Journal of Biological Chemistry

Solution structure of the calponin homology (CH)-domain from the smoothelin-like 1 protein: a unique Apo-calmodulin binding mode and the possible role of the C-terminal type 2 CH-domain in smooth muscle relaxation.

Publication Date: 2008 May 12 PMID: 18477568
Authors: Ishida, H. - Borman Meredith, A. - Ostrander, J. - Vogel Hans, J. - Macdonald Justin, A.
Journal: J Biol Chem

The smoothelin-like 1 protein (SMTNL1) is a recently discovered component of smooth muscle tissues [Borman M. A., MacDonald J. A., and Haystead T. A. J., (2004) FEBS Lett. 573, 207-213]. This 459-residue protein contains a single type-2 CH-domain at its C-terminus that shares sequence identity with the smoothelin family of smooth muscle specific proteins. In contrast to the smoothelins, SMTNL1 does not associate with F-actin in vitro, and although it is known to become phosphorylated during cGMP-mediated Ca(2+)-desensitization and relaxation, its specific role in smooth muscle remains unclear. In addition, the biological function of the C-terminal CH-domains found in the smoothelin proteins is also poorly understood. In this work, we have therefore determined the solution structure of the CH-domain of mouse SMTNL1 (SMTNL1-CH; residues 346-459). The secondary structure and the overall fold for the C-terminal type-2 CH-domain is very similar to that of other CH-domains. However, two clusters of basic residues form a unique surface structure that is characteristic of SMTNL1-CH. Moreover, the protein has an extended C-terminal a-helix, which contains a calmodulin (CaM)-binding IQ-motif, that is also a distinct feature of the smoothelins. We have characterized the binding of apo-CaM to SMTNL1-CH through its IQ-motif by isothermal titration calorimetry and NMR chemical shift perturbation studies. In addition, we have used the HADDOCK protein-protein docking approach to construct a model for the complex of apo-CaM and SMTNL1-CH. The model revealed a close interaction of SMTNL1-CH with the two Ca(2+)-binding loop regions of the C-domain of apo-CaM; this mode of apo-CaM binding is distinct from previously reported interactions of apo-CaM with IQ-motifs. Finally, we comment on the putative role of the CH-domain in the biological function of the SMTNL1 protein.

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NFATc2 is a necessary mediator of calcineurin-dependent cardiac hypertrophy and heart failure.

Publication Date: 2008 May 12 PMID: 18477567
Authors: Bourajjaj, M. - Armand, A. S. - da Costa Martins, P. A. - Weijts, B. - van der Nagel, R. - Heeneman, S. - Wehrens, X. H. - De Windt, L. J.
Journal: J Biol Chem

One major intracellular signaling pathway involved in heart failure employs the phosphatase calcineurin and its downstream transcriptional effector nuclear factor of activated T-cells (NFAT). In vivo evidence for the involvement of NFAT factors in heart failure development is still ill defined. Here we reveal that nfatc2 transcripts outnumber those from other nfat genes in the unstimulated heart by several folds. Transgenic mice with activated calcineurin in the postnatal myocardium crossbred with nfatc2-null mice revealed a significant abrogation of calcineurin-provoked cardiac growth, indicating that NFATc2 plays an important role downstream of calcineurin and validates the original hypothesis that calcineurin mediates myocyte hypertrophy through activation of NFAT transcription factors. In the absence of NFATc2, a clear protection against the geometrical, functional, and molecular deterioration of the myocardium following biomechanical stress was also evident. In contrast, physiological cardiac enlargement in response to voluntary exercise training was not affected in nfatc2-null mice. Combined, these results reveal a major role for the NFATc2 transcription factor in pathological cardiac remodeling and heart failure.

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PIASy represses CCAAT/enhancer binding protein delta (C/EBPdelta ) transcriptional activity by sequestering C/EBPdelta to the nuclear periphery.

Publication Date: 2008 May 12 PMID: 18477566
Authors: Zhou, S. - Si, J. - Liu, T. - Dewille, J. W.
Journal: J Biol Chem

CCAAT/Enhancer Binding Protein d (C/EBPd) plays a key role in mammary epithelial cell G0 growth arrest and "loss of function" alterations in C/EBPd have been reported in breast cancer and acute myeloid leukemia (AML). C/EBPd is regulated at the transcriptional, post-transcriptional and post-translational levels, suggesting tight control of C/EBPd content and function. Protein inhibitors of activated STATs (PIASs) regulate a growing number of transcription factors, including C/EBPs. HC11 nontransformed mammary epithelial cells express PIAS3, PIASxss and PIASy and all three PIAS family members repress C/EBPd transcriptional activity. PIASy is the most potent however, repressing C/EBPd transcriptional activity by >80%. PIASy repression of C/EBPd transcriptional activity is dependent upon interaction between the highly conserved PIASy N-terminal nuclear matrix binding domain (SAPD) and the C/EBPd transactivation domain (TAD). PIASy repression of C/EBPd transcriptional activity is independent of histone deacetylase activity, PIASy E3 SUMO ligase activity and C/EBPd sumoylation status. PIASy expression is associated with C/EBPd translocation from nuclear foci, where C/EBPd co-localizes with p300, to the nuclear periphery. PIASy-mediated translocation of C/EBPd is dependent upon the PIASy SAPD and C/EBPd TAD. PIASy reduces the expression of C/EBPd adhesion-related target genes and enhances repopulation of open areas within a cell monolayer in the in vitro "scratch" assay. These results demonstrate that PIASy represses C/EBPd by a mechanism that requires interaction between the PIASy SAPD and C/EBPd TAD and does not require PIASy SUMO ligase activity or C/EBPd sumoylation. PIASy alters C/EBPd nuclear localization, reduces C/EBPd transcriptional activity and enhances cell proliferation/migration.

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Functional effects of nemaline myopathy mutations on human skeletal alpha-actin.

Publication Date: 2008 May 12 PMID: 18477565
Authors: Miller, B. M. - Trybus, K. M.
Journal: J Biol Chem

Mutations in human a-skeletal actin have been implicated in causing congenital nemaline myopathy, a disease characterized histopathologically by nemaline bodies in skeletal muscle, and manifested in the patient as skeletal muscle weakness. Here we investigate the functional effects of three severe nemaline myopathy mutations (V43F, A138P, and R183G) in human a-skeletal actin. Wild-type and mutant actins were expressed and purified from the baculovirus/insect cell expression system. The mutations are located in different subdomains of actin: V43 is located in a flexible loop of subdomain 2, A138 is near a hydrophobic cleft in the "hinge" region between subdomains 1 and 3, and R183 is near the nucleotide-binding site. None of the three mutations affected the folding of the actin monomer, the velocity at which skeletal myosin moves actin in an in vitro motility assay, or the relative average isometric force supported by F-actin. Defects in fundamental actomyosin interactions are therefore unlikely to account for the muscle weakness observed in affected patients. There were, however, significant changes observed in the polymerization kinetics of V43F and A138P, and in the rate of nucleotide release for V43F. No detectable defect was found for R183G. If these subtle changes in polymerization observed in vitro are amplified in the context of the sarcomere, it could in principle be one of the primary insults that trigger the development of nemaline myopathy.

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Rho/ROCK signal regulates myogenic differentiation via MRTF-A/Smad-dependent transcription of the Id3 gene.

Publication Date: 2008 May 12 PMID: 18477564
Authors: Iwasaki, K. - Hayashi, K. - Fujioka, T. - Sobue, K.
Journal: J Biol Chem

RhoA is known to be involved in myogenic differentiation, but whether its acts as a positive or negative regulator are controversial. To resolve this issue, we investigated the differentiation-stage-specific roles of RhoA and its effector, Rho-associated kinase (ROCK) using C2C12 myoblasts. We found that proliferating myoblasts show high levels of RhoA and SRF activities and strong expression of RhoA's downstream target, myocardin-related transcription factor-A (MRTF-A or MAL); these activities and expression are markedly lower in differentiating myocytes. We further demonstrated that, in proliferating myoblasts, an increase in MRTF-A, which forms a complex with Smad 1/4, strikingly activates the expression level of the Id3 gene; the Id3 gene product is a potent inhibitor of myogenic differentiation. Finally, we found that during differentiation, one of the forkhead transcription factors (FKHR) translocates into the nucleus and suppresses Id3 expression by preventing the association of the MRTF-A/Smad complex with the Id3 promoter, which leads to the enhancement of myogenic differentiation. We conclude that RhoA/ROCK signaling plays positive and negative roles in myogenic differentiation, mediated by MRTF-A/Smad-dependent transcription of the Id3 gene in a differentiation-stage-specific manner.

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Phosphorylated heat shock protein 27 represses growth of hepatocellular carcinoma via inhibition of extracellular-signal-regulated kinase.

Publication Date: 2008 May 13 PMID: 18477563
Authors: Matsushima-Nishiwaki, R. - Takai, S. - Adachi, S. - Minamitani, C. - Yasuda, E. - Noda, T. - Kato, K. - Toyoda, H. - Kaneoka, Y. - Yamaguchi, A. - Kumada, T. - Kozawa, O.
Journal: J Biol Chem

Heat shock protein 27, one of the low-molecular-weight stress proteins, is recognized as a molecular chaperone, however, other functions have not yet been well established. Phosphorylated heat shock protein 27 levels inversely correlate with the progression of human hepatocellular carcinoma. This study shows that phosphorylated heat shock protein 27 interferes with cell growth of the hepatocellular carcinoma derived HuH7 cells in the presence of the proinflammatory cytokine, tumor necrosis factor-a, via inhibition of the sustained activation of the extracellular-signal-regulated kinase signal pathway. The activities of Raf/ extracellular-signal- regulated kinase and subsequent activator protein-1 transactivation, and the induction levels of cyclin D1 were lower in HuH7 cells transfected with phosphorylated heat shock protein 27 than those with unphosphorylated heat shock protein 27. Moreover, phosphorylated heat shock protein 27 up-regulated the levels of p38 mitogen-activated protein kinase and mitogen-activated protein kinase phosphatase-1, an inhibitory protein of extracellular-signal-regulated kinase. These results indicate that phosphorylated heat shock protein 27 might suppress the extracellular-signal-regulated kinase activity in the hepatocellular carcinoma cells via two separate pathways in an inflammatory state. The extracellular-signal-regulated kinase activity is inversely correlated with phosphorylated heat shock protein 27 at serine-15, also in human hepatocellular carcinoma tissues in vivo. Since, the extracellular-signal-regulated kinase signal pathway is a major proliferation signal of hepatocellular carcinoma, and activator protein-1 activation is an early event in hepatocarcinogenesis. These findings strongly suggest that the control of the phosphorylated heat shock protein 27 levels could be a new therapeutic strategy especially to counter the recurrence of hepatocellular carcinoma.

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Crystal structure of the GAF-B domain from human phosphodiesterase 10A complexed with its ligand, cAMP.

Publication Date: 2008 May 13 PMID: 18477562
Authors: Handa, N. - Mizohata, E. - Kishishita, S. - Toyama, M. - Morita, S. - Uchikubo-Kamo, T. - Akasaka, R. - Omori, K. - Kotera, J. - Terada, T. - Shirouzu, M. - Yokoyama, S.
Journal: J Biol Chem

Cyclic nucleotide phosphodiesterases (PDEs) catalyze the degradation of the cyclic nucleotides cAMP and cGMP, which are important second messengers. Five of the 11 mammalian PDE families have tandem GAF domains at their N-termini. PDE10A may be the only mammalian PDE for which cAMP is the GAF domain ligand, and it may be allosterically stimulated by cAMP. PDE10A is highly expressed in striatal medium spiny neurons (MSNs). Here we report the crystal structure of the C-terminal GAF domain (GAF-B) of human PDE10A complexed with cAMP at 2.1 A resolution. The conformation of the PDE10A GAF-B domain monomer closely resembles those of the GAF domains of PDE2A and the cyanobacterium Anabaena cyaB2 adenylyl cyclase, except for the helical bundle consisting of alpha1, alpha2, and alpha5. The PDE10A GAF-B domain forms a dimer in the crystal and in solution. The dimerization is mainly mediated by hydrophobic interactions between the helical bundles in a parallel arrangement, with a large buried surface area. In the PDE10A GAF-B domain, cAMP tightly binds to a cNMP-binding pocket. The residues in the alpha3 and alpha4 helices, the beta6 strand, the loop between 3(10) and alpha4, and the loop between alpha4 and beta5 are involved in the recognition of the phosphate and ribose moieties. This recognition mode is similar to those of the GAF domains of PDE2A and cyaB2. In contrast, the adenine base is specifically recognized by the PDE10A GAF-B domain in a unique manner, through residues in the beta1 and beta2 strands.

