: J Biol Chem

The YTH (YT521-B homology) domain was identified by sequence comparison and is found in 174 different proteins expressed in eukaryotes. It is characterized by 14 invariant residues within an alpha-helix/beta-sheet structure. Here we show that the YTH domain is a novel RNA binding domain which binds to a short, degenerated, single-stranded RNA sequence motif. The presence of the binding motif in alternative exons is necessary for YT521-B to directly influence splice site selection in vivo. Array analyses demonstrate that YT521-B predominantly regulates vertebrate-specific exons. An NMR titration experiment identified the binding surface for single stranded RNA on the YTH domain. Structural analyses indicate that the YTH domain is related to the pseudouridine synthase and archaeosine transglycosylase (PUA) domain. Our data show that the YTH domain conveys RNA-binding ability to a new class of proteins that are found in all eukaryotic organisms.

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Structural basis of membrane-targeting by the Dock180 family of Rho-family guanine exchange factors (RhoGEFs).

Publication Date: 2010 Feb 18 PMID: 20167601
Authors: Premkumar, L. - Bobkov, A. A. - Patel, M. - Jaroszewski, L. - Bankston, L. A. - Stec, B. - Vuori, K. - Cote, J. F. - Liddington, R. C.
Journal: J Biol Chem

The Dock180 family of atypical Rho-family guanine nucleotide exchange factors (Rho-GEFs) regulate a variety of processes involving cellular or sub-cellular polarization, including cell migration and phagocytosis. Each contains a Dock Homology Region-1 (DHR-1) domain that is required to localize its GEF activity to a specific membrane compartment where levels of phosphatidylinositol-(3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) are upregulated by the local activity of PtdIns 3-kinase. Here we define the structural and energetic bases of phosphoinositide specificity by the DHR-1 domain of Dock1 (a GEF for Rac1), and show that DHR-1 utilizes a C2 domain scaffold and surface loops to create a basic pocket on its upper surface for recognition of the PtdIns(3,4,5)P(3) head-group. The pocket has many of the characteristics of those observed in pleckstrin-homology (PH) domains. We show that point mutations in the pocket that abolish phospholipid binding in vitro ablate the ability of Dock1 to induce cell polarization, and propose a model that brings together recent mechanistic and structural studies to rationalize the central role of DHR-1 in dynamic membrane targeting of Rho-GEF activity.

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Activating transcription factor 3 activates p53 by preventing E6-associated protein from binding to E6.

Publication Date: 2010 Feb 18 PMID: 20167600
Authors: Wang, H. - Mo, P. - Ren, S. - Yan, C.
Journal: J Biol Chem

Genomic integration of human papillomavirus (HPV) DNA accounts for more than 90% of cervical cancers. High-risk genital HPVs encode E6 proteins that can interact with a cellular ubiquitin ligase E6-associated protein (E6AP) and target the tumor suppressor p53 for ubiquitin-mediated proteolysis. Currently, how this critical event is regulated is largely unknown. Here we report that activating transcription factor 3 (ATF3), a broad DNA-damage sensor whose expression is frequently downregulated in cervical cancer, interacted with E6 and prevented p53 from ubiquitination and degradation mediated by the viral protein. Consistent with its role as a potent E6 antagonist, ATF3 expressed enforcedly in HPV-positive SiHa cells activated p53, leading to expression of p53-target genes (e.g., p21 and PUMA), cell cycle arrest and apoptotic cell death. The leucine zipper domain of ATF3 appears indispensable for these effects as an ATF3 mutant lacking this domain failed to interact with E6 and activate p53 in the cervical cancer cells. The prevention of p53 degradation was unlikely caused by binding of ATF3 to the tumor suppressor, but rather was a consequence of disruption of the E6-E6AP interaction by ATF3. These results indicate that ATF3 plays a key role in a mechanism defending against HPV-induced carcinogenesis, and could serve as a novel therapeutic target for HPV-positive cancers.

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Rational design of Interleukin-21 antagonist through selective elimination of {gamma}C binding epitope.

Publication Date: 2010 Feb 18 PMID: 20167599
Authors: Kang, L. - Bondensgaard, K. - Li, T. - Hartmann, R. - Hjorth, S. A.
Journal: J Biol Chem

The cytokine Interleukin (IL)-21 exerts pleiotropic effects acting through innate as well as adaptive immune responses. The activities of IL-21 are mediated through binding to its cognate receptor complex composed of the IL-21 receptor private chain (IL-21Ralpha) and the common gamma-chain (gammaC), the latter being shared by IL-2, IL-4, IL-7, IL-9, and IL-15. The binding energy of the IL-21 ternary complex is predominantly provided by the high affinity interaction between IL-21 and IL-21Ralpha, while the interaction between IL-21 and gammaC, albeit essential for signalling, is rather weak. The design of IL-21 analogues, which have lost most or all affinity towards the signalling gammaC chain, while simultaneously maintaining a tight interaction with the private chain, would in theory represent candidates for IL-21 antagonists. We predicted the IL-21 residues which compose the gammaC binding epitope using homology modelling and alignment with the related cytokines, IL-2 and IL-4. Next we systematically analyzed the predicted binding epitope by a mutagenesis study. Indeed two mutants which have significantly impaired gammaC affinity, while undiminished IL-21Ralpha affinity were successfully identified. Functional studies confirmed that these two novel hIL-21 double-mutants do act as hIL-21 antagonists.

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Novel insights into the cellular mechanisms of the anti-inflammatory effects of NF-[kappa]B essential modulator (NEMO)binding domain peptides.

Publication Date: 2010 Feb 18 PMID: 20167598
Authors: Baima, E. T. - Guzova, J. A. - Mathialagan, S. - Nagiec, E. E. - Hardy, M. M. - Song, L. R. - Bonar, S. L. - Weinberg, R. A. - Selness, S. R. - Woodard, S. S. - Chrencik, J. - Hood, W. F. - Schindler, J. F. - Kishore, N. - Mbalaviele, G.
Journal: J Biol Chem

The classical nuclear factor kappa B (NF-[kappa]B) signaling pathway is under the control of the I[kappa]B kinase (IKK) complex, which consists of IKK-1, IKK-2 and NEMO (NF-[kappa]B essential modulator). This complex is responsible for the regulation of cell proliferation, survival and differentiation. Dysregulation of this pathway is associated with several human diseases, and as such, its inhibition offers an exciting opportunity for therapeutic intervention. NEMO binding domain (NBD) peptides inhibit the binding of recombinant NEMO to IKK-2 in vitro. However, direct evidence of disruption of this binding by NBD peptides in biological systems has not been provided. Using a cell system, we expanded on previous observations to show that NBD peptides inhibit inflammation-induced, but not basal cytokine production. We report that these peptides cause the release of IKK-2 from an IKK complex and disrupt NEMO-IKK-2 interactions in cells. We demonstrate that by interfering with NEMO-IKK-2 interactions, NBD peptides inhibit IKK-2 phosphorylation, without affecting signaling intermediates upstream of the IKK complex of the NF-[kappa]B pathway. Furthermore, in a cell-free system of IKK complex activation by TNF receptor-associated factor 6 (TRAF6), we show that these peptides inhibit the ability of this complex to phosphorylate downstream substrates such as p65 and inhibitor of [kappa]B[alpha] (I[kappa]B[alpha]). Thus, consistent with the notion that NEMO regulates IKK-2 catalytic activity by serving as a scaffold, appropriately positioning IKK-2 for activation by upstream kinase(s), our findings provide novel insights into the molecular mechanisms by which NBD peptides exert their anti-inflammatory effects in cells.

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Nucleosome formation activity of human sNASP.

Publication Date: 2010 Feb 18 PMID: 20167597
Authors: Osakabe, A. - Tachiwana, H. - Matsunaga, T. - Shiga, T. - Nozawa, R. S. - Obuse, C. - Kurumizaka, H.
Journal: J Biol Chem

NASP (nuclear autoantigenic sperm protein) is a member of the N1/N2 family, which is widely conserved among eukaryotes. Human NASP reportedly prefers to bind to histones H3/H4 and the linker histone H1, as compared to H2A/H2B, and is anticipated to function as an H3/H4 chaperone for nucleosome assembly. However, the direct nucleosome-assembly activity of human NASP has not been reported so far. In humans, two spliced isoforms, somatic and testicular NASPs (sNASP and tNASP, respectively), were identified. In the present study, we purified human sNASP, and found that sNASP efficiently promoted the assembly of nucleosomes containing the conventional H3.1, H3.2, H3.3, or centromere-specific CENP-A. On the other hand, sNASP inefficiently promoted nucleosome assembly with H3T, a testis-specific H3 variant. Mutational analyses revealed that the Met71 residue of H3T is responsible for this inefficient nucleosome formation by sNASP. Tetrasomes, composed of the H3/H4 tetramer and DNA without H2A/H2B, were efficiently formed by the sNASP-mediated nucleosome-assembly reaction. A deletion analysis of sNASP revealed that the central region, amino acid residues 26-325, of sNASP is responsible for nucleosome assembly in vitro. These experiments are the first demonstration that human NASP directly promotes nucleosome assembly, and provide compelling evidence that sNASP is a bona fide histone chaperone for H3/H4.

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Magnesium coordination controls the molecular switch function of DNA mismatch repair protein muts.

Publication Date: 2010 Feb 18 PMID: 20167596
Authors: Lebbink, J. H. - Fish, A. - Reumer, A. - Natrajan, G. - Winterwerp, H. H. - Sixma, T. K.
Journal: J Biol Chem

The DNA mismatch repair protein MutS acts as a molecular switch that toggles between ADP and ATP states, which is regulated by mismatched DNA. This is analogous to G-protein switches and regulation of their 'on' and 'off' states by guanine exchange factors (GEFs). While GDP release in monomeric GTPases is accelerated by GEF-induced removal of magnesium from the catalytic site, we find that release of ADP from MutS is not influenced in this manner by the metal ion. Rather, ADP release is induced by binding of mismatched DNA at the opposite end of the protein, a long-range allosteric response resembling the mechanism of activation of heterotrimeric GTPases. Magnesium does influence switching in MutS by inducing faster and tighter ATP binding, allowing rapid downstream responses. MutS mutants with decreased affinity for the metal ion are impaired in fast switching and in vivo mismatch repair. Thus, the G-proteins and MutS conceptually employ the same efficient use of the high-energy cofactor: slow hydrolysis in the absence of a signal and fast conversion to the active state when required.

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The N-terminal region of comparative gene identification-58 (CGI-58) is important for lipid droplet binding and activation of adipose triglyceride lipase.

Publication Date: 2010 Feb 17 PMID: 20164531
Authors: Gruber, A. - Cornaciu, I. - Lass, A. - Schweiger, M. - Poeschl, M. - Eder, C. - Kumari, M. - Schoiswohl, G. - Wolinski, H. - Kohlwein, S. D. - Zechner, R. - Zimmermann, R. - Oberer, M.
Journal: J Biol Chem

In mammals, excess energy is stored in the form of triacylglycerol (TG) primarily in lipid droplets of white adipose tissue. The first step of lipolysis, i.e. the mobilization of fat stores, is catalyzed by adipose triglyceride lipase (ATGL). The enzymatic activity of ATGL is strongly enhanced by CGI-58 (comparative gene identification-58) and the loss of either ATGL or CGI-58 function causes systemic triglyceride accumulation in humans and mice. However, the mechanism by which CGI-58 stimulates ATGL activity is unknown. To gain insight into CGI-58 function using structural features of the protein, we generated a 3D homology model based on sequence similarity with other proteins. Interestingly, the model of CGI-58 revealed that the N-terminus forms an extension of the otherwise compact structure of the protein. This N-terminal region (aa 1-30) harbors a lipophilic tryptophan-rich stretch, which affects the localization of the protein. 1H-NMR experiments revealed strong interaction between the N-terminal peptide and dodecylphosphocholine micelles as a lipid droplet-mimicking system. A role for this N-terminal region of CGI-58 in lipid droplet binding was further strengthened by localization studies in cultured cells. Whereas wild-type CGI-58 localizes to the lipid droplet, the N-terminally truncated fragments of CGI-58 are dispersed in the cytoplasm. Moreover, CGI-58 lacking the N-terminal extension loses the ability to stimulate ATGL, implying that CGI-58;s ability to activate ATGL is linked to correct localization. In summary, our study shows that the N-terminal, Trp-rich region of CGI-58 is essential for correct localization and ATGL-activating function of CGI-58.

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Structural determination and tryptophan fluorescence of heterokaryon incompatibility C2 protein (HET-C2), a fungal glycolipid transfer protein (GLTP), provide novel insights into glycolipid specificity and membrane interaction by the GLTP-fold.

Publication Date: 2010 Feb 17 PMID: 20164530
Authors: Kenoth, R. - Simanshu, D. K. - Kamlekar, R. K. - Pike, H. M. - Molotkovsky, J. G. - Benson, L. M. - Bergen, H. R. 3rd - Prendergast, F. G. - Malinina, L. - Venyaminov, S. Y. - Patel, D. J. - Brown, R. E.
Journal: J Biol Chem

HET-C2 is a fungal protein that transfers glycosphingolipids between membranes and has limited sequence homology with human glycolipid transfer protein (GLTP). The human GLTP-fold is unique among lipid binding/transfer proteins, defining the GLTP superfamily. Herein, GLTP-fold formation by HET-C2, its glycolipid transfer specificity, and the functional role(s) of its two Trp residues have been investigated. X ray diffraction (1.9A) revealed a GLTP-fold with all key sugar headgroup recognition residues (D66, N70, K73, W109, H147) conserved and properly oriented for glycolipid binding. Far UV circular dichroism (CD) showed secondary structure dominated by alpha-helices and a cooperative thermal unfolding transition of 49 degrees C, features consistent with a GLTP-fold. Environmentally-induced optical activity of Trp/Tyr/Phe (2/4/12) detected by near-UV-CD was unaffected by membranes containing glycolipid but was slightly altered by membranes lacking glycolipid. Trp fluorescence was maximal at ~355 nm and accessible to aqueous quenchers, indicating free exposure to the aqueous milieu and consistent with surface localization of the two Trps. Interaction with membranes lacking glycolipid triggered significant decreases in Trp emission intensity but lesser than decreases induced by membranes containing glycolipid. Binding of glycolipid (confirmed by electrospray injection mass spectrometry) resulted in a blue-shifted emission wavelength maximum (~6 nm) permitting determination of binding affinities. The unique positioning of W208 at the HET-C2 C-terminus revealed membrane-induced conformational changes that precede glycolipid uptake, while key differences in residues of the sugar headgroup recognition center accounted for altered glycolipid specificity and suggested evolutionary adaptation for the simpler glycosphingolipid compositions of filamentous fungi.

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Phosphorylation of cardiac troponin I at protein kinase C site threonine-144 depresses cooperative activation of thin filaments.