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How do enzymes really work?

Publication Date: 2008 May 13 PMID: 18477561
Authors: Hammes, G. G.
Journal: J Biol Chem

None.

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S6K1 phosphorylates and regulates FMRP with the neuronal protein synthesis-dependent mTOR signaling cascade.

Publication Date: 2008 May 12 PMID: 18474609
Authors: Narayanan, U. - Nalavadi, V. - Nakamoto, M. - Thomas, G. - Ceman, S. - Bassell, G. J. - Warren, S. T.
Journal: J Biol Chem

Fragile X syndrome is a common form of cognitive deficit caused by the functional absence of FMRP, a dendritic RNA-binding protein that represses translation of specific messages. Although FMRP is phosphorylated in a groupI mGluR activity-dependent manner following brief PP2A-mediated dephosphorylation, the kinase regulating FMRP function in neuronal protein synthesis is unclear. Here we identify ribosomal protein S6 kinase (S6K1) as a major FMRP kinase in the mouse hippocampus finding that activity-dependent phosphorylation of FMRP by S6K1 requires signaling inputs from mTOR, ERK1/2 and PP2A. Further, the loss of hippocampal S6K1, and the subsequent absence of phospho-FMRP, mimics FMRP loss in the increased expression of SAPAP3, a synapse-associated FMRP target mRNA. Together these data reveal a S6K1-PP2A signaling module regulating FMRP function and place FMRP phosphorylation in the mGluR-triggered signaling cascade required for protein-synthesis-dependent synaptic plasticity.

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All JNKs can Kill but Nuclear Localisation is Critical for Neuronal Death.

Publication Date: 2008 May 12 PMID: 18474608
Authors: Bjorkblom, B. - Vainio, J. C. - Hongisto, V. - Herdegen, T. - Courtney, M. J. - Coffey, E. T.
Journal: J Biol Chem

JNKs are implicated in a range of brain pathologies, and receive considerable attention as potential therapeutic targets. However, JNKs also regulate physiological and homeostatic processes. An attractive hypothesis from the drug development perspective is that distinct JNK isoforms mediate "physiological" and "pathological" responses. However, this lacks experimental evaluation. Here we investigate the isoforms, subcellular pools and c-Jun/ATF2 targets of JNK in death of CNS neurons following withdrawal of trophic support. We use gene knockouts, gene silencing, subcellularly targeted dominant negative constructs and pharmacological inhibitors. Combined siRNA knockdown of all JNK genes, 1, 2 and 3 provides substantial neuroprotection. In contrast, knockdown or knockout of individual JNKs or two JNKs together does not protect. This explains why the evidence for JNK in neuronal death has to date been largely been pharmacological. Complete knockdown of c-Jun and ATF2 using siRNA also fails to protect, casting doubt on c-Jun as a critical effector of JNK in neuronal death. Nonetheless, the death requires nuclear but not cytosolic JNK activity as nuclear dominant negative inhibitors of JNK protect while cytosolic inhibitors only block physiological JNK function. Thus any one of the three JNKs is capable of mediating apoptosis and inhibition of nuclear JNK is protective.

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Hsf-1 and POB1 induce drug-sensitivity and apoptosis by inhibiting Ralbp1.

Publication Date: 2008 May 12 PMID: 18474607
Authors: Singhal, S. S. - Yadav, S. - Drake, K. - Singhal, J. - Awasthi, S.
Journal: J Biol Chem

Heat shock factor-1 (Hsf-1) is a transcription factor which is known to regulate cellular heat-shock response through its binding with the multi-specific transporter protein, Ralbp1. Results of present studies demonstrate that Hsf-1 causes specific and saturable inhibition of the transport activity of Ralbp1, and that the combination of Hsf-1 and POB1 cause near complete inhibition through specific bindings with Ralbp1. Augmentation of cellular levels of Hsf-1 and POB1 caused dramatic apoptosis in NSCLC H358 through Ralbp1 inhibition. These findings indicate a novel model for mutual regulation of Hsf-1 and Ralbp1 through Ralbp1 mediated sequestration of Hsf-1 in the cellular cytoskeleton, and Hsf-1 mediated inhibition of the transport activity of membrane bound Ralbp1.

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SRC directly phosphorylates BIF-1 and prevents its interaction with Bax and the initiation of anoikis.

Publication Date: 2008 May 12 PMID: 18474606
Authors: Yamaguchi, H. - Woods, N. T. - Dorsey, J. F. - Takahashi, Y. - Gjertsen, N. R. - Yeatman, T. - Wu, J. - Wang, H. G.
Journal: J Biol Chem

Bif-1 interacts with Bax and enhances its conformational rearrangement resulting in apoptosis. However, the molecular mechanism governing the interaction between Bif-1 and Bax is poorly defined. Here we provide evidence that Bif-1 is phosphorylated, an event which can be repressed by apoptotic stimuli. The protein kinase c-Src binds to and directly phosphorylates Bif-1 on tyrosine 80. Moreover, Src phosphorylation of Bif-1 suppresses the interaction between Bif-1 and Bax resulting in the inhibition of Bax activation during anoikis. Together, these results suggest that phosphorylation of Bif-1 impairs its binding to Bax and represses apoptosis, providing another mechanism by which Src oncogenic signaling can prevent cell death.

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BDNF-TRKB signaling contributes to activity-dependent changes in synaptic proteins.

Publication Date: 2008 May 12 PMID: 18474605
Authors: Jia, J. M. - Chen, Q. - Zhou, Y. - Miao, S. - Zheng, J. - Zhang, C. - Xiong, Z. Q.
Journal: J Biol Chem

The ability of synapses to undergo changes in structure and function in response to alterations of neuronal activity is an essential property of neural circuits. One way that this is achieved is through global changes in the molecular composition of the synapse, however it is not clear how these changes are coupled to the dynamics of neuronal activity. Here we found that, in cultured rat cortical neurons, bi-directional changes of neuronal activity led to corresponding alterations in the expression of brain-derived neurotrophic factor (BDNF) and phosphorylation of its receptor TrkB, as well as in the level of synaptic proteins. Exogenous BDNF reversed changes in synaptic proteins induced by chronic activity blockade, while inhibiting Trk kinase activity or depleting endogenous BDNF abolished the concentration changes induced by chronic activity elevation. Both TTX and bicuculline had significant, but opposite, effects on synaptic protein ubiquitination in a time-dependent manner. Furthermore, exogenous BDNF was sufficient to increase ubiquitination of synaptic proteins, whereas scavenging endogenous BDNF or inhibiting Trk kinase activity prevented the ubiquitination of synaptic proteins induced by chronic elevation of neuronal activity. Inhibiting the proteasome or blocking protein polyubiquitination mimicked the effect of TTX on the levels of synaptic proteins and canceled the effects of BDNF. Our study indicates that BDNF-TrkB signaling acts upstream of the ubiquitin proteasome system, linking neuronal activity to protein turnover at the synapse.

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DR4 selective TRAIL variants obtained by structure based design.

Publication Date: 2008 May 12 PMID: 18474604
Authors: Tur, V. - van der Sloot, A. M. - Reis, C. R. - Szegezdi, E. - Cool, R. H. - Samali, A. - Serrano, L. - Quax, W. J.
Journal: J Biol Chem

Tumour necrosis factor-related apoptosis inducing ligand (TRAIL) is a potential anticancer agent that selectively induces apoptosis in a variety of cancer cells by interacting with death receptors DR4 and DR5. TRAIL can also bind to decoy receptors (DcR1, DcR2 and OPG receptor) that cannot induce apoptosis. Different tumour types respond either to DR4 or to DR5 activation and chemotherapeutic drugs can increase the expression of DR4 or DR5 in cancer cells. Thus, DR4 or DR5 receptor-specific TRAIL variants would permit new and tumour-selective therapies. Previous success in generating a DR5-selective TRAIL mutant using computer-assisted protein design prompted us to make a DR4 selective TRAIL variant. Technically, the design of DR4 receptor selective TRAIL variants is considerably more challenging compared to DR5 receptor selective variants, due to the lack of a crystal structure of the TRAIL-DR4 complex. A single amino acid substitution of Asp at residue position 218 of TRAIL to His or Tyr was predicted to have a favourable effect on DR4 binding specificity. Surface Plasmon Resonance based receptor binding tests showed a lowered DR5 affinity in concert with increased DR4 specificity for the designed variants, D218H and D218Y. Binding to DcR1, DcR2 and OPG was also decreased. Cell line assays confirmed that the variants could not induce apoptosis in DR5 responsive Jurkat and A2780 cells but were able to induce apoptosis in DR4 responsive EM-2 and ML-1 cells.

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Prothyrotropin releasing hormone targets its processing products to different vesicles of the secretory pathway.

Publication Date: 2008 May 12 PMID: 18474603
Authors: Perello, M. - Stuart, R. C. - Nillni, E. A.
Journal: J Biol Chem

ProThyrotropin Releasing Hormone (proTRH) is initially cleaved by the prohormone convertase 1/3 (PC1/3) in the trans-Golgi network (TGN) generating N- and C-terminal intermediate forms that are then packed into secretory vesicles. However, it is unknown if these peptides are differentially sorted within the Secretory Pathway. This is of key importance because the processing products of several prohormones fulfill different biological functions. Using AtT20 cells stably transfected with preproTRH cDNA, we found that two specific N- and C-terminal peptides were located in different vesicles. Furthermore, the C-terminal proTRH-derived peptides were more efficiently released in response to KCl and norepinephrine, a natural secretagogue of TRH. Similar sorting and secretion of N- and C-terminal peptides occurs in vivo. When we blocked the initial proteolytic processing by a mutagenic approach, the differential sorting and secretion of these peptides were prevented. In summary, these data show that proTRH-derived peptides are differentially sorted within the secretory pathway, and that the initial cleavage in the TGN is key to this process. This could be a common mechanism used by neuroendocrine cells to independently regulate the secretion of different bioactive peptides derived from the same gene product.

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CD20 homo-oligomers physically associate with the B cell antigen receptor; dissociation upon receptor engagement and recruitment of phosphoproteins and calmodulin binding proteins.

Publication Date: 2008 May 12 PMID: 18474602
Authors: Polyak, M. J. - Li, H. - Shariat, N. - Deans, J. P.
Journal: J Biol Chem

B cell antigen receptor (BCR) signaling initiates sustained cellular calcium influx necessary for the development, differentiation and activation of B-lymphocytes. CD20 is a B cell-restricted tetraspanning protein organized in the plasma membrane as multimeric molecular complexes involved in BCR-activated calcium entry. Using co-precipitation of native CD20 with tagged or truncated forms of the molecule, we provide here direct evidence of CD20 homo-oligomerization into tetramers. Additionally, the function of CD20 was explored by examining its association with surface-labeled and intracellular proteins before and after BCR signaling. Two major surface-labeled proteins that co-precipitated with CD20 were identified as the heavy and light chains of cell surface IgM, the antigen-binding components of the BCR. After activation, BCR-CD20 complexes dissociated and phosphoproteins and calmodulin-binding proteins were transiently recruited to CD20. These data provide new evidence of CD20's involvement in signaling downstream of the BCR, and, together with CD20's previously described involvement in calcium influx, the first evidence of physical coupling of the BCR to a calcium entry pathway.

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Basal ubiquitin-independent internalization of interferon alpha receptor is prevented by Tyk2-mediated masking of a linear endocytic motif.

Publication Date: 2008 May 12 PMID: 18474601
Authors: Kumar, K. G. - Varghese, B. - Banerjee, A. - Baker, D. P. - Constantinescu, S. N. - Pellegrini, S. - Fuchs, S. Y.
Journal: J Biol Chem

Linear endocytic motifs of signaling receptors as well as their ubiquitination determine the rate of ligand-induced endocytosis that mediates downregulation of these receptors and restricts the magnitude and duration of their respective signal transduction pathways. We previously hypothesized that, in the absence of its cognate ligand, type I interferon (IFN), the IFNAR1 receptor chain is protected from basal endocytosis by a hypothetical masking complex that prevents the Tyr-based endocytic motif within IFNAR1 from interacting with components of the adaptin protein complex 2 (AP2). Here we identify a member of the Janus kinase (Jak) family, Tyk2, as a component of such a masking complex. In the absence of ligand or of receptor chain ubiquitination, binding of Janus kinase Tyk2 within the proximity of the Tyr-based linear motif of IFNAR1 is required to prevent IFNAR1 internalization and to maintain its cell surface expression. Furthermore, interaction experiments revealed that Tyk2 physically shields this Tyr-based motif from the recognition by the AP50 sub-unit of AP2. These data delineate a long sought ligand- and ubiquitin-independent mechanism by which Tyk2 contributes to both the regulation of total IFNAR1 levels as well as of the and the cell surface density of this receptor chain.

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Characterization of the human alpha 1beta 1 soluble guanylyl cyclase promoter: Key role for NFkappa B(p50) and CCAAT binding factors in regulating expression of the nitric oxide receptor.

Publication Date: 2008 May 12 PMID: 18474600
Authors: Marro, M. L. - Peiro, C. - Panayiotou, C. M. - Baliga, R. S. - Meurer, S. - Schmidt, H. H. - Hobbs, A. J.
Journal: J Biol Chem

Soluble guanylyl cyclase (sGC) is the principal receptor for nitric oxide (NO) and plays a ubiquitous role in regulating cellular function. This is exemplified in the cardiovascular system where sGC governs smooth muscle tone and growth, vascular permeability, leukocyte flux and platelet aggregation. As a consequence, aberrant NO-sGC signalling has been linked to diseases including hypertension, atherosclerosis and stroke. Despite these key (patho)physiological roles, little is known about the expressional regulation of sGC. To address this deficit, we have characterised the promoter activity of human a1 and ss1 sGC genes in a cell-type relevant to cardiovascular (patho)physiology, primary human aortic smooth muscle cells. Luciferase reporter constructs revealed that the 0.3kb and 0.5kb regions upstream of the transcription start sites were optimal for a1 and ss1 sGC promoter activity, respectively. Deletion of consensus sites for c-Myb, GAGA, NFAT, NFB(p50) and CCAAT-binding factor(s) (CCAAT-BF) revealed that these are the principal transcription factors (TFs) regulating basal sGC expression. In addition, under pro-inflammatory conditions, the effects of the strongest a1 and ss1 sGC repressors were enhanced and enzyme expression and activity reduced; in particular, NFB(p50) is pivotal in regulating enzyme expression under such conditions. NO itself also elicited a cGMP-independent negative-feedback effect on sGC promoter activity that is mediated, in part, via CCAAT-BF activity. In sum, these data provide a systematic characterisation of the promoter activity of human sGC a1 and ss1 subunits and identify key TFs that govern subunit expression under basal and pro-inflammatory (i.e. atherogenic) conditions and in the presence of ligand NO.

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A novel mechanism for human K2P2.1 channel gating: facilitation of C-type gating by protonation of extra cellular histidine residues.

Publication Date: 2008 May 12 PMID: 18474599
Authors: Cohen, A. - Ben-Abu, Y. - Hen, S. - Zilberberg, N.
Journal: J Biol Chem

The mammalian K2P2.1 potassium channel (TREK-1, KCNK2) is highly expressed in excitable tissues, where it plays a key role in the cellular mechanisms of neuroprotection, anaesthesia, pain perception and depression. Here, we report that external acidification, within the physiological range, strongly inhibits the human K2P2.1 channel by inducing 'C-type' closure. We have identified two histidine residues (i.e., H87 and H141), located in the first external loop of the channel, which govern the response of the channel to external pH. We demonstrate that these residues are within physical proximity to glutamate 84, homologous to Shaker E418, KcsA E51 and KCNK0 E28 residues, all previously argued to stabilize the outer pore gate in the open conformation by forming hydrogen bonds with pore-adjacent residues. We thus propose a novel mechanism for pH sensing in which protonation of H141 and H87 generates a local positive charge that serves to draw E84 away from its natural interactions, facilitating the collapse of the selectivity filter region. In accordance with this proposed mechanism, low pH modified K2P2.1 selectivity towards potassium. Moreover, the proton-mediated effect was inhibited by external potassium ions and was enhanced by a mutation (S164Y), known to accelerate C-type gating. Furthermore, proton-induced current inhibition was more pronounced at negative potentials. Thus, voltage-dependent C-type gating acceleration by protons represents a novel mechanism for K2P2.1 outward rectification.

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RGR-opsin mediates light-dependent translocation of all-trans-retinyl esters for synthesis of visual chromophore in retinal pigment epithelial cells.

Publication Date: 2008 May 12 PMID: 18474598
Authors: Radu, R. A. - Hu, J. - Peng, J. - Bok, D. - Mata, N. L. - Travis, G. H.
Journal: J Biol Chem

Visual perception begins with the absorption of a photon by an opsin pigment, inducing isomerization of its 11-cis-retinaldehyde chromophore. After a brief period of activation, the resulting all-trans-retinaldehyde dissociates from the opsin apo-protein rendering it insensitive to light. Restoring light sensitivity to apo-opsin requires thermal re-isomerization of all-trans-retinaldehyde to 11-cis-retinaldehyde via an enzyme pathway called the visual cycle in retinal pigment epithelial (RPE) cells. Vertebrates can see over a 108-fold range of background illumination. This implies that the visual cycle can regenerate visual chromophore over a similarly broad range. However, nothing is known about how the visual cycle is regulated. Here we show that RPE cells, functionally or physically separated from photoreceptors, respond to light by mobilizing all-trans-retinyl esters. These retinyl esters are substrates for the retinoid isomerase and hence critical for regenerating visual chromophore. We show in knockout mice and by RNA-interference in human RPE cells, that this mobilization is mediated by a protein called 'RPE-retinal G protein receptor' (RGR) opsin. These data establish that RPE cells are intrinsically sensitive to light. Finally, we show that in the dark, RGR-opsin inhibits lecithin:retinol acyl-transferase and all-trans-retinyl-ester hydrolase in vitro, and that this inhibition is released upon exposure to light. The results of this study suggest that RGR-opsin mediates light-dependent translocation of all-trans-retinyl esters from a storage pool in lipid droplets to an 'isomerase pool' in membranes of the endoplasmic reticulum. This translocation permits insoluble all-trans-retinyl esters to be utilized as substrate for the synthesis of new visual chromophore.

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Hydrogen peroxide prolongs nuclear localisation of NF-kappa B in activated cells by suppressing negative regulatory mechanisms.

Publication Date: 2008 May 12 PMID: 18474597
Authors: Enesa, K. - Ito, K. - Luong, L. A. - Thorbjornsen, I. - Phua, C. - To, Y. - Dean, J. - Haskard, D. O. - Boyle, J. - Adcock, I. - Evans, P. C.
Journal: J Biol Chem

NF-kappaB transcription factors induce pro-inflammatory molecules (e.g. IL-8) in response to cytokines (e.g. TNFalpha, IL-1beta) or other stimuli. In the basal state, they are sequestered in the cytoplasm by inhibitory IkappaB proteins. Pro-inflammatory signalling triggers polyubiquitination of intermediaries (e.g. RIP1) which activate IkappaB kinases which trigger Ser phosphorylation and degradation of IkappaBalpha, thereby promoting nuclear translocation of NF-kappaB. A negative feedback loop exists whereby NF-kappaB drives re-synthesis of IkappaBalpha which promotes export of NF-kappaB from the nucleus to the cytoplasm. This process relies on Cezanne, a deubiquitinating cysteine protease that stabilizes re-synthesised IkappaBalpha by removing polyubiquitin from modified intermediaries. H(2)O(2) is generated during inflammation. Here we examined the effects of H(2)O(2) on NF-kappaB dynamics and pro-inflammatory activation in cultured cells co-stimulated with TNFalpha or IL-1beta. Quantitative RT-PCR and ELISA revealed that H(2)O(2) enhanced the induction of IL-8 by TNFalpha or IL-1beta. We demonstrated using assays of NF-kappaB nuclear localisation and by imaging of live cells expressing a fluorescent form of NF-kappaB that H(2)O(2) prolonged NF-kappaB nuclear localisation in cells co-stimulated with TNFalpha or IL-1beta by suppressing its export from the nucleus. We provide evidence that H(2)O(2) suppresses NF-kappaB export by prolonging polyubiquitination of signalling intermediaries which promotes Ser phosphorylation and destabilisation of newly synthesised IkappaBalpha proteins. Finally, we observed that the catalytic activity of Cezanne and its ability to suppress RIP1 polyubiquitination and NF-kappaB transcriptional activity were inhibited by H(2)O(2). We conclude that H(2)O(2) prolongs NF-kappaB activation in co-stimulated cells by suppressing the negative regulatory functions of Cezanne and IkappaBalpha.

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Unique small molecule entry inhibitors of hemorrhagic fever arena viruses.

Publication Date: 2008 May 12 PMID: 18474596
Authors: Lee, A. M. - Rojek, J. M. - Spiropoulou, C. F. - Gundersen, A. T. - Jin, W. - Shaginian, A. - York, J. - Nunberg, J. H. - Boger, D. L. - Oldstone, M. B. - Kunz, S.
Journal: J Biol Chem

Viral hemorrhagic fevers (VHF) caused by the arenaviruses Lassa virus (LASV) in Africa and Machupo (MACV), Guanarito (GTOV), Junin (JUNV), and Sabia virus (SABV) in South America are among the most devastating emerging human diseases with fatality rates of 15 to 35% and a limited antiviral therapeutic repertoire available. Here we used high-throughput screening of synthetic combinatorial small molecule libraries to identify inhibitors of arenavirus infection using pseudotyped virion particles bearing the glycoproteins (GPs) of highly pathogenic arenaviruses. Our screening efforts resulted in the discovery of a series of novel small molecule inhibitors of viral entry that are highly active against both Old World and New World hemorrhagic arenaviruses. We observed potent inhibition of infection of human and primate cells with live hemorrhagic arenaviruses (IC50 = 500-800 nM). Investigations of the mechanism of action revealed that the candidate compounds efficiently block pH-dependent fusion by the arenavirus GPs (IC50 of 200-350 nM). While our lead compounds were potent against phylogenetically distant arenaviruses, they did not show activity against other enveloped viruses with class I viral fusion proteins, indicating specificity for arenavirus GP-mediated membrane fusion.

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Structural determinants of Ca2+ permeability and conduction in the human 5-HT3A receptor.