Publication Date: 2010 Feb 17 PMID: 20164197
Authors: Lu, Q. W. - Hinken, A. C. - Patrick, S. E. - Solaro, R. J. - Kobayashi, T.
Journal: J Biol Chem

There is evidence for PKC-dependent multi-site phosphorylation of cardiac troponin I (cTnI) at Ser-23 and Ser-24 (also PKA sites) in the cardiac specific N-terminal extension and at Thr-144, a unique residue in the inhibitory region. The functional effect of these phosphorylations in combination is of interest in view of data indicating intra-molecular interaction between the N-terminal extension and the inhibitory region of cTnI. To determine the role of PKC-dependent phosphorylation of cTnI on sarcomeric function, we measured contractile regulation at multiple levels of complexity. Ca2+-binding to thin filaments reconstituted with either cTnI(wild-type) or pseudo-phosphorylated cTnI(S23D/S24D), cTnI(T144E), and cTnI(S23D/S24D/T144E) was determined. Compared with controls regulated by cTnI(wild-type), thin filaments with cTnI(S23D/S24D) and cTnI(S23D/S24D/T144E) exhibited decreased Ca2+ sensitivity. In contrast, there was no significant difference between Ca2+ binding to thin filaments with cTnI(wild-type) and with cTnI(T144E). Studies of the pCa-force relations in skinned papillary fibers regulated by these forms of cTnI yielded similar results. However, in both the Ca2+-binding measurements and the skinned fiber tension measurements, the presence of cTnI(S23D/S24D/T144E) induced a much lower Hill coefficient than either wild-type, S23D/S24D, or T144E. These data highlight the importance of thin filament based cooperative mechanisms in cardiac regulation, with implications for mechanisms of control of function in normal and pathological hearts.

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Dynamic regulation of CD45 lateral mobility by the spectrin-ankyrin cytoskeleton of T cells.

Publication Date: 2010 Feb 17 PMID: 20164196
Authors: Cairo, C. W. - Das, R. - Albohy, A. - Baca, Q. J. - Pradhan, D. - Morrow, J. S. - Coombs, D. - Golan, D. E.
Journal: J Biol Chem

The leukocyte common antigen, CD45, is a critical immune regulator whose activity is modulated by cytoskeletal interactions. Components of the spectrin-ankyrin cytoskeleton have been implicated in the trafficking and signaling of CD45. We have examined the lateral mobility of CD45 in resting and activated T lymphocytes using single particle tracking (SPT) and find that the receptor has decreased mobility due to increased cytoskeletal contacts in activated cells. Experiments with cells that have disrupted betaI spectrin interactions show decreased cytoskeletal contacts in resting cells and attenuation of receptor immobilization in activated cells. Applying two types of population analyses to SPT trajectories, we find good agreement between the diffusion coefficients obtained using either a mean-squared displacement (MSD) analysis or a hidden Markov model (HMM) analysis. HMM analysis also reveals the rate of association and dissociation of CD45-cytoskeleton contacts, demonstrating the importance of this analysis for measuring cytoskeleton binding events in live cells. Our findings are consistent with a model in which multiple cytoskeletal contacts, including those with spectrin and ankyrin, participate in the regulation of CD45 lateral mobility. These interactions are a major factor in CD45 immobilization in activated cells. Furthermore, cellular activation leads to CD45 immobilization by reduction of the CD45-cytoskeleton dissociation rate. Short peptides that mimic spectrin repeat domains alter the association rate of CD45 to the cytoskeleton, and cause an apparent decrease in dissociation rates. We propose a model for CD45-cytoskeleton interactions, and conclude that the spectrin-ankyrin-actin network is an essential determinant of immunoreceptor mobility.

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The phospholipid-binding protein SESTD1 is a novel regulator of the transient receptor potential channels TRPC4 and TRPC5.

Publication Date: 2010 Feb 17 PMID: 20164195
Authors: Miehe, S. - Bieberstein, A. - Arnould, I. - Ihdene, O. - Ruetten, H. - Strubing, C.
Journal: J Biol Chem

TRPC4 and TRPC5 are two closely related members of the mammalian transient receptor potential (TRP) cation channel family that have been implicated in important physiological functions such as growth cone guidance and smooth muscle contraction. To further unravel the role of TRPC4 and TRPC5 in these processes in vivo, detailed information about the molecular composition of native channel complexes and their association with cellular signaling networks is needed. We, therefore, searched a human aortic cDNA library for novel TRPC4 interacting proteins using a modified yeast two-hybrid assay. This screen identified SESTD1, a previously uncharacterized protein containing a lipid-binding SEC14-like domain as well as spectrin-type cytoskeleton-interaction domains. SESTD1 was found to associate with TRPC4 and TRPC5 via the channel's calmodulin- and inositol 1,4,5-trisphosphate receptor-binding domain. In functional studies we demonstrate that SESTD1 binds several phospholipid species in vitro and is essential for efficient receptor-mediated activation of TRPC5. Notably, phospholipid binding to SESTD1 was Ca(2+)-dependent. As TRPC4 and -5 conduct Ca(2+) SESTD1 - channel signaling may be bi-directional and also couple TRPC activity to lipid signaling through SESTD1. The modulation of TRPC channel function by specific lipid-binding proteins such as SESTD1 adds another facet to the complex regulation of these channels complementary to the previously described effects of direct channel-phospholipid interaction.

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Crystal structure of human REV7 in complex with a human REV3 fragment and structural implication of the interaction between DNA polymerase {zeta} and REV1.

Publication Date: 2010 Feb 17 PMID: 20164194
Authors: Hara, K. - Hashimoto, H. - Murakumo, Y. - Kobayashi, S. - Kogame, T. - Unzai, S. - Akashi, S. - Takeda, S. - Shimizu, T. - Sato, M.
Journal: J Biol Chem

DNA polymerase zeta (Polzeta) is an error-prone DNA polymerase involved in translesion DNA synthesis (TLS). Polzeta consists of two subunits: the catalytic REV3, which belongs to B-family DNA polymerase, and the non-catalytic REV7. REV7 also interacts with REV1 polymerase, which is an error-prone Y-family DNA polymerase and also involved in TLS. Cells deficient in one of the three REV proteins and those deficient in all three proteins show similar phenotype, indicating the functional collaboration of the three REV proteins. REV7 interacts with both REV3 and REV1 polymerases, but the structure of REV7 or REV3, as well as the structural and functional basis of the REV1-REV7 and REV3-REV7 interactions remains unknown. Here we show the first crystal structure of human REV7 in complex with a fragment of human REV3 polymerase (residues 1847-1898) and reveal the mechanism underlying REV7-REV3 interaction. The structure indicates that the interaction between REV7 and REV3 creates a structural interface for REV1-binding. Furthermore, we show that the REV7-mediated interactions are responsible for DNA-damage tolerance. Our results highlight the function of REV7 as an adapter protein to recruit Polzeta to a lesion site. REV7 is alternatively called MAD2B or MAD2L2 and also involved in various cellular functions such as signal transduction and cell-cycle regulation. Our results will provide a general structural basis for understanding the REV7-interaction.

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Hantavirus nucleocapsid protein has distinct m7G cap and RNA binding sites.

Publication Date: 2010 Feb 17 PMID: 20164193
Authors: Mir, M. A. - Sheema, S. - Haseeb, A. - Haque, A.
Journal: J Biol Chem

Hantaviruses, members of the Bunyaviridae family, are emerging category A pathogens that carry three negative stranded RNA molecules as their genome. Hantavirus nucleocapsid protein (N) is encoded by the smallest S segment genomic RNA (vRNA). N specifically binds mRNA caps and requires four nucleotides adjacent to the cap for high affinity binding. We shown that N peptide has distinct cap and RNA binding sites that independently interact with mRNA cap and viral genomic RNA respectively. In addition, N can simultaneously bind with both mRNA cap and vRNA. N undergoes distinct conformational changes after binding with either mRNA cap or vRNA or both mRNA cap and vRNA simultaneously. Hantavirus RNA dependent RNA polymerase (RdRp) uses a capped RNA primer for transcription initiation. The capped RNA primer is generated from host cell mRNA by the cap snatching mechanism and is supposed to anneal with the 3 terminus of vRNA template by single G-C base pairing. We show that capped RNA primer binds at the cap binding site and induces a conformational change in N. The conformationaly altered N with a capped primer loaded at the cap binding site, specifically binds the conserved 3 nine nucleotides of vRNA and assists the bound primer to anneal at the 3 terminus. We suggest that cap binding site of N in conjunction with RdRp plays a key role during transcription and replication initiation of vRNA genome.

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Cross-phosphorylation between Arabidopsis thaliana sucrose non-fermenting 1-related protein kinase 1 (AtSnRK1) and its activating kinase (AtSnAK) determines their catalytic activities.

Publication Date: 2010 Feb 17 PMID: 20164192
Authors: Crozet, P. - Jammes, F. - Valot, B. - Ambard-Bretteville, F. - Nessler, S. - Hodges, M. - Vidal, J. - Thomas, M.
Journal: J Biol Chem

Arabidopsis thaliana Sucrose Non-Fermenting 1-Related protein Kinase 1 complexes belong to the SNF1/AMPK/SnRK1 protein kinase family that share an ancestral function as central regulators of metabolism. In Arabidopsis thaliana, the products of AtSnAK1 and AtSnAK2, orthologous to yeast genes, have been shown to auto-phosphorylate and to phosphorylate/activate the AtSnRK1.1 catalytic subunit on Thr175. The phosphorylation of these kinases has been investigated by site-directed mutagenesis and tandem mass spectroscopy. The auto-phosphorylation site of AtSnAK2 was identified as Thr154 and it was shown to be required for AtSnAK catalytic activity. Interestingly, activated AtSnRK1 exerted a negative feedback phosphorylation on AtSnAK2 at Ser261 (Ser260 of AtSnAK1) that was dependent on AtSnAK auto-phosphorylation. The dynamics of these reciprocal phosphorylation events on the different kinases was established and structural modeling allowed clarification of the topography of the AtSnAK phosphorylation sites. A mechanism is proposed to explain the observed changes in the enzymatic properties of each kinase triggered by these phosphorylation events.

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A multifunctional mannosyltransferase family in candida albicans determines cell wall mannan structure and host-fungus interactions.

Publication Date: 2010 Feb 17 PMID: 20164191
Authors: Mora-Montes, H. M. - Bates, S. - Netea, M. G. - Castillo, L. - Brand, A. - Buurman, E. T. - Diaz-Jimenez, D. F. - Kullberg, B. J. - Brown, A. J. - Odds, F. C. - Gow, N. A.
Journal: J Biol Chem

The cell wall proteins of fungi are modified by N-linked, O-linked and phospho-mannosylation, resulting in modification of the physical and immunological properties of the cell. Glycosylation of cell wall proteins involves the activities of families of ER and Golgi-located glycosyl transferases whose activities are difficult to infer through bioinformatics. The Candida albicans MNT1/KRE2 mannosyl transferases family is represented by five members. We showed previously that Mnt1 and Mnt2 are involved in O-linked mannosylation and are required for virulence. Here, the role of C. albicans MNT3, MNT4, and MNT5 was determined by generating single and multiple MNT gene null mutants and by functional complementation experiments in Saccharomyces cerevisiae. Mnt3, Mnt4 and Mnt5 did not participate in O-linked mannosylation, but CaMnt3 and CaMnt5 had redundant activities in phosphomannosylation, and were responsible for attachment of approximately half of the phosphomannan attached to N-linked mannans. CaMnt4 and CaMnt5 participated in N-mannan branching. Deletion of CaMNT3, CaMNT4 and CaMNT5 affected growth rate and virulence of C. albicans, the recognition of the yeast by human monocytes and cytokine stimulation, and led to increased cell wall chitin content and exposure of beta-glucan at the cell wall surface. Therefore the MNT1/KRE2 gene family participates in three types of protein mannosylation in C. albicans, and these modifications play vital roles in fungal cell wall structure and cell surface recognition by the innate immune system.

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The acyclic 2,4-diaminopyrimidine nucleoside phosphonate acts as a purine mimetic in HIV-1 reverse transcriptase DNA polymerization.

Publication Date: 2010 Feb 17 PMID: 20164190
Authors: Herman, B. D. - Votruba, I. - Holy, A. - Sluis-Cremer, N. - Balzarini, J.
Journal: J Biol Chem

The acyclic pyrimidine nucleoside phosphonate (ANP) phosphonylmethoxyethoxy-diamino-pyrimidine (PMEO-DAPym) differs from other ANPs in that the aliphatic alkyloxy linker is bound to the C-6 of the 2,4-diaminopyrimidine base through an ether bond, instead of the traditional alkyl linkage to the N-1 or N-9 of the pyrimidine or purine base. In this study, we have analyzed the molecular interactions between PMEO-DAPym- diphosphate (PMEO-DAPym-pp) and the active sites of wild-type (WT) and drug-resistant HIV-1 reverse transcriptase (RT). Pre-steady-state kinetic analyses revealed that PMEO-DAPym-pp is a good substrate for WT HIV-1 RT: its catalytic efficiency of incorporation (kpol/Kd) is only 2- to 3-fold less than that of the corresponding prototype purine nucleotide analogues PMEA-pp or (R)PMPA-pp. HIV-1 RT recognizes PMEO-DAPym-pp as a purine base instead of a pyrimidine base and incorporates it opposite to thymine (in DNA) or uracil (in RNA). Molecular modeling demonstrates that PMEO-DAPym-pp fits into the active site of HIV-1 RT without significant perturbation of key amino acid residues and mimics an open incomplete purine ring that allows the canonical Watson-Crick base pairing to be maintained. PMEO-DAPym-pp is incorporated more efficiently than (R)PMPA-pp by mutant K65R HIV-1 RT and is not as efficiently excised as (R)PMPA by HIV-1 RT containing thymidine analog mutations (TAM). Overall, the data revealed that PMEO-DAPym represents the prototype compound of a novel class of pyrimidine acyclic nucleoside phosphonates that are recognized as a purine nucleotide and should form the rational basis for the design and development of novel purine nucleo(s)(t)ide mimetics as potential antiviral or antimetabolic agents.

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Perturbations in mitochondrial dynamics induced by human mutant PINK1 can be rescued by the mitochondrial division inhibitor mdivi-1.

Publication Date: 2010 Feb 17 PMID: 20164189
Authors: Cui, M. - Tang, X. - Christian, W. V. - Yoon, Y. - Tieu, K.
Journal: J Biol Chem

Mutations in the mitochondrial encoded protein PTEN induced putative kinase 1 (PINK1) cause autosomal recessive Parkinsons disease (PD). In mammalian cells, mutant PINK1 has been reported to promote fission or inhibit fusion in mitochondria; however, the mechanism by which this process occurs remains elusive. Using an ecdysone inducible expression system in mammalian dopaminergic neuronal cells, we report here that human mutant PINK1 (L347P and W437X) mediates an overall fission effect by increasing the ratio of mitochondrial fission over fusion proteins, leading to excessive dysfunctional fragmented mitochondria. Knocking down endogenous Pink1 produces similar effects. In contrast, overexpressing human wild type PINK1 produces a profusion effect by increasing the ratio of mitochondrial fusion / fission proteins, without resulting in functionally compromised mitochondria. Parkin knockdown blocks the imbalance in fission / fusion proteins. Furthermore, overexpressing parkin and ubiquitin increases degradation of the mitochondrial fission hFis1 protein, suggesting PINK1 and parkin maintain proper mitochondrial function and integrity via the fission / fusion machinery. Through genetic manipulations and treatment with the small molecule Mitochondrial Division Inhibitor (mdivi1), which inhibits DLP1/Drp1, both structural and functional mitochondrial defects induced by mutant PINK1 were attenuated, highlighting a potential novel therapeutic avenue for PD.