Publication Date: 2008 May 12 PMID: 18474595
Authors: Livesey, M. R. - Cooper, M. A. - Deeb, T. Z. - Carland, J. E. - Kozuska, J. - Hales, T. G. - Lambert, J. J. - Peters, J. A.
Journal: J Biol Chem

Cation selective cysteine (Cys)-loop transmitter-gated ion channels provide an important pathway for Ca2+ entry into neurones. We examined the influence on Ca2+ permeation of amino acids located at intra- and extra-cellular ends of the conduction pathway of the human 5-hydroxytryptamine-type 3A (5-HT3A) receptor. Mutation of cytoplasmic arginine residues 432, 436 and 440 to glutamine, aspartate and alanine (the aligned residues of the human 5-HT3B subunit (yielding 5-HT3A(QDA)) increased PCa/PCs from 1.4 to 3.7. The effect was attributable to the removal of an electrostatic influence of the R436 residue. Despite its relatively high permeability to Ca2+, the single channel conductance of the 5-HT3A(QDA) receptor was depressed in a concentration-dependent and voltage-independent manner by extracellular Ca2+. A conserved aspartate, located towards the extracellular end of the conduction pathway, is known to influence ionic selectivity and contributes to the inhibitory effect of Ca2+ on macroscopic currents mediated by 5-HT3A receptors. We introduced a D293A mutation into the 5-HT3A(QDA) receptor (yielding the 5-HT3A(QDA D293A) construct) to determine whether the aspartate is required for the suppression of single channel conductance by Ca2+. The D293A mutation decreased PCa/PCs to 0.25, reduced inwardly directed single channel conductance from 41 to 30 pS, but did not prevent suppression of single channel conductance by Ca2+. The D293A mutation also reduced PCa/PCs when engineered into the wild-type 5-HT3A receptor. The data identify key residues in the cytoplasmic domain (R436) and extracellular vestibule (D293) that markedly influence PCa/PCs and additionally directly demonstrate a depression of single channel conductance by Ca2+.

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Apical loop-internal loop RNA pseudoknots: a new type of stimulator of -1 translational frameshifting in bacteria.

Publication Date: 2008 May 12 PMID: 18474594
Authors: Mazauric, M. H. - Licznar, P. - Prere, M. F. - Canal, I. - Fayet, O.
Journal: J Biol Chem

Nearly all members of a widespread family of bacterial transposable elements related to Insertion Sequence 3 (IS3), therefore called the IS3 family, very likely use programmed -1 ribosomal frameshifting to produce their transposase, a protein required for mobility. Comparative analysis of the potential frameshift signals in this family suggested that most of the IS from the IS51 group contain in their mRNA an elaborate pseudoknot that could act as a recoding stimulator. It results from a specific intra-molecular interaction between an apical loop and an internal loop from two stem-loop structures. Directed mutagenesis, chemical probing and gel mobility assays of the frameshift region of one element from the IS51 group, IS3411, provided clear evidences of the existence of the predicted structure. Modeling was used to generate a 3D molecular representation of the apical loop-internal loop complex. We could demonstrate that mutations affecting the stability of the structure reduce both frameshifting and transposition, thus establishing the biological importance of this new type of RNA structure for the control of transposition level.

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Plasmin potentiates synaptic NMDA receptor function in hippocampal neurons through activation of PAR1.

Publication Date: 2008 May 12 PMID: 18474593
Authors: Mannaioni, G. - Orr, A. G. - Hamill, C. E. - Yuan, H. - Pedone, K. H. - McCoy, K. L. - Palmini, R. B. - Junge, C. E. - Lee, C. J. - Yepes, M. - Hepler, J. R. - Traynelis, S. F.
Journal: J Biol Chem

Protease-activated receptor-1 (PAR1) is activated by a number of serine proteases, including plasmin. Both PAR1 and plasminogen, the precursor of plasmin, are expressed in the central nervous system. In this study, we examined the effects of plasmin in astrocyte and neuronal cultures as well as in hippocampal slices. We find that plasmin evokes an increase in both phosphoinositide hydrolysis (EC50 64 nM) and Fura-2/AM fluorescence (195 +/- 6.7 % above baseline, EC50 65 nM) in cortical cultured murine astrocytes. Plasmin also activates extracellular signal-regulated kinase (ERK1/2) within cultured astrocytes. The plasmin-induced rise in intracellular Ca2+ concentration ([Ca2+]i), as well as the increase in phospho-ERK1/2 levels, was diminished in PAR1-/- astrocytes and was blocked by 1 microM BMS-200261, a selective PAR1 antagonist. However, plasmin had no detectable effect on ERK1/2 or [Ca2+]i signaling in primary cultured hippocampal neurons or in CA1 pyramidal cells in hippocampal slices. Plasmin (100-200 nM) application potentiated the NMDA receptor-dependent component of miniature excitatory postsynaptic currents recorded from CA1 pyramidal neurons, but had no effect on AMPA or GABA receptor-mediated synaptic currents. Plasmin also increased NMDA-induced whole cell receptor currents recorded from CA1 pyramidal cells (2.5 +/- 0.3 fold potentiation over control). This effect was blocked by BMS-200261 (1 microM; 1.02 +/- 0.09 fold potentiation over control). These data suggest that plasmin may serve as an endogenous PAR1 activator that can increase [Ca2+]i in astrocytes and potentiate NMDA receptor synaptic currents in CA1 pyramidal neurons.

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Reactive nitrogen species is required for the activation of the AMP-activated protein kinase by statin in vivo.

Publication Date: 2008 May 12 PMID: 18474592
Authors: Choi, H. C. - Song, P. - Xie, Z. - Wu, Y. - Xu, J. - Zhang, M. - Dong, Y. - Wang, S. - Lau, K. - Zou, M. H.
Journal: J Biol Chem

The AMP-activated protein kinase (AMPK) is reported to mediate the beneficial effects of statin on the vascular functions, but the biochemical mechanisms are incompletely understood. The aim of the study was to determine how statin activates AMPK. Exposure of confluent bovine aortic endothelial cells (BAEC) to simvastatin (statin) dose-dependently increased phosphorylation of AMPK at Thr172 and activities of AMPK, which was in parallel with increased detection of both LKB1 phosphorylation at Ser428 and LKB1 nuclear export. Furthermore, statin treatment was shown to increase protein kinase C (PKC)-zeta activity and PKC-zeta phosphorylation at Thr410/403. Consistently, inhibition of PKC-zeta either by pharmacological or genetic manipulations abolished statin-enhanced LKB1 phosphorylation at Ser428 but blocked LKB1 nucleus export, and prevented the subsequent activation of AMPK. Similarly, in vivo transfection of PKC-zeta-specific siRNA in C57B6/6 mice significantly attenuated statin-enhanced phosphorylation of AMPK-Thr172, ACC-Ser79, and LKB1-Ser428. In addition, Statin significantly increased reactive oxygen species whereas preincubation of mito-Tempol, a SOD mimetic, abolished statin-enhanced phosphorylation of both AMPK-Thr172 and ACC-Ser79. Finally, in vivo administration of statin increased 3-nitrotyrosine and the phosphorylation of AMPK and ACC in C57BL/6 mice but not in mice deficient of endothelial nitric oxide synthase. Taken together, our data suggest that AMPK activation by statin is peroxynitrite-mediated but PKC-zeta-dependent.

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Roles of the glycogen-binding domain and Snf4 in glucose inhibition of SNF1 protein kinase.

Publication Date: 2008 May 12 PMID: 18474591
Authors: Momcilovic, M. - Iram, S. H. - Liu, Y. - Carlson, M.
Journal: J Biol Chem

The SNF1/AMP-activated protein kinase (AMPK) family is required for adaptation to metabolic stress and energy homeostasis. The g subunit of AMPK binds AMP and ATP, and mutations that affect binding cause human disease. We have here addressed the role of the Snf4 gamma subunit in regulating SNF1 protein kinase in response to glucose availability in Saccharomyces cerevisiae. Previous studies of mutant cells lacking Snf4 suggested that Snf4 counteracts autoinhibition by the C-terminal sequence of the Snf1 catalytic subunit but is dispensable for glucose regulation, and AMP does not activate SNF1 in vitro. We first introduced substitutions at sites that, in AMPK, contribute to nucleotide binding and regulation. Mutations at several sites relieved glucose inhibition of SNF1, as judged by catalytic activity, phosphorylation of the activation-loop Thr-210, and growth assays, although analogs of the severe human mutations R531G/Q had little effect. We further showed that alterations of Snf4 residues that interact with the glycogen-binding domain (GBD) of the beta subunit strongly relieved glucose inhibition. Finally, substitutions in the GBD of the Gal83 beta subunit that are predicted to disrupt interactions with Snf4, and also complete deletion of the GBD, similarly relieved glucose inhibition of SNF1. Analysis of mutant cells lacking glycogen synthase showed that regulation of SNF1 is normal in the absence of glycogen. These findings reveal novel roles for Snf4 and the GBD in regulation of SNF1.

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Kex1 protease is involved in yeast cell death induced by defective N-glycosylation, acetic acid and during chronological aging.

Publication Date: 2008 May 12 PMID: 18474590
Authors: Hauptmann, P. - Lehle, L.
Journal: J Biol Chem

N-glycosylation in the endoplasmic reticulum is an essential protein modification and highly conserved in evolution from yeast to man. The key step of this pathway is the transfer of the lipid-linked core oligosaccharide to the nascent polypeptide chain, catalyzed by the oligosaccharyltransferase complex (OST). Temperature-sensitive OST mutants of S. cerevisiae at the restrictive temperature, such as wbp1-1, as well as wild-type cells in the presence of the N-glycosylation inhibitor tunicamycin display typical apoptotic phenotypes like nuclear condensation, DNA fragmentation, phosphatidylserine translo-cation, caspase-like activity as well as ROS accumulation. As deletion of the yeast metacaspase YCA1 did not abrogate this death pathway, we postulated a different proteolytic process to be responsible for. Here, we show that Kex1 protease is involved in the programmed cell death caused by defective N-glycosylation. Its disruption decreases caspase-like activity, production of reactive oxygen species (ROS), as well as fragmentation of mitochondria and, conversely, improves growth and survival of cells. Moreover, we demonstrate that Kex1 contributes also to the active cell death program induced by acetic acid stress or during chronological aging, suggesting that Kex1 plays a more general role in cellular suicide of yeast.

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Increased resistance of complex I mutants to phytosphingosine-induced programmed cell death.

Publication Date: 2008 May 12 PMID: 18474589
Authors: Castro, A. - Lemos, C. - Falcao, A. - Glass, N. L. - Videira, A.
Journal: J Biol Chem

We have studied the effects of phytosphingosine (PHS) on cells of the filamentous fungus Neurospora crassa. Highly reduced viability, impairment of asexual spore germination, DNA condensation and fragmentation and production of reactive oxygen species (ROS) were observed in conidia treated with the drug, suggesting that PHS induces an apoptosis-like death in this fungus. Interestingly, we found that complex I mutants are more resistant to PHS treatment than the wild type strain. This effect appears to be specific because it was not observed in mutants defective in other components of the mitochondrial respiratory chain, pointing to a particular involvement of complex I in cell death. The response of the mutant strains to PHS correlated with their response to hydrogen peroxide. The fact that complex I mutants generate less ROS than the wild type strain when exposed to PHS likely explains the PHS-resistant phenotype. Compared to the wild type strain, we also found that a strain containing a deletion in the gene encoding an AIF (apoptosis-inducing factor)-like protein is more resistant to PHS and H2O2. In contrast, a strain containing a deletion in a gene encoding an AMID (AIF-homologous mitochondrion-associated inducer of death)-like polypeptide is more sensitive to both drugs. These results indicate that N. crassa has the potential to be a model organism to investigate the molecular basis of programmed cell death in eukaryotic species.

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Myosin regulatory light chain phosphorylation attenuates cardiac hypertrophy.

Publication Date: 2008 May 12 PMID: 18474588
Authors: Huang, J. - Shelton, J. M. - Richardson, J. A. - Kamm, K. E. - Stull, J. T.
Journal: J Biol Chem

Hyperphosphorylation of myosin regulatory light chain (RLC) in cardiac muscle is proposed to cause compensatory hypertrophy. We therefore investigated potential mechanisms in genetically modified mice. Transgenic (TG) mice were generated to overexpress Ca2+/calmodulin-dependent myosin light chain kinase specifically in cardiomyocytes. Phosphorylation of sarcomeric cardiac RLC (cRLC) and cytoplasmic nonmuscle RLC (nRLC) increased markedly in hearts from TG mice compared to hearts from WT mice. Quantitative measures of RLC phosphorylation revealed no spatial gradients. No significant hypertrophy or structural abnormalities were observed up to 6 months of age in hearts of TG mice compared to WT animals. Hearts and cardiomyocytes from WT animals subjected to voluntary running exercise and isoproterenol treatment showed hypertrophic cardiac responses but the responses for TG mice were attenuated. Additional biochemical measurements indicated that overexpression of the Ca2+/calmodulin-binding kinase did not perturb other Ca2+/calmodulin-dependent processes involving Ca2+/calmodulin-dependent protein kinase II or the protein phosphatase, calcineurin. Thus, increased myosin RLC phosphorylation per se does not cause cardiac hypertrophy and probably inhibits physiological and pathophysiological hypertrophy by contributing to enhanced contractile performance and efficiency.

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Structural and biochemical basis for the binding selectivity of PPARgamma to PGC-1alpha.

Publication Date: 2008 May 9 PMID: 18469005
Authors: Li, Y. - Kovach, A. - Suino-Powell, K. - Martynowski, D. - Xu, H. E.
Journal: J Biol Chem

The functional interaction between the peroxisome proliferator-activated receptor gamma (PPARgamma) and its coactivator PGC-1alpha is crucial for the normal physiology of PPARgamma and its pharmacological response to antidiabetic treatment with rosiglitazone. Here we report the crystal structure of the PPARgamma ligand binding domain (LBD) bound to rosiglitazone and to a large PGC-1alpha fragment that contains two LXXLL-related motifs. The structure reveals critical contacts mediated through the first LXXLL motif of PGC-1alpha and the PPARgamma coactivator binding site. Through a combination of biochemical and structural studies, we demonstrate that the first LXXLL motif of is the most potent PPARgamma binding motif among all nuclear receptor coactivator motifs tested, and only this motif of the two LXXLL-related motifs in PGC-1alpha is capable of binding to PPARgamma. Our studies reveal that the strong interaction of PGC-1alpha and PPARgamma is mediated through both hydrophobic and specific polar interactions. Mutations within the context of the full-length PGC-1alpha indicate that the first PGC-1alpha motif is necessary and sufficient for PGC-1alpha to coactivate PPARgamma in the presence or absence of rosiglitazone. These results provide a molecular basis for specific recruitment and functional interplay between PPARgamma and PGC-1alpha in glucose homeostasis and adipocyte differentiation.

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Cysteine cathepsins trigger caspase-dependent cell death through cleavage of Bid and antiapoptotic Bcl-2 homologues.

Publication Date: 2008 May 9 PMID: 18469004
Authors: Droga Mazovec, G. - Bojic, L. - Petelin, A. - Ivanova, S. - Romih, R. - Repnik, U. - Salvesen, G. S. - Stoka, V. - Turk, V. - Turk, B.
Journal: J Biol Chem

As a model for defining the role of lysosomal cathepsins in apoptosis, we characterized the action of the lysosomotropic agent LeuLeuOMe using distinct cellular models. LeuLeuOMe induces lysosomal membrane permeabilization, resulting in release of lysosomal cathepsins that cleave the proapoptotic Bcl-2 family member Bid and degrade the antiapoptotic members Bcl-2, Bcl-xL or Mcl-1. The papain-like cysteine protease inhibitor E-64d largely prevented apoptosis, Bid cleavage and Bcl-2/Bcl-xL/Mcl-1 degradation. The pancaspase inhibitor z-VAD-fmk failed to prevent Bid cleavage and degradation of anti-apoptotic Bcl-2 homologues, but substantially decreased cell death suggesting that cathepsin-mediated apoptosis in these cellular models mostly follows a caspase-dependent pathway. Moreover, in vitro experiments showed that one or more of the cysteine cathepsins B, L, S, K, and H could cleave Bcl-2, Bcl-xL, Mcl-1, Bak and BimEL, whereas no Bax cleavage was observed. On the basis of inhibitor studies we demonstrate that lysosomal disruption triggered by LeuLeuOMe ocurrs prior to mitochondrial damage. We propose that degradation of anti-apoptotic Bcl-2 family members by lysosomal cathepsins synergizes with cathepsin-mediated activation of Bid to trigger a mitochondrial pathway to apoptosis. Moreover, XIAP was also found to be a target of cysteine cathepsins, suggesting that cathepsins can mediate caspase-dependent apoptosis also downstream of mitochondria.

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Regulation of plasminogen activator inhibitor-1 expression by tumor suppressor protein p53.

Publication Date: 2008 May 9 PMID: 18469003
Authors: Shetty, S. - Shetty, P. - Idell, S. - Velusamy, T. - Bhandary, Y. P. - Shetty, R. S.
Journal: J Biol Chem

H1299 lung carcinoma cells lacking p53 (p53-/-) express minimal amounts of plasminogen activator inhibitor-1 (PAI-1) protein as well as mRNA. p53-/- cells express highly unstable PAI-1 mRNA. Transfection of p53 in p53-/- cells enhanced PAI-1 expression and stabilized PAI-1 mRNA. On the contrary, inhibition of p53 expression by RNA silencing (SiRNA) in non-malignant human lung epithelial (Beas2B) cells decreased basal as well as uPA-induced PAI-1 expression due to accelerated degradation of PAI-1 mRNA. Purified p53 protein specifically binds to the PAI-1 mRNA 3'UTR and endogenous PAI-1 mRNA forms an immune-complex with p53. Treatment of purified p53 protein with anti-p53 antibody abolished p53 binding to the 3'UTR of PAI-1 mRNA. The p53 binding region maps to a 70 nt PAI-1 mRNA 3'UTR sequence and insertion of the p53 binding sequence into beta-globin mRNA destabilized the chimeric transcript. Deletion experiments indicate that the C-terminal region (amino acid residues 296-393) of p53 protein interacts with PAI-1 mRNA. These observations demonstrate a novel role for p53 as an mRNA binding protein that regulates increased PAI-1 expression and stabilization of PAI-1 mRNA in human lung epithelial and carcinoma cells.

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Phosphorylation of bad at Thr201 by JNK1 promotes glycolysis through activation of phosphofructokinase-1.

Publication Date: 2008 May 9 PMID: 18469002
Authors: Deng, H. - Yu, F. - Chen, J. - Zhao, Y. - Xiang, J. - Lin, A.
Journal: J Biol Chem

The mitogen-activated protein kinase JNK1 suppresses interleukin-3 (IL-3) withdrawal-induced cell death through phosphorylation of the BH3-only pro-apoptotic Bcl-2 family protein Bad at threonine 201 (Thr201). Yet, it is unknown whether JNK1 regulates glycolysis, an important metabolic process that is involved in cell survival and if so, whether the regulation depends on Thr201 phosphorylation of Bad. Here we report that phosphorylation of Bad by JNK1 is required for glycolysis through activation of phosphofructokinase-1 (PFK-1), one of the key enzymes that catalyze glycolysis. Genetic disruption of Jnk1 alleles or silencing of Jnk1 by siRNA abrogates glycolysis induced by growth/survival factors like serum or IL-3. Proteomic analysis identifies PFK-1 as a novel Bad-associated protein. Although the interaction between PFK-1 and Bad is independent of JNK1, Thr201 phosphorylation of Bad by JNK1 is required for PFK-1 activation. Thus, our results provide a novel molecular mechanism by which JNK1 promotes glycolysis for cell survival.

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Mutagenesis of the dengue virus type 2 NS5 methyltransferase domain.

Publication Date: 2008 May 9 PMID: 18469001
Authors: Kroschewski, H. - Lim, S. P. - Butcher, R. E. - Yap, T. L. - Lescar, J. - Wright, P. J. - Vasudevan, S. G. - Davidson, A. D.
Journal: J Biol Chem

The flavivirus NS5 protein possesses both (guanine-N7)-methyltransferase and nucleoside-2 -O methyltransferase activities required for sequential methylation of the cap structure present at the 5' end of the flavivirus RNA genome. Seventeen mutations were introduced into the Dengue virus type 2 NS5 methyltransferase domain, targeting amino acids either predicted to be directly involved in S-adenosyl-L-methionine binding or important for NS5 conformation and/or charged interactions. The effects of the mutations on i) (guanine-N7)-methyltransferase and nucleoside-2 -O methyltransferase activities using biochemical assays based on a bacterially expressed NS5 methyltransferase domain and ii) viral replication using a Dengue virus type 2 infectious cDNA clone were examined. Clustered mutations, targeting the S-adenosyl-L-methionine binding pocket, or an active site residue, abolished both methyltransferase activities and viral replication demonstrating that both methyltransferase activities utilise a single S-adenosyl-L-methionine binding pocket. Substitutions to single amino acids binding S-adenosyl-L-methionine decreased both methyltransferase activities to a range of extents. However, viruses that replicated at wild type levels could be recovered with mutations that reduced both activities by >75% suggesting that only a threshold level of methyltransferase activity was required for virus replication in vivo. Mutation of residues outside of regions directly involved in S-adenosyl-L-methionine binding or catalysis also affected methyltransferase activity and virus replication. The recovery of viruses containing compensatory second site mutations in the NS5 and NS3 proteins identified regions of the methyltransferase domain important for overall stability of the protein or likely to play a role in virus replication distinct from that of cap methylation.

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Cellular functions of human RPA1: Multiple roles of domains in replication, repair, and checkpoints.

Publication Date: 2008 May 9 PMID: 18469000
Authors: Haring, S. J. - Mason, A. C. - Binz, S. K. - Wold, M. S.
Journal: J Biol Chem

In eukaryotes, the single strand DNA (ssDNA) binding protein, Replication Protein A (RPA), is essential for DNA replication, repair, and recombination. RPA is composed of three subunits: RPA1, RPA2, and RPA3. The RPA1 subunit contains four structurally related domains and is responsible for high affinity ssDNA binding. This study uses a depletion/replacement strategy in human cells to reveal contributions of each domain to RPA cellular functions. Mutations that substantially decrease ssDNA binding activity do not necessarily disrupt cellular RPA function. Conversely, mutations that only slightly affect ssDNA binding can dramatically affect cellular function. The N terminus of RPA1 is not necessary for DNA replication in the cell; however, this region is important for the cellular response to DNA damage. Highly conserved aromatic residues in the high affinity ssDNA binding domains are essential for DNA repair and cell cycle progression. Our findings suggest that as long as a threshold of RPA ssDNA binding activity is met, DNA replication can occur, and that an RPA activity separate from ssDNA binding is essential for function in DNA repair.

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Regulation of FE65 nuclear translocation and function by APP in osmotically stressed cells.

Publication Date: 2008 May 9 PMID: 18468999
Authors: Nakaya, T. - Kawai, T. - Suzuki, T.
Journal: J Biol Chem

FE65, a neural adaptor protein, interacts with amyloid ss-protein precursor (APP) and is known to regulate amyloid-ss generation from APP. FE65 also associates with nuclear proteins; however, its physiological function in the nucleus remains unclear. A fixed population of cytoplasmic FE65 is tethered to membranes by binding APP. This membrane-tethered FE65 is liberated from membranes by APP phosphorylation, which is facilitated by a stress-activated protein kinase in sorbitol-treated cells. Here, we show that liberated FE65, which is distinct from "virgin" FE65 in the cytoplasm, translocates into the nucleus and accumulates in the nuclear matrix forming a patched structure. Targeting of FE65 into the nuclear matrix was suppressed by the APP intracellular domain fragment, which is generated by consecutive cleavages of APP. Thus, nuclear translocation of FE65 is under the regulation of APP. In the nucleus, FE65 induced H2AX, which plays an important role in DNA repair as a cellular response by stress-damaged cells. These observations suggest that APP-regulated FE65 plays an important role in the early stress response of cells, and that FE65 deregulated from APP induces apoptosis.