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The carboxyl terminal domain of connexin43 is a negative modulator of neuronal differentiation.

Publication Date: 2010 Feb 17 PMID: 20164188
Authors: Santiago, M. F. - Alcami, P. - Striedinger, K. M. - Spray, D. C. - Scemes, E.
Journal: J Biol Chem

Connexin43 (Cx43) is widely expressed in embryonic brain and its expression becomes restricted mainly to astrocytes in the mature brain. Recent studies have indicated that Cx43 plays important, non-channel, roles during CNS development by affecting neuronal cell migration. Here we evaluated the effects of Cx43 on neuronal differentiation. For that we used an in vitro model of neural cell development (neurospheres) to evaluate, through immunocytochemistry, electrophysiology and molecular biology, the degree of neuronal maturation from neurospheres derived from wild-type (WT) and Cx43-null mice. Our results indicate that Cx43 is a negative modulator of neuronal differentiation. The percent neurospheres containing differentiated neurons and the number of cells displaying inward currents was significantly higher in Cx43-null than in WT littermate neurospheres. Knockdown of Cx43 with siRNA increased the number of WT neurospheres generating differentiated neurons. Blockade of gap junctional communication with carbenoxolone did not induce neuronal differentiation in WT neurospheres. Transfection of Cx43-null neurospheres with Cx43 mutants revealed that Cx43 carboxyl terminus (CT) prevents neuronal maturation. In agreement with these in vitro data, in situ analysis of E16 brains revealed increased beta-III-tubulin expression in germinal zones of Cx43-null compared to that of WT littermates. These results indicate that Cx43, and specifically its carboxyl terminus is crucial for signaling mechanisms preventing premature neuronal differentiation during embryonic brain development.

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MicroRNA-27b contributes to lipopolysaccharide-mediated peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) mRNA destabilization.

Publication Date: 2010 Feb 17 PMID: 20164187
Authors: Jennewein, C. - von Knethen, A. - Schmid, T. - Bruene, B.
Journal: J Biol Chem

Peroxisome proliferator-activated receptor gamma (PPARgamma) gained considerable interest as a therapeutic target during chronic inflammatory diseases. Remarkably, the pathogenesis of diseases such as multiple sclerosis or Alzheimer is associated with impaired PPARgamma expression. Considering that regulation of PPARgamma expression during inflammation is largely unknown, we were interested in elucidating underlying mechanisms. To this end, we initiated an inflammatory response by exposing primary human macrophages to lipopolysaccharide (LPS) and observed a rapid decline of PPARgamma1 expression. Since promoter activities were not affected by LPS, we focused on mRNA stability and noticed a decreased mRNA half-life. As RNA stability is often regulated via 3'untranslated regions (UTRs), we analyzed the impact of the PPARgamma-3'UTR by reporter assays using specific constructs. LPS significantly reduced luciferase activity of the pGL3-PPARgamma-3'UTR, suggesting that PPARgamma1 mRNA is destabilized. Deletion or mutation of a potential microRNA-27a/b (miR-27a/b) binding site within the 3'UTR restored luciferase activity. Moreover, inhibition of miR-27b, which was induced upon LPS-exposure, partially reversed PPARgamma1 mRNA decay, while miR-27b overexpression decreased PPARgamma1 mRNA content. In addition, LPS further reduced this decay. The functional relevance of miR-27b-dependent PPARgamma1 decrease was proven by inhibition or overexpression of miR-27b, which affected LPS-induced expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNFalpha) and interleukin (IL)-6. We provide evidence that LPS-induced miR-27b contributes to destabilization of PPARgamma1 mRNA. Understanding molecular mechanisms decreasing PPARgamma might help to better appreciate inflammatory diseases.

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A novel glucosyltransferase is required for glycosylation of a serine-rich adhesin and biofilm formation by Streptococcus parasanguinis.

Publication Date: 2010 Feb 17 PMID: 20164186
Authors: Zhou, M. - Zhu, F. - Dong, S. - Pritchard, D. - Wu, H.
Journal: J Biol Chem

Fap1-like serine-rich glycoproteins are conserved in streptococci, staphylococci and lactobacilli, and required for bacterial biofilm formation and pathogenesis. Glycosylation of Fap1 is mediated by a gene cluster flanking the fap1 locus. The key enzymes responsible for the first step of Fap1 glycosylation have been identified and correspond to glycosyltransferases Gtf1 and Gtf2. They form a functional enzyme complex that catalyzes the transfer of N-acetylglucosamine(GlcNAc) residues to the Fap1 polypeptide. However, until now nothing was known about the subsequent step in Fap1 glycosylation. Here, we show that the second step in Fap1 glycosylation is catalyzed by nucleotide-sugar synthetase-like (Nss) protein. The nss gene located upstream of fap1 is also highly conserved in streptococci and lactobacilli. Nss deficient mutants failed to catalyze the second step of Fap1 glycosylation in vivo in Streptococcus parasanguinis and in a recombinant Fap1 glycosylation system. Nss catalyzed the direct transfer of glucosyl-residues to GlcNAc-modified Fap1 substrate in vitro, demonstrating that Nss is a glucosyltransferase. Thus we renamed Nss as glucosyltranferase-Gtf3. A gtf3 mutant exhibited biofilm defect. Taken together, we conclude that this new glucosyltransferase mediates the second step of Fap1 glycosylation and is required for biofilm formation.

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Interactions among HAMP domain repeats act as an osmosensing molecular switch in group III hybrid histidine kinases from fungi.

Publication Date: 2010 Feb 17 PMID: 20164185
Authors: Meena, N. - Kaur, H. - Mondal, A. K.
Journal: J Biol Chem

The members of group III hybrid histidine kinases (HHK) are ubiquitous in fungi. They have been implicated to function as osmosensors in the HOG pathway which is essential for fungal survival under high osmolarity stress. Recent literature suggests that they are also involved in conidia formation, virulence in several filamentous fungi and an excellent molecular target for antifungal agents. Thus, they constitute a very important group of sensor kinases. Structurally, they are distinct from Sln1p, the osmosensing HHK that regulate HOG pathway in S. cerevisiae. They lack any transmembrane domain and typically contain HAMP domain repeats at the N-terminus. Till now, it is not clear how they function as an osmosensor to regulate the HOG pathway. To investigate this, we undertook molecular characterization of DhNIK1, an ortholog from osmotolerant yeast Debaryomyces hansenii. We show here that DhNIK1 could complement sln1 mutation in Saccharomyces cerevisiae thereby confirming its role as a bona fide osmosensor. We further investigated the role of HAMP domains by deleting them systematically. Our results clearly indicate that HAMP4 domain is crucial for osmosensing by DhNik1p. Most importantly, we also show that the alternative interaction among the HAMP domains regulates the activity of DhNik1p like an "On-Off switch" and thus provides, for the first time, an insight into the molecular mechanism of osmosensing by this group of HHKs.

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HBx-induced expression of the CXC chemokine IP-10 is mediated through activation of NF-kappaB and increases migration of leukocytes.

Publication Date: 2010 Feb 17 PMID: 20164184
Authors: Zhou, Y. - Wang, S. - Ma, J. W. - Lei, Z. - Zhu, H. F. - Lei, P. - Yang, Z. S. - Zhang, B. - Yao, X. X. - Shi, C. - Sun, L. F. - Wu, X. W. - Ning, Q. - Shen, G. X. - Huang, B.
Journal: J Biol Chem

IFN-gamma inducible protein 10 (IP-10) involves the inflammatory cell recruitment and cellular immune damage during virus infection. Although the increase of peripheral IP-10 level is known in HBV-infected patients, the molecular basis of HBV infection inducing IP-10 expression has remained elusive. In the present study, we demonstrate that hepatitis B virus protein X (HBx) increases IP-10 expression in a dose-dependent manner. The transfection of HBx-expressing vector into HepG2 cells results in the nuclear translocation of NF-kappa B, which directly binds the promoter of IP-10 at the position from -122 to -113, thus facilitating the transcription. The addition of NF-kappa B inhibitor, blocks the effect of HBx on IP-10 induction. In parallel, the increase of NF-kappa B subunits p65 and p50 in HepG2 cells also augments IP-10 expression. Furthermore, we show that HBx induces the activation of NF-kappa B through TRAF2/TAK1 signaling pathway, leading to the upregulation of IP-10 expression. As a consequence, the upregulation of IP-10 may mediate the migration of peripheral blood leukocytes (PBLs) in a NF-kappa B-dependent manner. In conclusion, we report a novel molecular mechanism of HBV infection inducing IP-10 expression, which involves viral protein HBx affecting NF-kappa B pathway, leading to the transactivation of IP-10 promoter. Our study provides insight into the migration of leucocytes in response to HBV infection, thus causing immune pathological injury of liver.

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A novel par1-interactor, bicaudal-D1, regulates G protein signaling and internalization.

Publication Date: 2010 Feb 17 PMID: 20164183
Authors: Swift, S. - Xu, J. - Trivedi, V. - Austin, K. M. - Tressel, S. L. - Zhang, L. - Covic, L. - Kuliopulos, A.
Journal: J Biol Chem

Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor which plays critical roles in cancer, angiogenesis, inflammation and thrombosis. Proteolytic cleavage of the extracellular domain of PAR1 generates a tethered ligand that activates PAR1 in an unusual intramolecular mode. The signal emanating from the irreversibly cleaved PAR1 is terminated by G protein uncoupling and internalization; however, the mechanisms of PAR1 signal shut-off still remain unclear. Using a yeast two-hybrid screen, we identified Bicaudal D1 (BicD1) as a direct interactor with the C-terminal cytoplasmic domain of PAR1. BICD was originally identified as an essential developmental gene associated with mRNA and golgi-ER transport. We discovered a novel function of BicD1 in the modulation of G protein signaling, cell proliferation, and endocytosis downstream of PAR1. BicD1 and its carboxy-terminal CC3 domain inhibited PAR1 signaling to Gq-phospholipase C-beta through coiled-coil interactions with the cytoplasmic 8th helix of PAR1. Unexpectedly, BicD1 was also found to be a potent suppressor of PAR1-driven proliferation of breast carcinoma cells. The growth suppressing effects of BicD1 required the ability to interact with the 8th helix of PAR1. Silencing of BicD1 expression impaired endocytosis of PAR1, and BicD1 co-localized with PAR1 and tubulin implicating BicD1 as an important adapter protein involved in the transport of PAR1 from the plasma membrane to endosomal vesicles. Together, these findings provide a link between PAR1 signal termination and internalization through the non-G protein effector, BicD1.

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Rga4 modulates the activity of the fission yeast cell integrity MAPK pathway by acting as a Rho2 GAP.

Publication Date: 2010 Feb 17 PMID: 20164182
Authors: Soto, T. - Villar-Tajadura, M. A. - Madrid, M. - Vicente, J. - Gacto, M. - Perez, P. - Cansado, J.
Journal: J Biol Chem

Rho GTPase-activating proteins (GAPs) are responsible for the inactivation of Rho GTPases, which are involved in the regulation of critical biological responses in eukaryotic cells, ranging from cell cycle control to cellular morphogenesis. The genome of fission yeast Schizosaccharomyces pombe contains six genes coding for putative Rho GTPases, whereas nine genes code for predicted Rho GAPs (Rga1 to Rga9). One of them, Rga4, has been recently described as a Cdc42 GAP, involved in the control of cell diameter and symmetry in fission yeast. In this work we show that Rga4 is also a Rho2 GAP that negatively modulates the activity of the cell integrity pathway and its main effector, MAPK Pmk1. The DYRK-type protein kinase Pom1, which regulates both the localization and phosphorylation state of Rga4, is also a negative regulator of the Pmk1 pathway, but this control is not dependent upon the Rga4 role as a Rho2-GAP. Hence, two subsets of Rga4 negatively regulate Cdc42 and Rho2 functions in an specific and unrelated way. Finally, we show that Rga7, another Rho2 GAP, downregulates the Pmk1 pathway in addition to Rga4. These results reinforce the notion of the existence of complex mechanisms determining the selectivity of Rho GAPs toward Rho GTPases and their functions.

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Critical role of the platelet-derived growth factor receptor (PDGFR)-{beta} transmembrane domain in the TEL-PDGFR{beta} cytosolic oncoprotein.

Publication Date: 2010 Feb 17 PMID: 20164181
Authors: Toffalini, F. - Hellberg, C. - Demoulin, J. B.
Journal: J Biol Chem

TEL-PDGFRbeta (TPbeta) is found in a subset of patients with atypical myeloid neoplasms associated with eosinophilia and is the archetype of a larger group of hybrid receptors that are produced by rearrangements of platelet-derived growth factor receptor (PDGFR) genes. TPbeta is activated by oligomerization mediated by the pointed domain of TEL/ETV6, leading to constitutive activation of PDGFRbeta kinase domain. The receptor transmembrane (TM) domain is retained in TPbeta and in most of the described PDGFRbeta hybrids. Deletion of the TM domain (DeltaTM-TPbeta) strongly impaired the ability of TPbeta to sustain growth factor-independent cell proliferation. We confirmed that TPbeta resides in the cytosol, indicating that the PDGFRbeta TM domain does not act as a transmembrane domain in the context of the hybrid receptor but has a completely different function. The DeltaTM-TPbeta protein was expressed at a lower level due to increased degradation. It could form oligomers, was phosphorylated at a slightly higher level, co-immunoprecipitated with the p85 adaptor protein, but showed a much reduced capacity to activate STAT5 and ERK1/2 in Ba/F3 cells, compared to TPbeta. In an in vitro kinase assay, DeltaTM-TPbeta was more active than TPbeta and less sensitive to imatinib, a PDGFR inhibitor. In conclusion, we show that the TM domain is required for TPbeta-mediated signaling and proliferation, suggesting that the activation of the PDGFRbeta kinase domain is not enough for cell transformation.

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A novel LZAP-Binding Protein, NLBP, inhibits cell invasion.

Publication Date: 2010 Feb 17 PMID: 20164180
Authors: Kwon, J. - Cho, H. J. - Han, S. H. - No, J. G. - Kwon, J. Y. - Kim, H.
Journal: J Biol Chem

LZAP was recently shown to function as a tumor suppressor through inhibition of the NF-kB signaling pathway. LZAP is also known as a negative regulator of cell invasion, and its expression was demonstrated to be reduced in several tumor tissues. However, the molecular mechanism of the negative effect of LZAP on cell invasion is unclear. In this study, we identify NLBP as a novel LZAP-binding protein using tandem affinity purification. We demonstrate the negative effects of NLBP on cell invasion and the NF-kB signaling pathway. NLBP expression was not detected in hepatocellular carcinoma cells with strong invasive actitivity, while its expression was detected in a hepatocellular carcinoma cell line with no invasive activity. We also demonstrate that these two proteins mutually affect the stability of each other by inhibiting ubiquitination of the other protein. Based on these results, we suggest that NLBP may act as a novel tumor suppressor by inhibiting cell invasion, blocking NF-kB signaling and by increasing stability of the LZAP protein.

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Reaction mechanism and molecular basis for selenium/sulfur discrimination of selenocysteine lyase.