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Blood pressure is regulated by an alpha 1D-adrenergic receptor/dystrophin signalosome.

Publication Date: 2008 May 9 PMID: 18468998
Authors: Lyssand, J. S. - Defino, M. C. - Tang, X. B. - Hertz, A. L. - Feller, D. B. - Wacker, J. L. - Adams, M. E. - Hague, C.
Journal: J Biol Chem

Hypertension is a cardiovascular disease associated with increased plasma catecholamines, overactivation of the sympathetic nervous system, and increased vascular tone and total peripheral resistance. A key regulator of sympathetic nervous system function is the alpha(1D)-adrenergic receptor (AR), which belongs to the adrenergic family of G-protein coupled receptors (GPCRs). Endogenous catecholamines norepinephrine and epinephrine activate alpha(1D)-ARs on vascular smooth muscle to stimulate vasoconstriction, which increases total peripheral resistance and mean arterial pressure. Indeed, alpha(1D)-AR KO mice display a hypotensive phenotype and are resistant salt-induced hypertension. Unfortunately, little information exists about how this important GPCR functions, because of an inability to obtain functional expression in vitro. Here, we identified the dystrophin proteins, syntrophin, dystrobrevin and utrophin, as essential GPCR interacting proteins (GIPs) for alpha(1D)-ARs. We found dystrophins complex with alpha(1D)-AR both in vitro and in vivo to ensure proper functional expression. More importantly, we demonstrate knock-out of multiple syntrophin isoforms results in the complete loss of alpha(1D)-AR function in mouse aortic smooth muscle cells and abrogation of alpha(1D)-AR mediated increases in blood pressure. Our findings demonstrate that syntrophin and utrophin associate with alpha(1D)-ARs to create a functional signalosome, which is essential for alpha(1D)-AR regulation of vascular tone and blood pressure.

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Disulfide bond structure and domain organization of yeast beta (1,3)-glucanosyltransferases involved in cell wall biogenesis.

Publication Date: 2008 May 9 PMID: 18468997
Authors: Popolo, L. - Ragni, E. - Carotti, C. - Palomares, O. - Aardema, R. - Back, J. W. - Dekker, H. - de Koning, L. J. - de Jong, L. - de Koster, C. G.
Journal: J Biol Chem

The Gel/Gas/Phr family of fungal beta(1,3)-glucanosyltransferases plays an important role in cell wall biogenesis by processing the main component, beta (1,3)-glucan. Two subfamilies are distinguished depending on the presence or absence of a C-terminal cysteine-rich domain, denoted "Cys-box". The N-terminal domain (NtD) contains the catalytic residues for transglycosidase activity and is separated from the Cys-box by a linker region. To obtain a better understanding of the structure and function of the Cys-box-containing subfamily we identified the disulfide bonds in Gas2p from Saccharomyces cerevisiae by an improved mass spectrometric methodology. We mapped two separate intra-domain clusters of three and four disulfide bridges. One of the bonds in the first cluster connects a central Cys residue of the NtD with a single conserved Cys residue in the linker. Site-directed mutagenesis of the Cys residue in the linker resulted in an endoplasmic reticulum-precursor that was not matured and underwent a gradual degradation. The relevant disulfide bond has a crucial role in folding as it may stabilize the NtD and facilitate its interaction with the C-terminal portion of a Gas protein. The four disulfide bonds in the Cys-box are arranged in a manner consistent with a partial structural resemblance with the plant X8 domain, an independent carbohydrate binding module which possesses only three disulfide bonds. Deletion of the Cys-box in Gas2 or Gas1 proteins led to the formation of an NtD devoid of any enzymatic activity. The results suggest that the Cys-box is required for proper folding of the NtD and/or substrate binding.

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Promotion of BACE1 mRNA alternative splicing reduces amyloid beta -peptide production.

Publication Date: 2008 May 8 PMID: 18468996
Authors: Mowrer, K. R. - Wolfe, M. S.
Journal: J Biol Chem

Production of the amyloid ss-peptide (Ass) via sequential proteolytic cleavage of the amyloid precursor protein by ss- and - secretases is strongly implicated in the pathogenesis of Alzheimer disease. The ss- secretase that executes the first cleavage event is a transmembrane aspartyl protease known as ss-site APP cleaving enzyme 1 (BACE1). BACE1 pre-mRNA is alternatively spliced through the use of alternative splice sites in exons 3 and 4, although the significance of these splicing events is unclear. Here, we quantitatively measure relative levels of BACE1 transcripts and identify a novel splice variant of BACE1. We find a subtle but significant difference in BACE1 splicing between brain and pancreas, indicating the cellular environment can affect BACE1 alternative splicing. Furthermore, we show that BACE1 proteins translated from alternatively spliced transcripts have dramatically reduced ss-secretase activity and promotion of BACE1 alternative splicing reduces Ass production. These findings illustrate the importance of BACE1 alternative splicing in affecting the level of Ass produced in cells and suggest that targeting regulation of BACE1 alternative splicing is a potential therapeutic strategy for lowering ss-secretase activity.

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N-glycans and the N-terminus of protein C inhibitor affect the cofactor-enhanced rates of thrombin inhibition.

Publication Date: 2008 May 8 PMID: 18467335
Authors: Sun, W. - Parry, S. - Panico, M. - Morris, H. R. - Kjellberg, M. - Engstrom, A. - Dell, A. - Schedin-Weiss, S.
Journal: J Biol Chem

Protein C inhibitor (PCI) is a serine protease inhibitor, displaying broad protease specificity, found in blood and other tissues. In blood, it is capable of inhibiting both procoagulant and anticoagulant proteases. Mechanisms that provide specificity to PCI remain largely unrevealed. In this study we have for the first time provided a full explanation for the marked size heterogeneity of blood-derived PCI and identified functional differences between naturally occurring PCI variants. The heterogeneity was caused by differences in N-glycan structures, N-glycosylation occupancy and the presence of a 6-N-cleaved form. Bi-, tri- and tetra-antennary complex N-glycans were identified. Fucose residues were identified both on the core GlcNAc and as parts of sialyl Lea/x epitopes. Moreover, a glycan with a composition that implied a di-sialyl antenna was observed. PCI was N-glycosylated at all three potential N-glycosylation sites, N230, N243 and N319, but a small fraction of PCI lacked the N-glycan at N243. The overall removal of N-glycans affected the maximal heparin- and thrombomodulin-enhanced rates of thrombin inhibition differently in different solution conditions. In contrast, the 6-N-region increased both the heparin- and the thrombomodulin-enhanced rates of thrombin inhibition at all conditions examined. These results thus demonstrate that the N-linked glycans and the N-terminal region of blood-derived PCI in different ways affect the cofactor-enhanced rates of thrombin inhibition and provide information on the mechanisms by which this may be achieved. The findings are medically important, in view of the documented association of PCI with atherosclerotic plaques and the promising effect of PCI on reducing hypercoagulability states.

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Juxtaposition of the steroid binding domain-like I and II regions constitutes a ligand binding site in the sigma-1 receptor.

Publication Date: 2008 May 7 PMID: 18467334
Authors: Pal, A. - Chu, U. B. - Ramachandran, S. - Grawoig, D. - Guo, L. W. - Hajipour, A. R. - Ruoho, A. E.
Journal: J Biol Chem

Sigma-1 receptors represent unique binding sites that are capable of interacting with a wide range of compounds to mediate different cellular events. The composition of the ligand binding site of this receptor is unclear as no NMR or crystal structures are available. Recent studies in our laboratory using radiolabeled photoreactive ligands suggested that the steroid binding domain-like I (SBDLI) (amino acids 91-109) and the steroid binding domain-like II (SBDLII) (amino acids 176-194) regions are involved in forming the ligand binding site(s) (1,2). In this report, we have further addressed this issue by utilizing our previously developed sulfhydryl-reactive, cleavable, radioiodinated photo-crosslinking reagent: methanesulfonothioic acid, S-[[4-(4-amino-3-[(125)I]iodobenzoyl) phenyl] methyl] ester ([(125)I]IABM) (3). This photoprobe was shown to derivatize the single cysteine residues as mixed disulfides at position 94 in the SBDLI region of the WT guinea pig sigma-1 receptor (94C) and at position 190 in the SBDLII region of a mutant guinea pig sigma-1 receptor (C94A, V190C), both in sigma-ligand (haloperidol or (+)-pentazocine) sensitive manner. Significantly, photo-crosslinking followed by Endo Lys C cleavage under reducing conditions, intramolecular radiolabel transfer from SBDLI to the SBDLII region in the WT receptor, and reversely, from SBDLII to the SBDLI region in the mutant receptor was observed. These data support a model in which the SBDLI and SBDLII regions are juxtaposed to form, at least in part, a ligand binding site of the sigma-1 receptor.

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Elevated levels of oncogenic protein kinase Pim-1 induce the p53 pathway in cultured cells and correlate with increased Mdm2 in mantle cell lymphoma.

Publication Date: 2008 May 8 PMID: 18467333
Authors: Hogan, C. - Hutchison, C. - Marcar, L. - Milne, D. - Saville, M. - Goodlad, J. - Kernohan, N. - Meek, D.
Journal: J Biol Chem

Mutation of the p53 gene is a common event during tumor pathogenesis. Other mechanisms, such as mdm2 amplification, provide alternative routes through which dysfunction of the p53 pathway is promoted. Here, we address the hypothesis that elevated expression of pim oncogenes might suppress p53 by regulating Mdm2. At a physiological level, we show that endogenous Pim-1 and Pim-2 interact with endogenous Mdm2. Additionally, the Pim kinases phosphorylate Mdm2 in vitro and in cultured cells at Ser166 and Ser186, two previously identified targets of other signalling pathways including Aktthe Akt protein kinase. Surprisingly, at high levels of Pim expression as would occur in tumors, active, but not inactive, Pim-1 or Pim-2 blocks the degradation of both p53 and Mdm2 in a manner that is independent of Mdm2 phosphorylation, and promotesleading to increased p53 levels and, proportionately, p53-dependent transactivation. Additionally, Pim-1 induces endogenous ARF, p53, Mdm2 and p21 in primary murine embryo fibroblasts and stimulates SA-ss-galactosidase levels, consistent with the induction of senescence. Immunohistochemical analysis of a cohort of 33 human mantle cell lymphomas (MCL) shows that elevated expression of Pim-1 occurs in 42% of cases, with elevated Pim-2 occurring in 9% of cases all of which also express Pim-1. Notably, elevated Pim-1 correlates with elevated Mdm2 in MCL with a p value of 0.003. Taken together, our data are consistent with the idea that Pim normally interacts with the p53 pathway but, when expressed at pathological levels, behaves as a classic dominant oncogene that stimulates a protective response through induction of the p53 pathway.

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Phosphorylation regulates tau interactions with SH3 domains of phosphatidylinositol-3-kinase, phospholipase cgamma 1, GRB2 and SRC-family kinases.

Publication Date: 2008 May 8 PMID: 18467332
Authors: Reynolds, C. H. - Garwood, C. J. - Wray, S. - Price, C. - Kellie, S. - Perera, T. - Zvelebil, M. - Yang, A. - Sheppard, P. W. - Varndell, I. M. - Hanger, D. P. - Anderton, B. H.
Journal: J Biol Chem

The microtubule-associated protein tau can associate with various other proteins in addition to tubulin, including the SH3 domains of Src family tyrosine kinases. Tau is well known to aggregate to form hyperphosphorylated filamentous deposits in several neurodegenerative diseases (tauopathies) including Alzheimer's disease. We now report that tau can bind to SH3 domains derived from the p85alpha subunit of phosphatidylinositol-3-kinase, phospholipase Cgamma1, and the N-terminal (but not the C-terminal) SH3 of Grb2, as well as to the kinases Fyn, cSrc and Fgr. However, the short inserts found in neuron-specific isoforms of Src prevented the binding of tau. The experimentally determined binding of tau peptides is well accounted for when modeled into the peptide-binding cleft in SH3 domain of Fyn. After phosphorylation in vitro or in transfected cells, tau showed reduced binding to SH3 domains; no binding was detected with hyperphosphorylated tau isolated from Alzheimer brain, but SH3 binding was restored by phosphatase treatment. Tau mutants with serines and threonines replaced by glutamate, to mimic phosphorylation, showed reduced SH3 binding. These results strongly suggest that tau has a potential role in cell signaling, in addition to its accepted role in cytoskeletal assembly, with regulation by phosphorylation that may be disrupted in the tauopathies, including Alzheimer's disease.