Publication Date: 2010 Feb 17 PMID: 20164179
Authors: Omi, R. - Kurokawa, S. - Mihara, H. - Hayashi, H. - Goto, M. - Miyahara, I. - Kurihara, T. - Hirotsu, K. - Esaki, N.
Journal: J Biol Chem

Selenocysteine lyase (SCL) catalyzes the pyridoxal 5(')-phosphate-dependent removal of selenium from L-selenocysteine to yield L-alanine. The enzyme is proposed to function in the recycling of the micronutrient selenium from degraded selenoproteins containing selenocysteine residue as an essential component. The enzyme exhibits strict substrate specificity toward L-selenocysteine and no activity to its cognate L-cysteine. However, it remains unclear how the enzyme distinguishes between selenocysteine and cysteine. Here, we present mechanistic studies of selenocysteine lyase from rat. ESI-MS analysis of wild-type and C375A mutant SCL revealed that the catalytic reaction proceeds via the formation of an enzyme-bound selenopersulfide intermediate on the catalytically essential Cys375 residue. UV-Vis spectrum analysis and the crystal structure of SCL complexed with L-cysteine demonstrated that the enzyme reversibly forms a nonproductive adduct with L-cysteine. Cys375 on the flexible loop directed L-selenocysteine, but not L-cysteine, to the correct position and orientation in the active site to initiate the catalytic reaction. These findings provide, for the first time, the basis for understanding how trace amounts of a selenium-containing substrate is distinguished from excessive amounts of its cognate sulfur-containing compound in a biological system.

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A two-step process controls the formation of the bienzyme cysteine synthase complex.

Publication Date: 2010 Feb 17 PMID: 20164178
Authors: Salsi, E. - Campanini, B. - Bettati, S. - Raboni, S. - Roderick, S. L. - Cook, P. F. - Mozzarelli, A.
Journal: J Biol Chem

The regulation of enzyme activity through the transient formation of multi-protein assemblies plays an important role in the control of biosynthetic pathways. One of the first regulatory complexes to be discovered was cysteine synthase (CS), formed by the pyridoxal 5'-phosphate-dependent enzyme O-acetylserine sulfhydrylase (OASS) and serine acetyltransferase (SAT). These enzymes are at the branch point of the sulfur, carbon and nitrogen assimilation pathways. Understanding the mechanism of complex formation helps to clarify the role played by CS in the regulation of sulfur assimilation in bacteria and plants. To this goal, stopped-flow fluorescence spectroscopy was used to characterize the interaction of SAT with OASS, at different temperatures and pHs, and in the presence of the physiological regulators cysteine and bisulfide. Results shed light on the mechanism of complex formation and regulation, so far poorly understood. Cysteine synthase assembly occurs via a two-step mechanism involving rapid formation of an encounter complex between the two enzymes, followed by a slow conformational change. The conformational change likely results from the closure of the active site of OASS upon binding of the SAT C-terminal peptide. Bisulfide, the second substrate and a feed-back inhibitor of OASS, stabilizes the CS complex mainly by decreasing the back rate of the isomerization step. Cysteine, the product of the OASS reaction and a SAT inhibitor, slightly affects the kinetics of CS formation leading to destabilization of the complex.

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Nerve growth factor inhibits metalloproteinase-disintegrins and blocks ectodomain shedding of platelet glycoprotein VI.

Publication Date: 2010 Feb 17 PMID: 20164177
Authors: Wijeyewickrema, L. C. - Gardiner, E. E. - Gladigau, E. L. - Berndt, M. C. - Andrews, R. K.
Journal: J Biol Chem

Nerve growth factor (NGF) plays an important role in regulating mammalian neuronal/embryonic development, angiogenesis, and other physiological processes, and has recently been investigated as a potential treatment for the neurodegenerative disorder, Alzheimers disease. In this study, we provide evidence that human NGF may also function as a metalloproteinase inhibitor, based on studies of NGF from snake venom. Originally, our aim was to isolate snake venom metalloproteinases targeting platelet receptors and/or ligands relevant to hemostasis and thrombosis, using Ni(2+)-agarose as a purification step based on the conserved metal ion-coordination motif in venom metalloproteinases. However, subsequent analysis of cobra (Naja kaouthia) venom led to the unexpected discovery that cobra venom NGF bound to Ni(2+)-agarose, eluting at approximately 15 mM imidazole, enabling a one-step purification. The identity of the purified protein was confirmed by mass spectrometry and N-terminal sequence analysis. Partial co-purification of NGF within metalloproteinase-enriched venom fractions led us to test whether NGF affected metalloproteinase activity. Venom NGF potently inhibited metalloproteinases isolated from the same or different venom, and specifically bound to purified Nk metalloproteinase immobilized on agarose beads. Human NGF also interacted with human metalloproteinases, since it blocked metalloproteinase-mediated shedding of the platelet collagen receptor, glycoprotein (GP)VI, and associated with recombinant ADAM10 by surface plasmon resonance. Together, these results suggest NGF can function as a metalloproteinase inhibitor.

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The single-domain globin from the pathogenic bacterium campylobacter jejuni: Novel D-helix conformation, proximal hydrogen bonding that influences ligand binding, and peroxidase-like redox properties.

Publication Date: 2010 Feb 17 PMID: 20164176
Authors: Shepherd, M. - Barynin, V. - Lu, C. - Bernhardt, P. V. - Wu, G. - Yeh, S. R. - Egawa, T. - Sedelnikova, S. E. - Rice, D. W. - Wilson, J. L. - Poole, R. K.
Journal: J Biol Chem

The foodborne pathogen Campylobacter jejuni possesses a single-domain globin (Cgb) whose role in detoxifying nitric oxide has been unequivocally demonstrated through genetic and molecular approaches. The X-ray structure of cyanide-bound Cgb has been solved to a resolution of 1.35 A. The overall fold is a classic three-on-three alpha-helical globin fold, similar to that of myoglobin and Vgb from Vitreoscilla stercoraria. However, the D region (defined according to the standard globin fold nomenclature) of Cgb adopts a highly ordered alpha-helical conformation unlike any previously characterized members of this globin family, and the GlnE7 residue has an unexpected role in modulating the interaction between the ligand and the TyrB10 residue. The proximal hydrogen bonding network in Cgb demonstrates that the heme cofactor is ligated by an imidazolate, a characteristic of peroxidase-like proteins. Mutation of either proximal H-bonding residue (GluH23 or TyrG5) results in the loss of the high frequency nu(Fe-His) stretching mode (251 cm(-1)), indicating that both residues are important for maintaining the anionic character of the proximal histidine ligand. Cyanide binding kinetics for these proximal mutants demonstrate for the first time that proximal hydrogen bonding in globins can modulate ligand binding kinetics at the distal site. A low redox midpoint for the ferrous/ferric couple (-134 mV versus Normal Hydrogen Electrode (NHE) at pH 7) is consistent with the peroxidase-like character of the Cgb active site. These data provide a new insight into the mechanism via which Campylobacter may survive host-derived nitrosative stress.

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Reconstitution of a functional toll-like receptor 5 binding site in Campylobacter jejuni flagellin.

Publication Date: 2010 Feb 17 PMID: 20164175
Authors: de Zoete, M. R. - Keestra, A. M. - Wagenaar, J. A. - van Putten, J. P.
Journal: J Biol Chem

Bacterial flagellin is important for intestinal immune homeostasis. Flagellins from most species activate Toll-Like Receptor 5 (TLR5). The principal bacterial food-borne pathogen Campylobacter jejuni escapes TLR5 recognition, probably due to an alternate flagellin subunit structure. We investigated the molecular basis of TLR5 evasion by aiming to reconstitute TLR5 stimulating activity in live C. jejuni. Both native glycosylated C. jejuni flagellins (FlaA and FlaB) and recombinant proteins purified from E. coli failed to activate NF-kappaB in HEK293 cells expressing TLR5. Introduction of multiple defined regions from Salmonella flagellin into C. jejuni FlaA via a recombinatorial approach revealed three regions critical for the activation of human and mouse TLR5, including a beta-hairpin structure not previously implicated in TLR5 recognition. Surprisingly, this domain was not required for the activation of chicken TLR5, indicating a selective requirement for the beta-hairpin in the recognition of mammalian TLR5. Expression of the active chimeric protein in C. jejuni resulted in secreted glycosylated flagellin that induced a potent TLR5 response. Overall, our results reveal a novel structural requirement for TLR5 recognition of bacterial flagellin and exclude flagellin glycosylation as an additional mechanism of bacterial evasion of the TLR5 response.

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Impairment of embryonic cell division and glycosaminoglycan biosynthesis in glucuronyltransferase-I-deficient mice.

Publication Date: 2010 Feb 17 PMID: 20164174
Authors: Izumikawa, T. - Kanagawa, N. - Watamoto, Y. - Okada, M. - Saeki, M. - Sakano, M. - Sugahara, K. - Sugihara, K. - Asano, M. - Kitagawa, H.
Journal: J Biol Chem

We have revealed that in Caenorhabditis elegans, non-sulfated chondroitin is required for normal cell division and cytokinesis at an early developmental stage, whereas heparan sulfate is essential for embryonic morphogenesis in the later stages of development. To clarify the roles of chondroitin sulfate and heparan sulfate in early embryogenesis in mammals, we generated glucuronyltransferase-I (GlcAT-I) knockout mice by gene targeting. GlcAT-I is an enzyme required for the synthesis of both chondroitin sulfate and heparan sulfate. Here we report that mice with a deletion of GlcAT-I showed remarkable reduction of the synthesis of chondroitin sulfate and heparan sulfate and embryonic lethality before the 8-cell stage due to failed cytokinesis. In addition, treatment of wild-type 2-cell embryos with chondroitinase ABC had marked effects on cell division, although many heparitinase-treated embryos normally developed to blastocysts. Taken together, these results suggest that chondroitin sulfate in mammals, as with of non-sulfated chondroitin in C. elegans, is indispensable for embryonic cell division.

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Structural and functional characterization of SporoSAG: A SAG2 related surface antigen from Toxoplasma gondii.

Publication Date: 2010 Feb 17 PMID: 20164173
Authors: Crawford, J. - Lamb, E. - Wasmuth, J. - Grujic, O. - Grigg, M. E. - Boulanger, M. J.
Journal: J Biol Chem

Toxoplasma gondii, the etiological agent of toxoplasmosis, utilizes stage specific expression of antigenically distinct glycosylphosphatidylinositol-tethered surface coat proteins to promote and establish chronic infection. Of the three infective stages of T. gondii, sporozoites are encapsulated in highly infectious oocysts that have been linked to large scale outbreaks of toxoplasmosis. SporoSAG is the dominant surface coat protein expressed on the surface of sporozoites. Using a bioinformatic approach, we show that SporoSAG (Surface Antigen Glycoprotein) clusters with the SAG2 subfamily of the SAG1 related superfamily (SRS) and is nonpolymorphic among the 11 haplogroups of T. gondii strains. In contrast to the immunodominant SAG1 protein expressed on tachyzoites, SporoSAG is non-immunogenic during natural infection. We report the 1.60 A resolution crystal structure of SporoSAG solved using cadmium single anomalous dispersion. SporoSAG crystallized as a monomer and displays unique features of the SRS beta sandwich fold relative to SAG1 and BSR4. Intriguingly, the structural diversity is localized to the upper sheets of the beta sandwich fold and may have important implications for dimerization and host cell ligand recognition. The structure of SporoSAG also reveals an unexpectedly acidic surface that contrasts with the previously determined SAG1 and BSR4 structures where a basic surface is predicted to play a role in binding negatively charged glycosaminoglycans. Our structural and functional characterization of SporoSAG provides a rationale for the evolutionary divergence of this key SRS family member.

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RHO kinase-2 activation in human endothelial cells drives LPA-mediated expression of cell adhesion molecules via NF-{kappa}B p65.

Publication Date: 2010 Feb 17 PMID: 20164172
Authors: Shimada, H. - Rajagopalan, L. E.
Journal: J Biol Chem

Endothelial cells play an important role in the recruitment of immune cells to a disease locus through the induced expression of chemokines and cell adhesion molecules (CAMs)(1). The proinflammatory lysophospholipid, lysophosphatidic acid (LPA) that is elevated in multiple inflammatory diseases, is a potent activator of the RhoA/Rho kinase signaling pathway and has been shown to induce the expression of CAMs in endothelial cells. The present study was undertaken to map signal transduction downstream of LPA and to investigate the contributions of the Rho kinase isoforms ROCK1 and ROCK2 to adhesion molecule expression in human umbilical vein endothelial cells (HUVEC). LPA activated Rho kinase within minutes and subsequently the NF-kappaB pathway through phosphorylation of the p65 subunit. The lipid also induced the late expression of intracellular adhesion molecule-1(ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Pharmacologic inhibition of Rho kinase signaling blocked LPA-induced p65 phosphorylation and suppressed ICAM-1 and VCAM-1 expression. Inhibition of the NF-kappaB pathway had no impact on LPA-induced Rho kinase activation, but inhibited adhesion molecule expression. siRNA-facilitated knockdown of each isoform identified ROCK2 as the mediator of LPA-driven phosphorylation of NF-kappaB p65 and of ICAM-1 and VCAM-1 mRNA and protein induction. Taken collectively, our data are consistent with Rho kinase being upstream of NF-kappaB in driving LPA-mediated adhesion molecule expression. This study also provides the first evidence of the critical involvement of ROCK2 in LPA-induced CAMs expression through activation of the NF-kappaB pathway in human endothelial cells.

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NF-{kappa}B activation in T cells requires discrete control of IKK{alpha}/{beta} phosphorylation and IKK{gamma} ubiquitination by the ADAP adapter protein.

Publication Date: 2010 Feb 17 PMID: 20164171
Authors: Srivastava, R. - Burbach, B. J. - Shimizu, Y.
Journal: J Biol Chem

NF-kB activation following engagement of the antigen-specific T cell receptor involves PKCtheta-dependent assembly of the CARMA1-BCL10-MALT1 (CBM) signalosome, which coordinates downstream activation of IKK. We previously identified a novel role for the adapter protein ADAP in regulating the assembly of the CBM complex via an interaction of ADAP with CARMA1. In this study, we identify a novel site in ADAP that is critical for association with the TAK1 kinase. ADAP is critical for recruitment of TAK1 and the CBM complex, but not IKK, to PKCtheta. ADAP is not required for TAK1 activation. While both the TAK1 and CARMA1 binding sites in ADAP are essential for IkBalpha phosphorylation and degradation, and NF-kB nuclear translocation, only the TAK1 binding site in ADAP is necessary for IKK phosphorylation. In contrast, only the CARMA1 binding site in ADAP is required for ubiquitination of IKKgamma. Thus, distinct sites within ADAP control two key activation responses that are required for NF-kB activation in T cells.

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Ubiquitin chain elongation enzyme Ufd2 regulates a subset of Doa10 substrates.

Publication Date: 2010 Feb 16 PMID: 20159987
Authors: Liu, C. - van Dyk, D. - Xu, P. - Choe, V. - Pan, H. - Peng, J. - Andrews, B. - Rao, H.
Journal: J Biol Chem

Ufd2 is the founding member of E4 enzymes that are specifically involved in ubiquitin chain elongation but whose roles in proteolysis remain scarce. Here, using a genome-wide screen, we identified one cellular target of yeast Ufd2 as the membrane protein Pex29. The ubiquitin chains assembled on Pex29 in vivo by Ufd2 mainly contain K48 linkages. We found that the ubiquitin-protein E3 ligase for overexpressed Pex29 is Doa10, which is known to be involved in protein quality control. Interestingly, not all Doa10 substrates are regulated by Ufd2, suggesting that E4 involvement is not specific to a particular E3, but may depend on the spatial arrangement of the E3-substrate interaction. Cells lacking UFD2 elicit an unfolded protein response, expanding the physiological function of Ufd2. Our results lead to novel insights into the biological role of Ufd2 and further underscore the significance of Ufd2 in proteolysis.

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Methylation of ribosomal protein s10 by protein arginine methyltransferase 5 regulates ribosome biogenesis.

Publication Date: 2010 Feb 16 PMID: 20159986
Authors: Ren, J. - Wang, Y. - Liang, Y. - Zhang, Y. - Bao, S. - Xu, Z.
Journal: J Biol Chem

Modulation of ribosomal assembly is a fine tuning mechanism for cell number and organ size control. Many ribosomal proteins undergo post-translational modification, but their exact roles remain elusive. Here, we report that ribosomal protein s10 (RPS10) is a novel substrate of an oncoprotein, protein arginine methyltransferase 5 (PRMT5). We show that PRMT5 interacts with RPS10 and catalyzes its methylation at the Arg158 and Arg160 residues. The methylation of RPS10 at Arg158 and Arg160 plays a role in the proper assembly of ribosomes, protein synthesis and optimal cell proliferation. The RPS10-R158/160K mutant is not efficiently assembled into ribosomes and is unstable and prone to degradation by the proteasomal pathway. In nucleoli, RPS10 interacts with Nucleophosmin (NPM)/B23 and is predominantly concentrated in the granular component (GC) region which is required for ribosome assembly. The RPS10 methylation mutant interacts weakly with NPM/B23 and fails to concentrate in the GC region. Our results suggest that PRMT5 is likely to regulate cell proliferation through the methylation of ribosome proteins, and thus reveal a novel mechanism for PRMT5 in tumorigenesis.

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Caspase-7 cleavage of Kaposi sarcoma-associated herpesvirus ORF57 confers a cellular function against viral lytic gene expression.

Publication Date: 2010 Feb 16 PMID: 20159985
Authors: Majerciak, V. - Kruhlak, M. - Dagur, P. K. - McCoy, J. P. Jr - Zheng, Z. M.
Journal: J Biol Chem

Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 is a viral early protein essential for KSHV multiplication. We found that B cells derived from cavity-based B-cell lymphoma with lytic KSHV infection display activation of caspase-8 and cleavage of ORF57 in the cytoplasm by caspase-7 at the aspartate residue at position 33 from the N-terminus. Caspase-7 cleavage of ORF57 is prevented by pan-caspase inhibitor z-VAD, caspase-3 and caspase-7 inhibitor z-DEVD, and caspase-7 siRNAs. The caspase-7 cleavage site 30DETD33 in ORF57 is not cleavable by caspase-3, although both enzymes use DEXD as a common cleavage site. B cells with lytic KSHV infection and caspase-7 activation exhibited a greatly reduced level of ORF57. Majority of the cells expressing active caspase-7 appeared no detectable ORF57 or vice versa. Upon cleavage with caspase-7, ORF57 was deficient in promoting the expression of viral lytic genes. Inhibiting caspase-7 cleavage of ORF57 in KSHV+ BCBL-1 cells by z-VAD, z-DEVD, or caspase-7 siRNA led to increased expression of viral lytic genes and production of cell-free virus particles. Collectively, our data provide the first compelling evidence that caspase cleavage of ORF57 may represent a cellular function against lytic KSHV infection.

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Chemotherapeutic drugs inhibit ribosome biogenesis at various levels.

Publication Date: 2010 Feb 18 PMID: 20159984
Authors: Burger, K. - Muhl, B. - Harasim, T. - Rohrmoser, M. - Malamoussi, A. - Orban, M. - Kellner, M. - Gruber-Eber, A. - Kremmer, E. - Holzel, M. - Eick, D.
Journal: J Biol Chem

Drugs for cancer therapy belong to different categories of chemical substances. The cellular targets for the therapeutic efficacy are often not unambiguously identified. Here, we describe the process of ribosome biogenesis as a target of a large variety of chemotherapeutic drugs. We determined the inhibitory concentration of 36 chemotherapeutic drugs for transcription and processing of ribosomal RNA by in vivo labeling experiments. Inhibitory drug concentrations were correlated to the loss of nucleolar integrity. The synergism of drugs inhibiting ribosomal RNA synthesis at different levels was studied. Drugs inhibited ribosomal RNA synthesis either at the level of (i) rRNA transcription (e.g. Oxaliplatin, Doxorubicin, Mitoxantrone, Methotrexate), (ii) early rRNA processing (e.g. Camptothecin, Flavopiridol, Roscovitine), or (iii) late rRNA processing (e.g. 5-Fluorouracil, MG-132, Homoharringtonine). Blockage of rRNA transcription or early rRNA processing steps caused nucleolar disintegration, while blockage of late rRNA processing steps left the nucleolus intact. Flavopiridol and 5-Fluorouracil showed a strong synergism for inhibition of rRNA processing. We conclude that inhibition of ribosome biogenesis by chemotherapeutic drugs potentially may contribute to the efficacy of therapeutic regimens.

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Structural diversity of triplet repeat RNAs.

Publication Date: 2010 Feb 16 PMID: 20159983
Authors: Sobczak, K. - Michlewski, G. - de Mezer, M. - Kierzek, E. - Krol, J. - Olejniczak, M. - Kierzek, R. - Krzyzosiak, W. J.
Journal: J Biol Chem

Tandem repeats of various trinucleotide motifs are present in the human transcriptome, but the functions of these regular sequences, which likely depend on the structures they form, are still poorly understood. To gain new insight into the structural and functional properties of triplet repeats in RNA, we have performed a biochemical structural analysis of the complete set of triplet repeat transcripts, each composed of a single sequence repeated 17 times. We show that these transcripts fall into four structural classes. The repeated CAA, UUG, AAG, CUU, CCU, CCA and UAA motifs did not form any higher order structure under any analyzed conditions. The CAU, CUA, UUA, AUG and UAG repeats are ordered according to their increasing tendency to form semi-stable hairpins. The repeated CGA, CGU and all CNG motifs form fairly stable hairpins, whereas AGG and UGG repeats fold into stable G-quadruplexes. The triplet repeats that formed the most stable structures were characterized further by biophysical methods. UV-monitored structure melting revealed that CGG and CCG repeats form, respectively, the most and least stable hairpins of all CNG repeats. Circular dichroism spectra showed that the AGG and UGG repeat quadruplexes are formed by parallel RNA strands. Furthermore, we demonstrated that the different susceptibility of various triplet repeat transcripts to serum nucleases can be explained by the sequence and structural features of the tested RNAs. The results of this study provide a comprehensive structural foundation for the functional analysis of triplet repeats in transcripts.

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Protection against cytokine toxicity through ER and mitochondrial stress prevention by prostacyclin synthase overexpression in insulin-producing cells.

Publication Date: 2010 Feb 16 PMID: 20159982
Authors: Gurgul-Convey, E. - Lenzen, S.
Journal: J Biol Chem

Proinflammatory cytokines play a crucial role in the pathogenesis of type 1 diabetes mellitus. One of the cytokine-regulated pathways mediating inflammation in this autoimmune disease is the arachidonic acid metabolism pathway, comprising both the induction of cyclooxygenases and the production of different prostaglandins. Cytokine toxicity is mediated in many cell types, including pancreatic beta cells through this pathway. Interestingly, some cell types have been shown to be insensitive to such toxicity and this correlated with a high expression of prostacyclin synthase (PGIS). Using insulin-producing RINm5F cells as a model for pancreatic beta cells PGIS was overexpressed and exhibited a large protective effect against cytokine toxicity. This protective effect of PGIS against cytokine toxicity correlated with a decreased activation of the transcription factor NFkappaB and the iNOS promoter as well as a reduced iNOS protein expression and nitrite production. A reduction in the cytokine-stimulated ER and mitochondrial stress was also found in the PGIS overexpressing cells. Moreover, cytokine-induced caspase-3 activation and reduction of glucose oxidation and cell proliferation were suppressed. Thus, PGIS overexpression apparently protects insulin-producing cells against cytokine toxicity via suppression of ER and mitochondrial stress-mediated cell death pathways.

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MUTANT FERRITIN L-CHAINS THAT CAUSE NEURODEGENERATION ACT IN A DOMINANT NEGATIVE MANNER TO REDUCE FERRITIN IRON INCORPORATION.

Publication Date: 2010 Feb 16 PMID: 20159981
Authors: Luscieti, S. - Santambrogio, P. - Langlois d'Estaintot, B. - Granier, T. - Cozzi, A. - Poli, M. - Gallois, B. - Finazzi, D. - Cattaneo, A. - Levi, S. - Arosio, P.
Journal: J Biol Chem

Neuroferritinopathies are inherited movement disorders with dominant transmission associated with one or two nucleotide insertions in the forth exon of L-ferritin gene that modify the protein's C-terminus. The disorder is characterized by abnormal accumulation of iron in the brain due to local iron deregulation. Here we describe a biochemical and crystallographic characterization of the pathogenic mutant p.Phe167SerfsX26 showing an altered conformation of its C-terminus. Moreover we analyze functional and stability properties of ferritin heteropolymers made of 20-23 H chains and 1-4 L chains, either wild types or representing the pathogenic mutations or truncated of the last 10-28 residues. The results show no major differences in ferritin iron incorporation between L wild type and the mutant homopolymers. However, all the heteropolymers containing the pathogenic or the truncated mutants showed a strongly reduced capacity to incorporate iron, both when expressed in E. coli and in vitro when iron was supplied as Fe(III) in the presence of ascorbate. The mutations reduced also the physical stability of the heteropolymers. The data indicate that the disruption of even one or two channels on the four fold axes caused by the mutations is sufficient to alter ferritin capacity to incorporate iron. The dominant negative manner of action of the mutations explains the dominant transmission of the disorder. The data support the hypothesis that hereditary ferritinopathies are due to alterations of ferritin functionality and provide new input on the mechanism of the function of isoferritins.

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Three homologous subunits form a high-affinity peptide-gated ion channel in hydra.

Publication Date: 2010 Feb 16 PMID: 20159980
Authors: Durrnagel, S. - Kuhn, A. - Tsiairis, C. D. - Williamson, M. - Kalbacher, H. - Grimmelikhuijzen, C. J. - Holstein, T. W. - Grunder, S.
Journal: J Biol Chem

Recently, three ion channel subunits of the degenerin/epithelial Na+ channel (DEG/ENaC) gene family have been cloned from the freshwater polyp Hydra magnipapillata, the Hydra Na+ channels (HyNaCs) 2 - 4. Two of them, HyNaC2 and HyNaC3, co-assemble to form an ion channel that is gated by the neuropeptides Hydra-RFamides I and II. The HyNaC2/3 channel is so far the only cloned ionotropic receptor from cnidarians and, together with the related ionotropic receptor FMRFamide-activated Na+ channel (FaNaC) from snails, the only known peptide-gated ionotropic receptor. The HyNaC2/3 channel has pore properties, like a low Na+ selectivity and a low amiloride affinity, that are different from other channels of the DEG/ENaC gene family, suggesting that a component of the native Hydra channel might still be lacking. Here we report the cloning of a new ion channel subunit from Hydra, HyNaC5. The new subunit is closely related to HyNaC2 and 3 and co-localizes with HyNaC2 and 3 to the base of the tentacles. Co-expression in Xenopus oocytes of HyNaC5 with HyNaC2 and 3 largely increases current amplitude after peptide stimulation and affinity of the channel to Hydra-RFamides I and II. Moreover, the HyNaC2/3/5 channel has altered pore properties and amiloride affinity, more similar to other DEG/ENaC channels. Collectively, our results suggest that the three homologous subunits HyNaC2, 3, and 5 form a peptide-gated ion channel in Hydra that could contribute to fast synaptic transmission.

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AMSH interacts with ESCRT-0 to regulate the stability and trafficking of CXCR4.

Publication Date: 2010 Feb 16 PMID: 20159979
Authors: Sierra, M. I. - Wright, M. H. - Nash, P.
Journal: J Biol Chem

Reversible ubiquitination is essential for the endocytic sorting and down-regulation of G protein-coupled receptors, such as the chemokine receptor CXCR4. The deubiquitinating enzyme AMSH has been implicated in the endocytic sorting of both G protein-coupled receptors and receptor tyrosine kinases. Herein, we examine the role of AMSH in the regulation of CXCR4 stability and trafficking and characterize protein-protein interactions critical for this function. Loss of AMSH catalytic activity or depletion by RNAi results in increased steady-state levels of CXCR4 under basal conditions. Analysis of truncation and point mutation of AMSH reveal the importance of an RXXK motif for CXCR4 degradation. The RXXK motif of AMSH interacts with the SH3 domains of the STAM and Grb2 families of adaptor proteins with high affinity. Cells expressing a catalytically inactive mutant of AMSH show basal hyperubiquitination, but not increased degradation, of the ESCRT-0 components STAM1 and Hrs. This is dependant on the RXXK motif of AMSH. Ubiquitination of endocytic machinery modulates their activity suggesting that AMSH may directly regulate endocytic adaptor protein function. This is reflected in CXCR4 trafficking and provides a mechanism by which AMSH specifies the fate of endocytosed receptors. Taken together, these studies implicate AMSH as a key modulator of receptor fate determination through its action on components of the endocytic machinery.

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HSP90 directly modulates the spatial distribution of AF9/MLLT3 and affects target gene expression.

Publication Date: 2010 Feb 16 PMID: 20159978
Authors: Lin, J. J. - Hemenway, C. S.
Journal: J Biol Chem

AF9/MLLT3 contributes to the regulation of the gene encoding the epithelial sodium channel alpha, ENaCalpha, in renal tubular cells. Specifically, increases in AF9 protein lead to a reduction in ENaCalpha expression and changes in AF9 activity appear to be important component of aldosterone signaling in the kidney. While AF9 is found in the nucleus where it interacts with the histone H3 lysine 79 methyltransferase, Dot1, AF9 is also present in the cytoplasm. Data presented in this report indicate that the heat shock protein Hsp90 directly and specifically interacts with AF9 as part of an Hsp90-Hsp70-p60/Hop chaperone complex. Experimental manipulation of Hsp90 function by the inhibitor novobiocin, but not 17-AAG, results in redistribution of AF9 from primarily nuclear to cytoplasmic distribution. Knockdown of Hsp90 with siRNA mimics the effect elicited by novobiocin. As expected, a shift in AF9 from the nucleus to the cytoplasm in response to Hsp90 interference leads to increased ENaCalpha expression. This is accompanied by a decrease in AF9 occupancy at the ENaCalpha promoter. Our data suggest that the interaction of Hsp90, Hsp70, and p60/Hop with AF9 is necessary for the proper subnuclear localization and activity of AF9. AF9 is among a growing number of nuclear proteins recognized to rely on the Hsp90 complex for nuclear targeting.

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Deciphering the catalytic domain of colicin M, a peptidoglycan lipid II degrading enzyme.