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Crystal structure of a prolactin receptor antagonist bound to the extracellular domain of the prolactin receptor.

Publication Date: 2008 May 8 PMID: 18467331
Authors: Svensson, L. A. - Bondensgaard, K. - Norskov-Lauritsen, L. - Christensen, L. - Becker, P. - Andersen, M. D. - Maltesen, M. J. - Rand, K. D. - Breinholt, J.
Journal: J Biol Chem

The crystal structure of the complex between a N-terminally truncated G129R human prolactin (PRL) variant and the extracellular domain (ECD) of the human prolactin receptor (PRLR) was determined at 2.5 A resolution by X-ray crystallography. This structure represents the first experimental structure reported for a PRL variant bound to its cognate receptor. The binding of PRL variants to PRLR-ECD was furthermore characterized by the solution state techniques, hydrogen exchange mass spectrometry (HX-MS) and nuclear magnetic resonance (NMR) spectroscopy. Compared to the binding interface derived from mutagenesis studies, the structural data imply that the definition of PRL binding site 1 (BS1) should be extended to include residues situated in the N-terminal part of loop 1 and in the C-terminus. Comparison of the structure of the receptor bound PRL variant with the structure reported for the unbound form of a similar analogue (Jomain et al. J. Biol. Chem. 282 (45), 33118-33131 (2007)) demonstrates that receptor induced changes in the back-bone of the four helix bundle are subtle, whereas large scale rearrangements and structuring occur in the flexible N-terminal part of loop 1. HX-MS data imply that the dynamics of the four-helix bundle in solution generally become stabilized upon receptor interaction at BS1.

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Regulation of IKK-related kinases and antiviral responses by tumor suppressor CYLD.

Publication Date: 2008 May 8 PMID: 18467330
Authors: Zhang, M. - Wu, X. - Lee, A. J. - Jin, W. - Chang, M. - Wright, A. - Imaizumi, T. - Sun, S. C.
Journal: J Biol Chem

The IKK-related kinases, IKKe and TBK1, participate in the induction of type I interferons (IFNs) during viral infections. Deregulated activation of IKKe and TBK1 also contributes to the abnormal cell survival and transformation. However, how these kinases are negatively regulated remains unclear. We show here that the tumor suppressor CYLD has an essential role in preventing aberrant activation of IKKe/TBK1. CYLD deficiency causes constitutive activation of IKKe/TBK1, which is associated with hyper-induction of IFNs in virus-infected cells. We further show that CYLD targets a cytoplasmic RNA sensor, RIG-I, and inhibits the ubiquitination of this IKKe/TBK1 stimulator. Consistent with the requirement of ubiquitination in RIG-I function, CYLD potently inhibits RIG-I mediated activation of IFN-ss promoter. These findings establish CYLD as a key negative regulator of IKKe/TBK1 and suggest a role for CYLD in the control of RIG-I ubiquitination.

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Pathway for heme uptake from human methemoglobin by the iron-regulated surface determinants (Isd) system of staphylococcus aureus.

Publication Date: 2008 May 8 PMID: 18467329
Authors: Zhu, H. - Xie, G. - Liu, M. - Olson, J. S. - Fabian, M. - Dooley, D. M. - Lei, B.
Journal: J Biol Chem

The iron-regulated surface proteins IsdA, IsdB and IsdC and transporter IsdDEF of Staphylococcus aureus are involved in heme acquisition. To establish an experimental model of heme acquisition by this system, we have investigated hemin transfer between the various couples of human methemoglobin (metHb), IsdA, IsdB, IsdC and IsdE by spectroscopic and kinetic analyses. The efficiencies of hemin transfer from hemin-containing donors (holo-protein) to different hemin-free acceptors (apo-protein) were examined, and the rates of the transfer reactions were compared to that of indirect loss of hemin from the relevant donor to H64Y/V68F apomyoglobin. The efficiencies, spectral changes, and kinetics of the transfer reactions demonstrate that: (1) metHb directly transfers hemin to apo-IsdB, but not to apo-IsdA, apo-IsdC and apo-IsdE; (2) holo-IsdB directly transfers hemin to apo-IsdA and apo-IsdC, but not to apo-IsdE; (3) apo-IsdE directly acquires hemin from holo-IsdC, but not from holo-IsdB and holo-IsdA; and (4) IsdB and IsdC enhance hemin transfer from metHb to apo-IsdC and from holo-IsdB to apo-IsdE, respectively. Taken together with our recent finding that holo-IsdA directly transfers its hemin to apo-IsdC, these results provide direct experimental evidence for a model in which IsdB acquires hemin from metHb and transfers it directly or through IsdA to IsdC. Hemin is then relayed to IsdE, the lipoprotein component of the IsdDEF transporter.

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Decidual cells produce a heparin-binding prolactin family cytokine with putative intrauterine regulatory actions.

Publication Date: 2008 May 8 PMID: 18467328
Authors: Alam, S. K. - Konno, T. - Sahgal, N. - Lu, L. - Soares, M. J.
Journal: J Biol Chem

Pregnancy in the mouse and rat are associated with the production of a large family of hormones/cytokines related to prolactin (PRL). The hormones/cytokines are hypothesized to coordinate maternal and fetal adaptations to pregnancy. In this study, PRL-like protein-J (PLP-J, also known as PRL family 3, subfamily c, member 1, Prl3c1) is shown to be a product of the uterine decidua and a regulator of postimplantation intrauterine events. PLP-J specific antibodies and a series of recombinant PLP-J proteins were generated and used to investigate PLP-J expression and as ligands for investigating biological targets. Decidual PLP-J migrates as a 29 kDa protein and localizes to a band of decidual cells surrounding the trophoblast cell layer on gestation day 8.5. PLP-J ligands specifically bound in situ to the surrounding uterine stromal cells and vasculature within the decidua of gestation day 8.5 implantation sites. We then investigated the in vitro actions of PLP-J on uterine stromal cells and endothelial cells. PLP-J specifically interacted with both cell populations. PLP-J promoted uterine stromal cell proliferation and inhibited endothelial cell proliferation. We determined that PLP-J does not interact with PRL receptors. Instead, PLP-J interacts with heparin-containing molecules, including syndecan-1, which is expressed in gestation day 8.5 pregnant uteri, as well as in uterine stromal cells and endothelial cells. The restricted expression of PLP-J and its specific interactions with uterine stromal cells and endothelial cells suggests that it acts locally and regulates decidual cell development and the endometrial vasculature.

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The ubiquitin-like protein Plic-1 enhances the membrane insertion of GABAA receptors by increasing their stability within the endoplasmic reticulum.

Publication Date: 2008 May 8 PMID: 18467327
Authors: Saliba, R. S. - Pangalos, M. - Moss, S. J.
Journal: J Biol Chem

-aminobutyric acid receptors (GABAAR) are the major sites of fast inhibitory neurotransmission in the brain, and a critical determinant for the efficacy of neuronal inhibition is the number of these receptors that are expressed on the neuronal cell surface. GABAARs are hetero-pentamers that can be constructed from 7 subunit classes with multiple members; a, ss, (1-3), d, e(1-3) and p. Receptor assembly occurs within the endoplasmic reticulum (ER) and it is evident that transport competent combinations exiting this organelle can access the cell surface, while unassembled subunits are ubiquitinated and subject to proteasomal degradation. Here we have explored the role that the ubiquitin-like protein Plic-1, which directly interacts with GABAAR subunits plays in regulating their membrane trafficking. Using both recombinant and neuronal preparations it was apparent that Plic-1 increased the stability of ER resident GABAARs together, with an increase in the abundance of poly-ubiquitinated receptor subunits. Furthermore Plic-1 promoted cell surface expression levels by selectively increasing their rates of membrane insertion. Thus Plic-1 may play a significant role in regulating the strength of synaptic inhibition by increasing the stability of GABAARs within the secretory pathway and thereby promoting their insertion into the neuronal plasma membrane.

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Phosphorylation of murine caspase-9 by the protein kinase CK2 regulates its cleavage by caspase-8.

Publication Date: 2008 May 8 PMID: 18467326
Authors: McDonnell, M. A. - Abedin, M. J. - Melendez, M. - Platikanova, T. N. - Ecklund, J. R. - Ahmed, K. - Kelekar, A.
Journal: J Biol Chem

Previous studies from our laboratory had indicated that cytochrome c-independent processing and activation of caspase-9 by caspase-8 contributed to early amplification of the caspase cascade in TNF-a-treated murine cells. Here we show that murine caspase-9 is phosphorylated by casein kinase 2 (CK2) on a serine near the site of caspase-8 cleavage. CK2 has been shown to regulate cleavage of the pro-apoptotic Bid protein by phosphorylating serine residues near its caspase-8 cleavage site. Similarly, CK2 modification of serine348 on caspase-9 appears to render the protease refractory to cleavage by active caspase-8. This phosphorylation did not affect the ability of caspase-9 to autoprocess. Substitution of S348 abolished phosphorylation but not cleavage, and a phospho-site mutant promoted apoptosis in TNF-a-treated caspase-9 knockout mouse embryo fibroblasts. Furthermore, inhibition of CK2 activity and RNA interference-mediated knockdown of the kinase accelerated caspase-9 activation, while phosphatase inhibition delayed both caspase-9 activation and death in response to TNF receptor occupation. Taken together, these studies show that TNF receptor cross-linking promotes dephosphorylation of caspase-9 rendering it susceptible to processing by activated caspase-8 protein. Thus, our data suggest that modification of procaspase-9 to protect it from inappropriate cleavage and activation is yet another mechanism by which the oncogenic kinase CK2 promotes survival.

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The solution structure of Emilin1 gC1q domain reveals a disordered insertion necessary for interaction with the alpha 4beta 1 integrin.

Publication Date: 2008 May 7 PMID: 18463100
Authors: Verdone, G. - Doliana, R. - Corazza, A. - Colebrooke, S. A. - Spessotto, P. - Bot, S. - Bucciotti, F. - Capuano, A. - Silvestri, A. - Viglino, P. - Campbell, I. D. - Colombatti, A. - Esposito, G.
Journal: J Biol Chem

The extracellular matrix protein EMILIN1 (Elastin Microfibril Interface Located Protein 1) is implicated in maintaining blood pressure homeostasis via the N-terminal EMI domain and in trophoblast invasion of the uterine wall via the globular C1q (gC1q) domain. Here, we describe the first NMR-based homology-model structure of the human 52 kDa homotrimer of the EMILIN1 gC1q domain. In contrast to all the gC1q (crystal) structures solved to date the ten-stranded ss-sandwich fold of the gC1q domain is reduced to nine ss strands with a consequent increase in the size of the central cavity lumen. An unstructured loop, resulting from an insertion unique to EMILIN family members and located at the trimer apex, upstream of the missing strand, specifically engages the a4ss1 integrin. Using both Jurkat T and EA.hy926 endothelial cells as well as site directed mutagenesis we demonstrate that the ability of a4ss1 integrins to recognize the trimeric EMILIN1 gC1q domain mainly depends on a single glutamic acid residue (E933). Static and flow adhesion of T cells and haptotactic migration of endothelial cells on gC1q is fully dependent on this residue. Thus, EMILIN1 gC1q-a4ss1 represents a unique ligand/receptor system, with a requirement for a three-fold arrangement of the interaction site.