Publication Date: 2010 Feb 16 PMID: 20159977
Authors: Barreteau, H. - Bouhss, A. - Gerard, F. - Duche, D. - Boussaid, B. - Blanot, D. - Lloubes, R. - Mengin-Lecreulx, D. - Touze, T.
Journal: J Biol Chem

Colicin M inhibits Escherichia coli peptidoglycan synthesis through cleavage of its lipid-linked precursors. It has a compact structure, while other related toxins are organized in three independent domains, each devoted to a particular function: translocation though the outer membrane, receptor binding, and toxicity, from the N- to the C-terminus, respectively. In order to establish whether colicin M displays such an organization in spite of its structural characteristics, protein dissection experiments were performed which allowed us to delineate an independent toxicity domain encompassing exactly the C-terminal region conserved among colicin M-like proteins and covering about half of colicin M (residues 124-271). Surprisingly, the in vitro activity of the isolated domain was 45-fold higher than that of the full-length protein, suggesting a mechanism by which the toxicity of this domain is revealed following primary protein maturation. In vivo, the isolated toxicity domain appeared as toxic as the full-length protein under conditions where the reception and translocation steps were by-passed. Contrary to the full-length colicin M, the isolated domain did not require the presence of the periplasmic FkpA protein to be toxic under these conditions, demonstrating that FkpA is involved in the maturation process. Mutational analysis further identified five residues that are essential for cytotoxicity as well as in vitro lipid II degrading activity: D229, H235, D226, Y228 and R236. Most of these residues are surface-exposed and located relatively close to each other, hence suggesting they belong to the colicin M active site.

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Transmembrane domain 6 of the human serotonin transporter contributes to an aqueously accessible binding pocket for serotonin and the psychostimulant methylenedioxymethamphetamine(MDMA).

Publication Date: 2010 Feb 16 PMID: 20159976
Authors: Field, J. R. - Henry, L. K. - Blakely, R. D.
Journal: J Biol Chem

The plasma membrane serotonin (5-HT) transporter (SERT, SLC6A4) clears 5-HT following release at nerve terminals and is targeted by both antidepressant medications and psychostimulants (e.g. MDMA, cocaine). Homology modeling of human SERT (hSERT) based on high-resolution structures of the microbial SLC6 family member LeuT(Aa), along with biochemical studies of wild type and mutant transporters, predicts transmembrane (TM) domains 1, 3, 6, and 8 comprise the 5-HT binding pocket. We utilized the substituted cysteine (Cys) accessibility method (SCAM), along with surface and site-specific biotinylation, to probe TM6 for aqueous accessibility and differential interactions with 5-HT and psychostimulants. Our results are consistent with TM6 being comprised of an aqueous-accessible, alpha-helical extracellular domain (TM6a) that is separated by a central, unwound section from a cytoplasmically localized domain (TM6b) with limited aqueous accessibility. The substitution G338C appears to lock hSERT in an outward-facing conformation that, though accessible to MTSEA-biotin, 5-HT and citalopram, is incapable of inward 5-HT transport. Transport of 5-HT by G338C can be partially restored by the TM1 mutation Y95F. With regard to methanethiosulfonate (MTS) inactivation of uptake, TM6a Cys mutants demonstrate Na(+) dependent MTSET sensitivity. Studies with the centrally located substitution S336C reveal features of a common binding pocket for 5-HT and MDMA. Interestingly, the substitution I333C reveals an MDMA-induced conformation not observed with 5-HT. In the context of prior studies on TM1, our findings document shared and unique features of TM6 contributing to hSERT aqueous accessibility, ligand recognition and conformational dynamics.

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Rapid modulation of the organic anion transporting polypeptide 2B1 (OATP2B1, SLCO2B1) function by protein kinase C-mediated internalization.

Publication Date: 2010 Feb 16 PMID: 20159975
Authors: Kock, K. - Koenen, A. - Giese, B. - Fraunholz, M. - May, K. - Siegmund, W. - Hammer, E. - Volker, U. - Jedlitschky, G. - Kroemer, H. K. - Grube, M.
Journal: J Biol Chem

Members of the organic anion transporting polypeptide (OATP) family are involved in various pharmacological, pathophysiological and physiological processes like hepatic drug uptake, progress of cancer or transport of hormones. While variability in expression and function of OATPs has been investigated in detail, data concerning regulation are rather limited. Here, we report a novel mechanism for rapid regulation of OATP2B1 mediated by protein kinase C (PKC) resulting in significant changes of transport activity. PKC activation by the phorbol ester PMA resulted in increased phosphorylation of OATP2B1 as well as reduced OATP2B1 transport activity with a decrease in Vmax of E1S uptake [288 +/- 21 (control) vs. 165 +/- 16 pmol/min/mg protein (PMA)]. This effect was sensitive to the PKC inhibitor bisindolylmaleimide I (BIM-I). Confocal microscopy, fluorescence-based internalization assay, and live-cell imaging using GFP-tagged OATP2B1 revealed that transport inhibition was due to internalization of the transporter. Furthermore, colocalization with LAMP-2 and chloroquine-sensitive degradation of OATP2B1 suggest that the internalized protein is targeted to a lysosomal degradation pathway. With regard to the underlying mechanism inhibition of caveolin/lipid-raft-mediated endocytosis failed to prevent OATP2B1 internalization, while inhibition of clathrin-mediated processes blocked OATP2B1 sequestration. However, siRNA-mediated clathrin knock-down affected general trafficking of OATP2B1 and resulted in intracellular accumulation in absence of PMA. In conclusion, our data demonstrate that OATP2B1 function is regulated by a PKC-mediated, clathrin-dependent internalization and followed by lysosomal degradation. Furthermore, internalization could be shown in an ex vivo placenta perfusion. Our findings represent a new, rapid mechanism in regulation of human OATPs.

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Identification of a linear epitope in sortilin that partakes in pro-neurotrophin binding.

Publication Date: 2010 Feb 16 PMID: 20159974
Authors: Andersen, O. S. - Boisguerin, P. - Glerup, S. - Skeldal, S. - Volkmer, R. - Willnow, T. E. - Nykjaer, A. - Andersen, O. M.
Journal: J Biol Chem

Sortilin acts as cell surface receptor for proneurotrophins (proNT) that upon complex formation with the p75 neurotrophin receptor (p75NTR) is able to signal neuronal cell death. Here we screened a sortilin peptide library comprising 16-mer overlapping sequences for binding of the pro-domains of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). We find that a linear surface exposed sequence, R163IFRSSDFAKNF174, constitutes an important proNT binding epitope in sortilin. Systematic mutational analysis revealed residues R163, F165, R166 and F170 to be critical for the interaction. Expression of a sortilin mutant in which these four amino acids were substituted by alanines disrupted proNT binding without affecting receptor heterodimerization with p75NTR or binding of ligands that selectively engages the centrally located tunnel in the b-propeller of sortilin. We furthermore demonstrate that a peptide comprising the ligand-binding epitope can prevent proNT-induced apoptosis in RN22 schwannoma cells.

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Sumoylation and the structural maintenance of chromosomes (Smc) 5/6 complex slow senescence through recombination intermediate resolution.

Publication Date: 2010 Feb 16 PMID: 20159973
Authors: Chavez, A. - George, V. - Agrawal, V. - Johnson, F. B.
Journal: J Biol Chem

Telomeres are repetitive nucleoprotein structures that cap the ends of chromosomes. Without telomerase, telomeres shorten with replication and eventually signal cell cycle arrest (cell senescence). Homologous recombination (HR)-based mechanisms slow senescence, and distinct HR mechanisms support the growth of the rare survivors of senescence. Here we report novel roles for the post-translational modification SUMO in regulating the rate of senescence in S. cerevisiae telomerase mutants. We identify Mms21 as the relevant E3 SUMO ligase and demonstrate that cells lacking Mms21-dependent sumoylation accumulate HR intermediates selectively at telomeres during senescence. One target of Mms21-dependent sumoylation is the cohesin-and condensin-related Smc5-Smc6 complex (Smc5/6). We show that hypomorphic smc5 or smc6 alleles exhibit phenotypes similar to mms21 sumoylation deficient mutants with regard to senescence and the accumulation of unresolved HR intermediates. Further, we provide evidence that Mms21 and Smc5/6 prevent aberrant recombination between sister telomeres and also globally facilitate resolution of sister chromatid HR intermediates.

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Functional significance of FRH in regulating the phosphorylation and stability of the neurospora circadian clock protein FRQ.

Publication Date: 2010 Feb 16 PMID: 20159972
Authors: Guo, J. - Cheng, P. - Liu, Y.
Journal: J Biol Chem

FREQUENCY (FRQ) is the central component of the Neurospora circadian clock. All FRQ proteins form the FFC complex with FRH (FRQ-interacting RNA helicase) that acts as the negative element in the circadian negative feedback loop by repressing frq mRNA levels. To understand the function of FRQ-FRH interaction, we mapped and identified the minimal FRQ region that is required for FRQ-FRH interaction. We demonstrated that the FRQ-FRH complex formation is required for the interaction between FRQ and the WHITE COLLAR complex (WCC) and clock function. On the other hand, in the FRQ-FRH complex, FRQ is also required for the FRH-WCC interaction. Disruption of FRQ-FRH interaction or down-regulation of FRH results in hypophosphorylation, rapid degradation of FRQ, as well as low levels of WC-1 (white collar 1) and WC-2. Furthermore, we showed that the rapid FRQ degradation in the absence of FRH is independent of FWD-1, the ubiquitin E3 ligase of FRQ under normal conditions, thus uncovering an alternative pathway for FRQ degradation.

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Protocadherin of the liver, kidney and colon associates with detergent-resistant membranes during cellular differentiation.

Publication Date: 2010 Feb 16 PMID: 20159971
Authors: Krahn, M. P. - Rizk, S. - Alfalah, M. - Behrendt, M. - Naim, H. Y.
Journal: J Biol Chem

Protocadherin LKC (PLKC) is a member of the heterogeneous subgroup of protocadherins, which was identified and described as a potential tumor-suppressor gene involved in contact inhibition (1,2). Several aspects of the structure, posttranslational processing, targeting and function of this new protocadherin are still not known. Here, we demonstrate that the expression of PLKC at the apical membrane domain and its concentration at regions of cell-cell contacts occur concomitantly with significant elevation of PLKC-mRNA levels. Furthermore, it can be found within the adherens junctions, but it does not colocalize with tight junctions proteins ZO-1 and occludin, respectively. Additionally, unlike E-cadherin, PLKC is not redistributed upon Ca2+ removal. Biosynthetic labeling revealed N- and O-glycosylation as posttranslational modifications as well as a fast transport to the cell surface and a low turnover rate. During differentiation, PLKC associates with detergent-resistant membranes that trigger its redistribution from intracellular membranes to the cell surface. This association occurs concomitant with alterations in the glycosylation pattern. We propose a role for PLKC in the establishment of a proper epithelial cell polarity that requires O-linked glycosylation and association of the protein with detergent-resistant membranes.

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MYST-family lysine acetyltransferase facilitates ataxia telangiectasia mutated (ATM) kinase-mediated dna damage response in toxoplasma gondii.

Publication Date: 2010 Feb 16 PMID: 20159970
Authors: Vonlaufen, N. - Naguleswaran, A. - Coppens, I. - Sullivan, W. J. Jr
Journal: J Biol Chem

The MYST family of lysine acetyltransferases (KATs) function in a wide variety of cellular operations, including gene regulation and the DNA damage response. Here we report the characterization of the second MYST family KAT in the protozoan parasite Toxoplasma gondii (TgMYST-B). Toxoplasma causes birth defects and is an opportunistic pathogen in the immunocompromised, the latter due to its ability to convert into a latent cyst (bradyzoite). We demonstrate that TgMYST-B can gain access to the parasite nucleus and acetylate histones. Over-expression of recombinant, tagged TgMYST-B reduces growth rate in vitro and confers protection from a DNA alkylating agent. Expression of mutant TgMYST-B produced no growth defect and failed to protect against DNA damage. We demonstrate that cells over-expressing TgMYST-B have increased levels of ATM kinase and phosphorylated H2AX, and that TgMYST-B localizes to the ATM kinase gene. Pharmacological inhibitors of ATM kinase or KATs reverse the slow growth phenotype seen in parasites over-expressing TgMYST-B. These studies are the first to show that a MYST KAT contributes to ATM kinase gene expression, further illuminating the mechanism of how ATM kinase is upregulated to respond to DNA damage.

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A novel role for PIAS proteins in modulating the activity of Zimp7, a novel PIAS-like protein, on the androgen receptor-mediated transcription.

Publication Date: 2010 Feb 16 PMID: 20159969
Authors: Peng, Y. - Lee, J. T. - Zhu, C. - Sun, Z.
Journal: J Biol Chem

The PIAS proteins (protein inhibitor of activated STAT) were originally identified as inhibitors of the JAK-STAT pathway. Subsequently, their roles on transcriptional regulation have been identified in modulation of the androgen receptor (AR) and other nuclear hormone receptor mediated actions. Zimp7, also named Zmiz2, is a novel PIAS-like protein and functions as a transcriptional co-activator. In this study, we demonstrate an interaction between Zimp7 and PIAS proteins with higher preference for PIAS3. A modified mammalian one-hybrid assay showed that the N-terminal proline-rich domain of Zimp7 and the region spanning amino acids 321-486 of PIAS3 were the primary interaction segments. The interaction between Zimp7 and PIAS3 proteins was further confirmed by in vitro protein pulldown and co-immunoprecipitation assays with both exogenous and endogenous proteins. Expression of exogenous PIAS3 further enhances Zimp7-mediated augmentation of AR transcription. Knockdown of endogenous PIAS3 protein using a specific PIAS3 small hairpin RNA reduced the augmentation of Zimp7 on AR-mediated transcription. Co-localization of Zimp7 and PIAS3 proteins was observed in the nuclei of cells by immunostaining. Exogenous PIAS3 expression enhances the stability of the Zimp7 protein. Using chromatin immunoprecipitation assays, we showed that PIAS3 is involved in the AR and Zimp7 formed proteins complex(es) in the AR downstream target promoter to facilitate androgen-induced transcription. Finally, we further demonstrated that loss of Zimp7 significantly impaired PIAS3 mediated enhancement on AR activity in mouse Zimp7 null (zimp7-/-) embryonic fibroblasts. Taken together, these results demonstrate a novel interaction between PIAS and PIAS like proteins and elucidate a novel regulatory mechanism for PIAS proteins on AR mediated transcription.

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MccE provides resistance to protein synthesis inhibitor Microcin C by acetylating the processed form of the antibiotic.

Publication Date: 2010 Feb 16 PMID: 20159968
Authors: Novikova, M. - Kazakov, T. - Vondenhoff, G. H. - Semenova, E. - Rozenski, J. - Metlytskaya, A. - Zukher, I. - Tikhonov, A. - Van Aerschot, A. - Severinov, K.
Journal: J Biol Chem

The heptapeptide-nucleotide Microcin C (McC) is a potent inhibitor of enteric bacteria growth. McC is excreted from producing cells by the MccC transporter. The residual McC that remains in the producing cell can be processed by cellular aminopeptidases with the release of a non-hydrolysable aspartyl-adenylate, a strong inhibitor of aspartyl-tRNA synthetase. Accumulation of processed McC inside producing cells should therefore lead to translation inhibition and cessation of growth. Here, we show that a product of another gene of the McC biosynthetic cluster, mccE, acetylates processed McC and converts it into a non-toxic compound. MccE also makes E. coli resistant to albomycin, a Trojan-horse inhibitor unrelated to McC that, upon processing, gives rise to a serine coupled to a thioxylofuranosyl pyrimidine, an inhibitor of seryl-tRNA synthetase. We speculate that MccE and related cellular acetyltransferases of the Rim family may detoxify various aminoacyl nucleotides, either exogenous or those generated inside the cell.

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Selenium compounds activate early barriers of tumorigenesis.

Publication Date: 2010 Feb 15 PMID: 20157118
Authors: Wu, M. - Kang, M. M. - Schoene, N. W. - Cheng, W. H.
Journal: J Biol Chem

Selenium (Se) chemoprevention by apoptosis has been well studied, but it is not clear whether Se can activate early barriers of tumorigenesis, namely senescence and DNA damage response. To test this hypothesis, we treated normal and cancerous cells with a gradient concentration of sodium selenite, methylseleninic acid (MSeA) and methylselenocysteine (MSeC) for 48 hours, followed by a recovery of 1-7 days. Here we show that Se compounds at doses
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Widespread genomic instability mediated by a pathway involving glycoprotein Ib alpha and aurora B kinase.

Publication Date: 2010 Feb 15 PMID: 20157117
Authors: Li, Y. - Xu, F. L. - Lu, J. - Saunders, W. S. - Prochownik, E. V.
Journal: J Biol Chem

The c-Myc (Myc) oncoprotein's induction of genomic instability (GI) contributes to its initial transforming function and subsequent tumor cell evolution. We describe here a pathway by which Myc, via its target protein glycoprotein Ib alpha (GpIb-alpha), mediates GI. Proteomic profiling revealed that the serine/threonine kinase Aurora B is down-regulated by GpIb-alpha in p53-deficient primary human fibroblasts. The phenotypes of Aurora B deficiency are strikingly reminiscent of Myc or GpIb-alpha over-expression and include double-stranded DNA breaks, altered nuclear size and morphology, chromatin bridges, cleavage furrow regression, and tetraploidy. During mitosis, GpIb-alpha and Aurora B redistribute to the cleavage furrow along with other cleavage furrow proteins. GpIb-alpha over-expression at levels comparable to those seen in some tumor cells causes the dispersal of these proteins but not Aurora B, resulting in furrow regression and cytokinesis failure. Aurora B normalization redirects the mis-localized furrow proteins to their proper location, corrects the cleavage furrow abnormalities, and restores genomic stability. Aurora B thus appears necessary for a previously unrecognized function in guiding and positioning a number of key proteins, including GpIb-alpha to the cleavage furrow. These findings underscore the importance of maintaining a delicate balance among cleavage furrow-associated proteins during mitosis. Suppression of Aurora B via GpIb-alpha provides a unifying and mechanistic explanation for several types of Myc-mediated GI.

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A new asst-type sulfotransferase involved in liponucleoside antibiotic biosynthesis in streptomycetes.

Publication Date: 2010 Feb 15 PMID: 20157116
Authors: Kaysser, L. - Eitel, K. - Tanino, T. - Siebenberg, S. - Matsuda, A. - Ichikawa, S. - Gust, B.
Journal: J Biol Chem

Sulfotransferases are involved in a variety of physiological processes and typically use 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as sulfate donor. In contrast, microbial arylsulfate sulfotransferases (ASSTs) are PAPS-independent and utilize arylsulfates as sulfate donors. Yet, their genuine acceptor substrates are unknown. In this study we demonstrate that Cpz4 from Streptomyces sp. MK730-62F2 is an ASST-type sulfotransferase responsible for the formation of sulfated liponucleoside antibiotics. Gene deletion mutants showed that cpz4 is required for the production of sulfated caprazamycin derivatives. Cloning, overexpression and purification of Cpz4 resulted in a 58 kDa soluble protein. The enzyme catalyzed the transfer of a sulfate group from p-nitrophenolsulfate (pNS; Km 48.1 microM, kcat 0.14 s-1) and methylumbelliferone sulfate (MUS; Km 34.5 microM, kcat 0.15 s-1) onto phenol (Km 25.9 mM and 29.7 mM, respectively). The Cpz4 reaction proceeds by a ping pong bi-bi mechanism. Several structural analogs of intermediates of the caprazamycin biosynthetic pathway were synthesized and tested as substrates of Cpz4. Des-N-methyl-acyl-caprazol was converted with highest efficiency 100 times faster than phenol. The fatty acyl side chain and the uridyl moiety seem to be important for substrate recognition by Cpz4. Liponucleosides, partially purified from various mutant strains, were readily sulfated by Cpz4 using pNS. No product formation could be observed with PAPS as the donor substrate. Sequence homology of Cpz4 to the previously examined ASSTs is low. However, numerous orthologs are encoded in microbial genomes and represent interesting subjects for future investigations.

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Down-regulation of protein tyrosine phosphatases activates an immune receptor in the absence of its translocation into lipid rafts.

Publication Date: 2010 Feb 15 PMID: 20157115
Authors: Heneberg, P. - Draberova, L. - Bambouskova, M. - Pompach, P. - Draber, P.
Journal: J Biol Chem

The earliest known biochemical step that occurs after ligand binding to the multichain immune recognition receptor is tyrosine phosphorylation of the receptor subunits. In mast cells and basophils activated by multivalent antigen-IgE complexes this step is mediated by Src family kinase Lyn, which phosphorylates the high affinity IgE receptor (FcepsilonRI). However, the exact molecular mechanism of this phosphorylation is incompletely understood. In this study we tested the hypothesis that changes in activity and/or topography of protein tyrosine phosphatases (PTPs) could play a major role in the FcepsilonRI triggering. We found that exposure of rat basophilic leukemia cells or mouse bone marrow-derived mast cells to PTP inhibitors, like H(2)O(2) or pervanadate, induced phosphorylation of the FcepsilonRI subunits, similarly as FcepsilonRI triggering. Interestingly, and in sharp contrast to FcepsilonRI-induced activation, neither H(2)O(2) nor pervanadate induced any changes in the association of FcepsilonRI with detergent-resistant membranes (DRMs) and in the topography of FcepsilonRI detectable by electron microscopy on isolated plasma membrane sheets. In cells stimulated with pervanadate, H(2)O(2) or antigen, enhanced oxidation of active site cysteine was detected in several PTPs. Unexpectedly, most of oxidized phosphatases bound to the plasma membrane were associated with actin cytoskeleton. Several PTPs (SHP-1, SHP-2, HePTP and PTP-MEG2) showed changes in their enzymatic activity and/or oxidation state during activation. Based on these and other data we propose that down-regulation of enzymatic activity of PTPs and/or changes in their accessibility to the substrates play a key role in initial tyrosine phosphorylation of the FcepsilonRI and other multichain immune receptors.

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A structural determinant that renders G{alpha}i sensitive to activation by GIV/Girdin is required to promote cell migration.

Publication Date: 2010 Feb 15 PMID: 20157114
Authors: Garcia-Marcos, M. - Ghosh, P. - Ear, J. - Farquhar, M. G.
Journal: J Biol Chem

Although several non-receptor activators of heterotrimeric G proteins have been identified, the structural features of G proteins that determine their interaction with such activators and the subsequent biological effects are poorly understood. Here we investigated the structural determinants in Galphai3 necessary for its regulation by GIV/Girdin, a guanine-nucleotide exchange factor (GEF) that activates Galphai-subunits. Using G protein activity and in vitro pull-down assays we demonstrate that Galphai3 is a better substrate for GIV than the highly homologous Galphao. We identified W258 in the Galphai-subunit as a novel structural determinant for GIV binding by comparing GIV binding to Galphai3/Galphao chimeras. Mutation of W258 to the corresponding Phe in Galphao decreased GIV binding in vitro and in cultured cells but did not perturb interaction with other Galpha-binding partners, i. e., Gbetagamma, AGS3 (a GDI), GAIP/RGS19 (a GAP) and LPAR1 (a GPCR). Activation of Galphai3 by GIV was also dramatically reduced when W258 was replaced with Tyr, Leu, Ser, His, Asp or Ala, highlighting that Trp is required for maximal activation. Moreover, when mutant Galphai3 W258F was expressed in HeLa cells they failed to undergo cell migration and to enhance Akt signaling after growth factor or GPCR stimulation. Thus activation of Galphai3 by GIV is essential for biological functions associated with Galphai3 activation. In conclusion, we have discovered a novel structural determinant on Galphai that plays a key role in defining the selectivity and efficiency of GIV's GEF activity on Galphai and which represents an attractive target site for designing small molecules to disrupt the Galphai-GIV interface for therapeutic purposes.

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Dynamic regulation of a metabolic multi-enzyme complex by protein kinase CK2.

Publication Date: 2010 Feb 15 PMID: 20157113
Authors: An, S. - Kyoung, M. - Allen, J. J. - Shokat, K. M. - Benkovic, S. J.
Journal: J Biol Chem

The reversible association and dissociation of a metabolic multi-enzyme complex participating in de novo purine biosynthesis, the purinosome, was demonstrated in live cells to respond to the levels of purine nucleotides in the culture media. We also took advantage of in vitro proteomic-scale studies of cellular substrates of human protein kinases (e.g. casein kinase II (CK2) and Akt), that implicated several de novo purine biosynthetic enzymes as kinase substrates. Here, we successfully identified that purinosome formation in vivo was significantly promoted in HeLa cells by the addition of small-molecule CK2-specific inhibitors (i.e. 4,5,6,7-tetrabromo-1H-benzimidazole (TBI), 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), tetrabromocinammic acid (TBCA), 4,4('),5,5('),6,6(')-hexahydroxydiphenic acid 2,2('),6,6(')-dilactone (ellagic acid) as well as by silencing the endogenous hCK2alpha catalytic subunit with small interfering RNA. However, 4,5,6,7-tetrabromobenzotriazole (TBB), another CK2-specific inhibitor, triggered the dissociation of purinosome clusters in HeLa cells. Although the mechanism by which TBB affects purinosome clustering is not clear, we were capable of chemically reversing purinosome formation in cells by the sequential addition of two CK2 inhibitors. Collectively, we provide compelling cellular evidence that CK2-mediated pathways reversibly regulate purinosome assembly and thus the purinosome may be one of the ultimate targets of kinase inhibitors.

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Coordinate down-regulation of adenylyl cyclase isoforms and stimulatory G protein (Gs) in intestinal epithelial cell differentiation.

Publication Date: 2010 Feb 15 PMID: 20157112
Authors: Choi, L. J. - Jenikova, G. - Hanson, E. - Spehlmann, M. E. - Boehling, N. S. - Kirstein, S. L. - Bundey, R. A. - Smith, J. R. - Insel, P. A. - Eckmann, L.
Journal: J Biol Chem

The intestinal epithelium is dynamic, with proliferation of undifferentiated crypt cells balanced by terminal differentiation and cell death at the colon surface or small intestinal villus tips. Cyclic AMP, induced by agonists such as PGE2, vasoactive intestinal polypeptide (VIP) and cholera toxin, promotes ion secretion and proliferation, and suppresses apoptosis in intestinal epithelial cells. Here we show that cell differentiation in a model intestinal epithelium causes marked attenuation of cAMP production in response to G-protein-coupled receptor and receptor-independent agonists. Concomitantly, key components of the cAMP cascade, the alpha subunit of the stimulatory G protein, Gs, and the adenylyl cyclase (AC) isoforms 3, 4, 6 and 7, are down-regulated. By contrast, AC1, AC2, AC8, and AC9 are either not expressed or not affected by differentiation, nor are the PGE2 receptors, EP2 and EP4, or the VIP receptor, VAPC1R, expressed at lower levels. Suppression of AC isoforms and Gsalpha is functionally important, since their constitutive, differentiation-independent expression completely reverses the differentiation-induced cAMP attenuation. Thus, down-regulation of certain AC isoforms and Gsalpha is an integral part of the intestinal epithelial differentiation program, perhaps serving to release cells from cAMP-promoted anti-apoptosis as a prerequisite for cell death upon terminal differentiation.

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PROTEIN COMPLEX OF DROSOPHILA ATRX/XNP AND HP1A IS REQUIRED FOR THE FORMATION OF PERICENTRIC BETA-HETEROCHROMATIN IN VIVO.

Publication Date: 2010 Feb 13 PMID: 20154359
Authors: Emelyanov, A. V. - Konev, A. Y. - Vershilova, E. - Fyodorov, D. V.
Journal: J Biol Chem

ATRX belongs to the family of SWI2/SNF2-like ATP-dependent nucleosome remodeling molecular motor proteins. Mutations of the human ATRX gene result in a severe genetic disorder termed X-linked alpha-thalassemia mental retardation (ATR-X) syndrome. Here we perform biochemical and genetic analyses of the Drosophila melanogaster ortholog of ATRX. The loss of function allele of the Drosophila ATRX/XNP gene is semilethal. Drosophila ATRX is expressed throughout development in two isoforms, p185 and p125. ATRX185 and ATRX125 form distinct multi-subunit complexes in fly embryo. The ATRX185 complex comprises p185 and heterochromatin protein HP1a. Consistently, ATRX185 but not ATRX125 is highly concentrated in pericentric beta-heterochromatin of the X chromosome in larval cells. HP1a strongly stimulates biochemical activities of ATRX185 in vitro. Conversely, ATRX185 is required for HP1a deposition in pericentric beta-heterochromatin of the X chromosome. The loss of function allele of the ATRX/XNP gene and mutant allele that does not express p185 are strong suppressors of position effect variegation. These results provide evidence for essential biological functions of Drosophila ATRX in vivo and establish ATRX as a major determinant of pericentric beta-heterochromatin identity.

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MutL{alpha} and proliferating cell nuclear antigen share binding sites on Muts{beta}

Publication Date: 2010 Feb 16 PMID: 20154325
Authors: Iyer, R. R. - Pluciennik, A. - Genschel, J. - Tsai, M. S. - Beese, L. S. - Modrich, P.
Journal: J Biol Chem

MutSbeta (MSH2-MSH3) mediates repair of insertion-deletion heterologies but also triggers triplet repeat expansions that cause neurological diseases. Like other DNA metabolic activities, MutSbeta interacts with proliferating cell nuclear antigen (PCNA) via a conserved motif (QxxL/IxxFF). We demonstrate that MutSbeta-PCNA complex formation occurs with an affinity of ~ 0.1 muM, and a preferred stoichiometry of 1:1. However, upto 20% of complexes are multivalent under conditions where MutSbeta is in molar excess over PCNA. Conformational studies indicate that the two proteins associate in an end-to-end fashion in solution. Surprisingly, mutation of the PCNA-binding motif of MutSbeta not only abolishes PCNA binding, but unlike MutSalpha, also dramatically attenuates MutSbeta-MutLalpha interaction, MutLalpha endonuclease activation, and bidirectional mismatch repair. As predicted by these findings, PCNA competes with MutLalpha for binding to MutSbeta, an effect that is blocked by the cell cycle regulator p21(CIP1). We propose that MutSbeta-MutLalpha interaction is mediated in part by residues (L/ISRFF) embedded within the MSH3 PCNA-binding motif. To our knowledge this is the first case where residues important for PCNA-binding also mediate interaction with a second protein. These findings also indicate that MutSbeta and MutSalpha-initiated repair events differ in fundamental ways.