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Lysosome dispersion in osteoblasts accommodates enhanced collagen production during differentiation.

Publication Date: 2008 May 7 PMID: 18463099
Authors: Nabavi, N. - Urukova, Y. - Cardelli, M. - Aubin, J. E. - Harrison, R. E.
Journal: J Biol Chem

Lysosomes are essential organelles for intracellular degradation and are generally sequestered near the cell center to receive vesicles with contents targeted for destruction. During ascorbic acid (AA)-induced differentiation of osteogenic cells (1) we saw a marked increase in total lysosome organelles in osteoblastic cells, in addition to an enhanced endocytic rate. Interestingly, lysosomes were dispersed towards the cell periphery in differentiating osteoblasts. We determined that lysosome dispersion in differentiated osteoblasts required intact microtubules for long-range transport, was dependent on kinesin motors, but did not involve cytosolic acidification. Impairment of lysosome dispersion markedly reduced AA-induced osteoblast differentiation. Lysosomes were not secreted in differentiated osteoblasts implicating them instead in intracellular degradation. We assayed the degradative capacity and saw a significant increase in DQ-ovalbumin fluorescence in differentiated osteogenic cells compared to undifferentiated control cells. Osteogenic cells are specialized for type I collagen production, and we noted enhanced secreted and intracellular collagen in AA-differentiated osteoblasts versus control cells. Importantly, osteoblasts displayed procollagen-containing vesicles that were distributed throughout the cytoplasm, a portion of which colocalized with lysosomes. Treatment of cells with 2,2'-dipyridyl to inhibit procollagen trimerization, enhanced colocalization of lysosomes with procollagen-containing organelles, implicating dispersed lysosomes in collagen processing in osteogenic cells.

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Overexpression of neprilysin reduces Alzheimer's amyloid-beta 42 (Abeta 42)-induced neuron loss and intraneuronal Abeta 42 deposits, but causes a reduction in CREB-mediated transcription, age-dependent axon pathology and premature death in Drosophila.

Publication Date: 2008 May 7 PMID: 18463098
Authors: Iijima-Ando, K. - Hearn, S. A. - Granger, L. - Shenton, C. - Gatt, A. - Chiang, H. C. - Hakker, I. - Zhong, Y. - Iijima, K.
Journal: J Biol Chem

The amyloid-beta42 (Abeta42) peptide has been suggested to play a causative role in Alzheimer's disease (AD). Neprilysin (NEP) is one of the rate-limiting Abeta-degrading enzymes whose genetic deficiencies promote increased Abeta levels in the brain. Enhancement of NEP activity ameliorates extracellular amyloid pathology, synaptic dysfunction, and memory defects in mouse models of Abeta amyloidosis, suggesting that NEP is an attractive therapeutic target for AD. In addition to accumulation in the extracellular space, intraneuronal Abeta42 may contribute to AD pathogenesis. However, the effects of neuronal NEP expression on intraneuronal Assbeta42 accumulation and neurodegeneration in the brain remain elusive. In addition, sustained NEP activation in neurons may be detrimental because NEP can degrade many physiological peptides, but the consequences of chronic NEP expression in the brain are not fully understood. Using transgenic Drosophila expressing human NEP and Abeta42, we demonstrated that NEP efficiently degraded Abeta42 expressed in the secretory pathway of neurons, suppressed the formation of intraneuronal TS-positive Abeta42 deposits, and reduced Abeta42-induced neuron loss. However, neuronal NEP overexpression reduced CREB-mediated transcription, caused age-dependent axon degeneration and shortened lifespan of the flies. Interestingly, the mRNA levels of endogenous fly NEP genes and phosphoramidon-sensitive NEP activity declined during aging in fly brains, as observed in mammals. Taken together, these data suggest both protective and detrimental effects of chronically high NEP activity in the brain. Downregulation of mRNA levels and activity of NEP in aging brains may be an evolutionarily conserved phenomenon, which could predispose humans to developing late-onset AD.

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Hu antigen R (HuR) functions as an alternative pre-mRNA splicing regulator of Fas apoptosis-promoting receptor on exon definition.

Publication Date: 2008 May 7 PMID: 18463097
Authors: Izquierdo, J. M.
Journal: J Biol Chem

Exclusion of exon 6 by alternative RNA splicing of the primary transcript of the apoptosis receptor Fas produces a soluble isoform that prevents programmed cell death. We report that anti-apoptotic regulator Hu antigen R (HuR, ELAVL1), a member of the embryonic lethal, abnormal vision, Drosophila-like (ELAVL) family, promotes Fas exon 6 skipping by binding to an exonic splicing silencer. HuR inhibits the association of U2 snRNP auxiliary factor 65 kDa (U2AF65) with the upstream 3' splice site, without decreasing recognition of the downstream 5' splice site by U1 snRNP, but by antagonizing the role of TIA-1 (T-cell intracellular antigen 1) / TIAR (TIA-1 related protein) on exon definition. Remarkably, U1 snRNP-mediated recognition of the 5' splice site is partially required for efficient U2AF65 inhibition. Further, the silencing capacity of HuR as splicing regulator resides in the RRM1 and hinge-RRM3 domains. Taken together, our results support a functional link between HuR as repressor of alternative Fas splicing and the molecular mechanisms modulating programmed cell death.

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How protein recognizes ladder-like polycyclic Ethers: Interactions between ciguatoxin (CTX3C) fragments and its specific antibody 10C9.

Publication Date: 2008 May 7 PMID: 18463096
Authors: Ui, M. - Tanaka, Y. - Tsumuraya, T. - Fujii, I. - Inoue, M. - Hirama, M. - Tsumoto, K.
Journal: J Biol Chem

Ciguatoxins are a family of marine toxins composed of trans-fused polycyclic ethers. It has not yet been clarified at the atomic level on the pathogenic mechanism of these toxins or the interaction between a polycyclic ether compounds and a protein. Using the crystal structures of anti-ciguatoxin antibody 10C9 Fab in ligand-free form and in complexes with ABCD-ring (CTX3C-ABCD) and ABCDE-ring (CTX3C-ABCDE) fragments of the antigen CTX3C at resolutions of 2.6, 2.4,and 2.3 A, respectively, we elucidated the mechanism of the interaction between the polycyclic ethers and the antibody. 10C9 Fab has an extraordinary large and deep binding pocket at the center of the variable region, and both antigen bind longitudinally in the pocket via hydrogen bonds and van der Waals interactions. Upon antigen-antibody complexation, 10C9 Fab conforms to the antigen fragments by means of rotational motion in the variable region. In addition, the antigen fragment lacking the E-ring induces a large motion in the constant region. Consequently, the thermostability of 10C9 Fab is enhanced by 10 masculineC upon complexation with CTX3C-ABCDE, and not with CTX3C-ABCD. 10C9 Fab recognizes the antigens with making use of its whole structure. These results further the fundamental understanding of the mechanism by which ladder-like polycyclic ether compound are recognized and may be useful for the design of therapeutic agents for some sorts of the marine toxins or reagents in the detection targeting a polycyclic ether family.

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An enzyme-linked receptor mechanism for nitric oxide-activated guanylyl cyclase.

Publication Date: 2008 May 7 PMID: 18463095
Authors: Roy, B. - Halvey, E. J. - Garthwaite, J.
Journal: J Biol Chem

Nitric oxide (NO) exerts physiological effects by activating specialized receptors that are coupled to guanylyl cyclase activity, resulting in cGMP synthesis from GTP. Despite its widespread importance as a signal transduction pathway, the way it operates is still understood only in descriptive terms. The present work aimed to elucidate a formal mechanism for NO receptor activation and its modulation by GTP, ATP, and allosteric agents such as YC-1 and BAY 41-2272. The model comprised a module in which NO, the nucleotides and allosteric agents bind, dovetailing with a catalytic module where GTP is converted to cGMP and pyrophosphate. Experiments on NO-activated guanylyl cyclase purified from bovine lung allowed values for all the binding and isomerization constants to be derived. The catalytic module was a modified version of one describing the kinetics of adenylyl cyclase. The resulting enzyme-linked receptor mechanism faithfully reproduces all the main functional properties of NO-activated guanylyl cyclase reported to date and provides a thermodynamically sound interpretation of those properties. With appropriate modification, it also replicates activation by carbon monoxide and the remarkable enhancement of that activity brought about by the allosteric agents. In addition, the mechanism progresses understanding of the behavior of the receptor in a cellular setting.

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A second GDP-L-galactose phosphorylase in Arabidopsis en route to vitamin C: Covalent intermediate and substrate requirements for the conserved reaction.

Publication Date: 2008 May 7 PMID: 18463094
Authors: Linster, C. L. - Adler, L. N. - Webb, K. - Christensen, K. C. - Brenner, C. - Clarke, S. G.
Journal: J Biol Chem

The Arabidopsis thaliana VTC2 gene encodes an enzyme that catalyzes the conversion of GDP-L-galactose to L-galactose 1-phosphate in the first committed step of the Smirnoff-Wheeler pathway to plant vitamin C synthesis. Mutations in VTC2 had previously been found to lead to only partial vitamin C deficiency. Here we show that the Arabidopsis gene At5g55120 encodes an enzyme with high sequence identity to VTC2. Designated VTC5, this enzyme displays substrate specificity and enzymatic properties that are remarkably similar to those of VTC2, suggesting that it may be responsible for residual vitamin C synthesis in vtc2 mutants. The exact nature of the reaction catalyzed by VTC2/VTC5 is controversial on account of reports that kiwifruit and Arabidopsis VTC2 utilize hexose 1-phosphates as phosphorolytic acceptor substrates. Using LC-MS and a VTC2-H238N mutant, we provide evidence that the reaction proceeds through a covalent guanylylated histidine residue within the histidine triad motif. Moreover, we show that both the Arabidopsis VTC2 and VTC5 enzymes catalyze simple phosphorolysis of the guanylylated enzyme, forming GDP and L-galactose 1-phosphate from GDP-L-galactose and phosphate, with poor reactivity of hexose 1-phosphates as phosphorolytic acceptors. Indeed, the endogenous activities from Japanese mustard spinach, lemon, and spinach have the same substrate requirements. These results show that Arabidopsis VTC2 and VTC5 proteins and their homologs in other plants are enzymes that guanylylate a conserved active site His residue with GDP-L-galactose, forming L-galactose 1-phosphate for vitamin C synthesis, and regenerate the enzyme with phosphate to form GDP.

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Crystal structure of squid rhodopsin with intracellularly extended cytoplasmic region.

Publication Date: 2008 May 6 PMID: 18463093
Authors: Shimamura, T. - Hiraki, K. - Takahashi, N. - Hori, T. - Ago, H. - Masuda, K. - Takio, K. - Ishiguro, M. - Miyano, M.
Journal: J Biol Chem

G protein-coupled receptors (GPCRs) play a key step in cellular signal transduction cascades by transducing various extracellular signals via G-proteins. Rhodopsin is a prototypical GPCR involved in the retinal visual signaling cascade. We determined the structure of squid rhodopsin at 3.7 A resolution, which transduces signals through the G(q) protein to the phosphoinositol cascade. The structure showed seven transmembrane helices and an amphipathic helix H8 has similar geometry to structures from bovine rhodopsin, coupling to G(t), and human beta(2)-adrenergic receptor, coupling to G(s). Notably, squid rhodopsin contains a well-structured cytoplasmic region involved in the interaction with G-proteins, and this region is flexible or disordered in bovine rhodopsin and human beta(2)-adrenergic receptor. The transmembrane helices 5 and 6 are longer and extrude into the cytoplasm. The distal C-terminal tail contains a short hydrophilic alpha-helix CH after the palmitoylated cysteine residues. The residues in the distal C-terminal tail interact with the neighboring residues in the second cytoplasmic loop, the extruded transmembrane helices 5 and 6, and the short helix H8. Additionally, the Tyr111, Asn87, and Asn185 residues are located within hydrogen bonding distances from the nitrogen atom of the Schiff base.

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