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Real-time structural transitions are coupled to chemical steps in ATP hydrolysis by Eg5 kinesin.

Publication Date: 2010 Feb 12 PMID: 20154092
Authors: Jun, B. - Kim, S.
Journal: J Biol Chem

At the biochemical level, motor proteins are enzymatic molecules that function by converting chemical energy into mechanical motion. The key element for energy transduction and a major unresolved question common for all motor proteins is the coordination between the chemical and conformational steps in ATP hydrolysis. Here we show time-lapse monitoring of an in vitro ATP hydrolysis reaction by the motor domain of a human Kinesin-5 protein (Eg5), using difference Fourier-transform infrared (FTIR) spectroscopy and UV-photolysis of caged ATP. In this first continuous observation of a biological reaction coordinate from substrate to product, direct spectral markers for two catalytic events are measured: proton abstraction from nucleophilic water by the catalytic base and formation of the inorganic phosphate leaving group. Simultaneous examination of conformational switching in Eg5, in parallel with catalytic steps, shows structural transitions in solution consistent with published crystal structures of the prehydrolytic and ADP-bound states. In addition, we detect structural modifications in the Eg5 motor domain during bond cleavage between the beta- and gamma-phosphates of ATP. This conclusion challenges mechanochemical models for motor proteins that utilize only two stages of the catalytic cycle to drive force and motion.

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The connecdenn family: Rab35 guanine nucleotide exchange factors interfacing with the clathrin machinery.

Publication Date: 2010 Feb 12 PMID: 20154091
Authors: Marat, A. L. - McPherson, P. S.
Journal: J Biol Chem

Rabs constitute the largest family of monomeric GTPases, yet for the majority of Rabs relatively little is known about their activation and recruitment to vesicle trafficking pathways. We recently identified connecdenn (DENND1A), which contains an N-terminal DENN (differentially expressed in neoplastic versus normal cells) domain, a common and evolutionarily ancient protein module. Through its DENN domain, connecdenn functions enzymatically as guanine-nucleotide exchange factor (GEF) for Rab35. Here we identify two additional connecdenn family members and demonstrate that all connecdenns function as Rab35 GEFs, albeit with different levels of activity. The DENN domain of connecdenn 1 and 2 binds Rab35 whereas connecdenn 3 does not, indicating that Rab35 binding and activation are separable functions. Through their highly divergent C-termini, each of the connecdenns binds to clathrin and to the clathrin adaptor AP-2. Interestingly, all three connecdenns use different mechanisms to bind AP-2. Characterization of connecdenn 2 reveals binding to the beta2-ear of AP-2 on a site that overlaps with that used by the autosomal recessive hypercholesteremia protein and betaarrestin, although the sequence used by connecdenn 2 is unique. Loss of connecdenn 2 function through siRNA knockdown results in an enlargement of early endosomes, similar to what is observed upon loss of Rab35 activity. Our studies reveal connecdenn DENN domains as generalized GEFs for Rab35 and identify a new AP-2-binding motif, demonstrating a complex link between the clathrin machinery and Rab35 activation.

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Ubiquilin modifies toxicity of the 43 kilodalton TAR-DNA binding protein (TDP-43) in a Drosophila model of amyotrophic lateral sclerosis (ALS).

Publication Date: 2010 Feb 12 PMID: 20154090
Authors: Hanson, K. A. - Kim, S. H. - Wassarman, D. A. - Tibbetts, R. S.
Journal: J Biol Chem

TDP-43 (43 kDa TAR DNA-binding protein) is a major constituent of ubiquitin-positive cytosolic aggregates present in neurons of patients with amyotrophic lateral sclerosis (ALS) and ubiquitin-positive fronto-temporal lobular degeneration (FTLD-U). Inherited mutations in TDP-43 have been linked to familial forms of ALS, indicating a key role for TDP-43 in disease pathogenesis. Here, we describe a Drosophila melanogaster model of TDP-43 proteinopathy. Expression of wild-type human TDP-43 protein in Drosophila motor neurons led to motor dysfunction and dramatic reduction of lifespan. Interestingly, coexpression of Ubiquilin 1, a previously identified TDP-43-interacting protein with suspected functions in autophagy and proteasome targeting, reduced steady-state TDP-43 expression, but enhanced the severity of TDP-43 phenotypes. Finally, ectopically expressed TDP-43 was largely localized to motor neuron nuclei, suggesting that expression of wild-type TDP-43 alone is detrimental even in the absence of cytosolic aggregation. Our findings demonstrate that TDP-43 exerts cell autonomous neurotoxicity in Drosophila and further imply that dose-dependent alterations of TDP-43 nuclear function may underlie motor neuron death in ALS.

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Endogenous proteolytic cleavage of disease-associated prion protein to produce C2 fragments is strongly cell- and tissue-dependent.

Publication Date: 2010 Feb 12 PMID: 20154089
Authors: Dron, M. - Moudjou, M. - Chapuis, J. - Salamat, M. K. - Bernard, J. - Cronier, S. - Langevin, C. - Laude, H.
Journal: J Biol Chem

The abnormally folded form of the prion protein (PrPSc) accumulating in nervous and lymphoid tissues of prion-infected individuals can be naturally cleaved to generate a N-terminally truncated fragment called C2. Information about the identity of the cellular proteases involved in this process and its possible role in prion biology has remained limited and controversial. We investigated PrPSc N-terminal trimming in different cell lines and primary cultured nerve cells, and in the brain and spleen tissue from transgenic mice infected by ovine and mouse prions. We found the following: i) the full length-to-C2 ratio varies considerably depending on the infected cell or tissue. Thus, in primary neurons and in brain tissue, PrPSc accumulated predominantly as untrimmed species, whereas efficient trimming occurred in Rov and MovS cells, and in spleen tissue. ii) While C2 is generally considered to be the counterpart of the PrPSc proteinase K-resistant core, the N-termini of the fragments cleaved in vivo and in vitro can actually differ, as evidenced by a different reactivity towards the Pc248 anti-octarepeat antibody. iii) In lysosome-impaired cells, the ratio of full-length versus C2 species dramatically increased, yet efficient prion propagation could occur. Moreover, cathepsin but not calpain inhibitors markedly inhibited C2 formation, and in vitro cleavage by cathepsins B and L produced PrPSc fragments lacking the Pc248 epitope, strongly arguing for the primary involvement of acidic hydrolases of the endo-lysosomal compartment. These findings have implications on the molecular analysis of PrPSc and cell pathogenesis of prion infection.

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Distinct role of Kruppel-Like factor 11 in the regulation of prostaglandin E2 biosynthesis.

Publication Date: 2010 Feb 12 PMID: 20154088
Authors: Buttar, N. S. - Demars, C. J. - Lomberk, G. - Rizvi, S. - Bonilla-Velez, J. - Achra, S. - Rashtak, S. - Wang, K. K. - Fernandez-Zapico, M. E. - Urrutia, R.
Journal: J Biol Chem

Kruppel-like factor (KLF) proteins are emerging as key regulators of lipid metabolism, diabetes, and the biosynthesis of immunological cytokines. However, their role in the synthesis of prostaglandins, widely known biochemical mediators that act in a myriad of cell biological processes remain poorly understood. Consequently, in this study a comprehensive investigation at the cellular, biochemical, and molecular level reveals that KLF11 inhibits prostaglandin E2 (PGE2) synthesis via transcriptional silencing of the promoter of its biosynthetic enzyme, cytosolic phospholipase A2alpha (cPLA2alpha). Mechanistically, KLF11 accomplishes this function by binding to the promoter via specific GC-rich sites and recruiting the Sin3a-histone deacetylase (HDAC) chromatin remodeling complex. Further functional characterization reveals that this function of KLF11 can be reversed by epidermal growth factor receptor (EGFR)-AKT-mediated post-translational modification of threonine 56, a residue within its Sin3 binding domain. This is the first evidence supporting a relevant role for any KLF protein in doing both; transcriptionally inhibiting prostaglandin biosynthesis and its reversibility by an EGFR-AKT signaling-mediated posttranslational mechanisms.

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Celastrol, a triterpene, enhances TRAIL-induced apoptosis through the downregulation of cell survival proteins and upregulation of death receptors.

Publication Date: 2010 Feb 12 PMID: 20154087
Authors: Sung, B. - Park, B. - Yadav, V. R. - Aggarwal, B. B.
Journal: J Biol Chem

Whether celastrol, a triterpene from traditional Chinese medicine, can modulate the anticancer effects of TRAIL, the cytokine that is currently in clinical trial, was investigated. As indicated by assays that measure plasma membrane integrity, phosphotidylserine exposure, mitochondrial activity, and activation of caspase-8, caspase-9 and caspase-3, celastrol potentiated the TRAIL-induced apoptosis in human breast cancer cells, and converted TRAIL-resistant cells to TRAIL-sensitive. When examined for the mechanism, we found that the triterpene downregulated the expression of cells survival proteins including cFLIP, IAP-1, Bcl-2, Bcl-xL, survivin, and XIAP; and upregulated Bax expression. In addition, we found that celastrol induced the cell surface expression of both the TRAIL receptors DR4 and DR5. This increase in receptors was noted in a wide variety of cancer cells including breast, lung, colorectal, prostate, esophageal, and pancreatic cancer cells, and myeloid and leukemia cells. Gene silencing of the death receptor abolished the effect of celastrol on TRAIL-induced apoptosis. Induction of the death receptor by the triterpenoid was found to be p53-independent but required the induction of CAAT/enhancer binding protein homologous protein (CHOP), inasmuch as gene-silencing of CHOP abolished the induction of DR5 expression by celastrol and associated enhancement of TRAIL-induced apoptosis. We found that celastrol also induced reactive oxygen species (ROS) generation, and ROS sequestration inhibited celastrol-induced expression of CHOP and DR5, and consequent sensitization to TRAIL. Overall our results demonstrate that celastrol can potentiate the apoptotic effects of TRAIL through downregulation of cell survival proteins and upregulation of death receptors via the ROS-mediated up-regulation of CHOP pathway.

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Activation and stiffness of the inhibited states of F1-ATPase probed by single-molecule manipulation.

Publication Date: 2010 Feb 12 PMID: 20154086
Authors: Saita, E. I. - Iino, R. - Suzuki, T. - Feniouk, B. A. - Kinosita, K. Jr - Yoshida, M.
Journal: J Biol Chem

F(1)-ATPase (F(1)), a soluble portion of FoF(1)-ATP synthase (F(o)F(1)), is an ATP-driven motor in which gammaepsilon subunits rotate in the alpha (3)beta(3) cylinder. Activity of F(1) and F(o)F(1) from Bacillus PS3 is attenuated by the epsilon subunit in an inhibitory extended form. In this study, we observed [ATP]-dependent transition of epsilon in single F(1) molecules from extended form to hairpin form by fluorescence resonance energy transfer. The results justify the previous bulk experiments and ensure that fraction of F(1) with hairpin epsilon directly determines the fraction of active F(1) at any [ATP]. Next, mechanical activation and stiffness of epsilon-inhibited F(1) were examined by the forced rotation of magnetic beads attached to gamma. Compared with ADP-inhibition, which is another manner of inhibition, rotation by larger angle was required for the activation from epsilon-inhibition when the beads were forced to rotate to ATP hydrolysis direction, and more torque was required to reach the same rotation angle when beads were forced to rotate to ATP synthesis direction. The results implicate that if F(o)F(1) is resting in epsilon-inhibited state, F(o) motor must transmit to gamma a torque larger than expected from thermodynamic equilibrium to initiate ATP synthesis.

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Self-assembly of severe acute respiratory syndrome coronavirus membrane protein.

Publication Date: 2010 Feb 12 PMID: 20154085
Authors: Tseng, Y. T. - Wang, S. M. - Huang, K. J. - Lee, A. I. - Chiang, C. C. - Wang, C. T.
Journal: J Biol Chem

Coronavirus membrane (M) protein can form virus-like particles (VLPs) when coexpressed with nucleocapsid (N) or envelope (E) proteins, suggesting a pivotal role for M in virion assembly. Here we demonstrate the self-assembly and release of severe acute respiratory syndrome coronavirus (SARS-CoV) M protein in medium in the form of membrane-enveloped vesicles with densities lower than those of VLPs formed by M plus N. While efficient N-N interactions require the presence of RNA, we found that M-M interactions were RNA-independent. SARS-CoV M was observed in both the Golgi area and plasma membranes of a variety of cells. Blocking M glycosylation does not appear to significantly affect M plasma membrane labeling intensity, M-containing vesicle release, or VLP formation. Results from a genetic analysis indicate involvement of the third transmembrane domain of M in plasma membrane targeting signal. Fusion proteins containing M amino-terminal 50 residues encompassing the first transmembrane domain were found to be sufficient for membrane binding, multimerization, and Golgi retention. Surprisingly, we found that fusion proteins lacking all three transmembrane domains were still capable of membrane binding, Golgi retention, and interacting with M. The data suggest that multiple SARS-CoV M regions are involved in M self-assembly and subcellular localization.

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The roles of the lipid-binding motifs of Atg18 and Atg21 in the cytoplasm to vacuole targeting pathway and autophagy.

Publication Date: 2010 Feb 12 PMID: 20154084
Authors: Nair, U. - Cao, Y. - Xie, Z. - Klionsky, D. J.
Journal: J Biol Chem

Atg18 and Atg21 are homologous WD-40 repeat proteins that bind phosphoinositides via a novel conserved Phe-Arg-Arg-Gly (FRRG) motif and function in autophagy-related pathways. Atg18 is required for the cytoplasm to vacuole targeting (Cvt) pathway and autophagy, whereas Atg21 is only required for the Cvt pathway. Currently, the functions of both proteins are poorly understood. Here we examined the relationship between the phosphatidylinositol 3-phosphate (PtdIns(3)P)-binding abilities of Atg18 and Atg21 and autophagy, by expressing variants of these proteins that have mutations in their phosphoinositide-binding motifs. Cells expressing PtdIns(3)P-binding mutants of both these proteins showed highly reduced autophagy. Furthermore, the localization of components of two related ubiquitin-like protein conjugation systems, Atg8 and Atg16, to the phagophore assembly site (PAS) is affected. Consistent with the aberrant localization of the above Atg proteins, precursor Ape1, a cargo of the Cvt pathway and autophagy, is partially protease-sensitive in starvation conditions, suggesting a role for Atg18, and Atg21, PtdIns(3)P-binding in efficient completion of the sequestering autophagic vesicles. Finally, using a multiple knockout strain we found that Atg18 and Atg21 facilitate the recruitment of Atg8-PE to the site of autophagosome formation and protect it from premature cleavage by Atg4, which represents a key aspect of posttranslational autophagy regulation. Taken together, our results suggest that PtdIns(3)P-binding by at least Atg18 or Atg21 is required for robust autophagic activity, and that the PtdIns(3)P-binding motifs of Atg18 and Atg21 can compensate for one another in the recruitment of Atg components that are dependent on PtdIns(3)P for their PAS association.

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