Journal of Biological Chemistry Impact Factor & Information

Publisher: American Society for Biochemistry and Molecular Biology, American Society for Biochemistry and Molecular Biology

Journal description

Complete content of the Journal of Biological Chemistry as of April 1995.

Current impact factor: 4.60

Impact Factor Rankings

2015 Impact Factor Available summer 2015
2013 / 2014 Impact Factor 4.6
2012 Impact Factor 4.651
2011 Impact Factor 4.773
2010 Impact Factor 5.328
2009 Impact Factor 5.328
2008 Impact Factor 5.52
2007 Impact Factor 5.581
2006 Impact Factor 5.808
2005 Impact Factor 5.854
2004 Impact Factor 6.355
2003 Impact Factor 6.482
2002 Impact Factor 6.696
2001 Impact Factor 7.258
2000 Impact Factor 7.368
1999 Impact Factor 7.666
1998 Impact Factor 7.199
1997 Impact Factor 6.963
1996 Impact Factor 7.452
1995 Impact Factor 7.385
1994 Impact Factor 7.716
1993 Impact Factor 6.793
1992 Impact Factor 6.733

Impact factor over time

Impact factor
Year

Additional details

5-year impact 5.02
Cited half-life 10.00
Immediacy index 0.94
Eigenfactor 0.68
Article influence 1.95
Website Journal of Biological Chemistry website
Other titles Journal of biological chemistry (Online), Journal of biological chemistry, JBC online, JBC
ISSN 1083-351X
OCLC 32808313
Material type Document, Periodical, Internet resource
Document type Internet Resource, Computer File, Journal / Magazine / Newspaper

Publisher details

American Society for Biochemistry and Molecular Biology

  • Pre-print
    • Author cannot archive a pre-print version
  • Post-print
    • Author cannot archive a post-print version
  • Restrictions
    • 12 months embargo
  • Conditions
    • Authors accepted peer-reviewed manuscript may be posted on an institutional repository
    • Publisher copyright and source must be acknowledged with set phrase: "This research was originally published in Journal Name. Author(s). Title. Journal Name. Year. Vol:pp-pp. © the American Society for Biochemistry and Molecular Biology"
    • On a non-profit server
    • Publisher's version/PDF cannot be used
  • Classification
    ​ white

Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: Core fucosylation is catalyzed by α1,6-fucosyltransferase (Fut8), which transfers a fucose residue to the innermost GlcNAc residue via α1,6-linkage on N-glycans in mammals. We previously reported that Fut8 knockout (Fut8-/-) mice showed a schizophrenia-like phenotype and a decrease in working memory. To understand the underlying molecular mechanism, we analyzed early-form long-term potentiation (E-LTP), which is closely related to learning and memory in the hippocampus. The scale of E-LTP induced by high frequency stimulation was significantly decreased in Fut8-/- mice. Tetraethylammonium-induced LTP showed no significant differences, suggesting that the decline in E-LTP was caused by post-synaptic events. Unexpectedly, the phosphorylation levels of calcium/calmodulin-dependent protein kinase II (CaMKII), an important mediator of learning and memory in post-synapses, were greatly increased in Fut8-/- mice. The expression levels of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) in the postsynaptic density were enhanced in Fut8-/- mice, although there were no significant differences in the total expression levels, implicating that AMPARs without core fucosylation might exist in an active state. The activation of AMPARs was further confirmed by Fura-2 calcium imaging using primary cultured neurons. Taken together, loss of core fucosylation on AMPARs enhanced their heteromerization, which might increase sensitivity for postsynaptic depolarization, and persistently activate N-methyl-D-aspartate receptors as well as Ca2+ influx and CaMKII, and then impair LTP. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 05/2015; DOI:10.1074/jbc.M114.579938
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    ABSTRACT: Parathyroid hormone (PTH) induces osteoclast formation and activity by increasing the ratio of RANKL/OPG in osteoblasts. The proteasome inhibitor carfilzomib (CFZ) has been used as an effective therapy for multiple myeloma via the inhibition of pathologic bone destruction. But the effect of combination of PTH and CFZ on osteoclastogenesis is unknown. We now reported that CFZ inhibits PTH-induced RANKL expression and secretion without affecting PTH inhibition of OPG expression, and it does so by blocking HDAC4 proteasomal degradation in osteoblasts. Furthermore, we used different types of culture systems, including co-culture, indirect co-culture and transactivation, to assess the effect of CFZ on PTH action to induce osteoclastogenesis. Our results demonstrated that CFZ blocks PTH-induced osteoclast formation and bone resorption by its additional effect to inhibit RANKL-mediated I kappa B degradation and NF-kappa B activation in osteoclasts. This study showed for the first time that CFZ targets both osteoblasts and osteoclasts to suppress PTH-induced osteoclast differentiation and bone resorption. These findings warrant further investigation of this novel combination in animal models of osteoporosis and in patients. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 05/2015; DOI:10.1074/jbc.M115.663963
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    ABSTRACT: Neuronal Calcium Sensor-1 (NCS-1) is the primordial member of the Neuronal Calcium Sensor (NCS) family of EF-hand Ca2+-binding proteins. It interacts with both the G-protein coupled receptor (GPCR) dopamine D2 receptor (D2R), regulating its internalisation and surface expression, and the cognate kinases, GRK1 and GRK2. Determination of the crystal structures of Ca2+/NCS-1 alone and in complex with peptides derived from D2R and GRK1 reveals that the differential recognition is facilitated by the conformational flexibility of the C-lobe binding site. We find that two copies of the D2R peptide bind within the hydrophobic crevice on Ca2+/NCS-1, but only one copy of the GRK1 peptide. The different binding modes are made possible by the C-lobe binding site of NCS-1, which adopts alternate conformations in each complex. C-terminal residues S178-V190 act in concert with the flexible EF3/EF4 loop region to effectively form different peptide binding sites. In the Ca2+/NCS-1-D2R peptide complex, the C-terminal region adopts a 310 helix-turn-310 helix, whereas in the GRK1 peptide complex it forms an alpha-helix. Removal of S178-V190 generated a C-terminal truncation mutant which formed a dimer, indicating that the NCS-1 C-terminal region prevents NCS-1 oligomerization. We propose that the flexible nature of the C-terminal region is essential to allow it to modulate its protein binding sites and adapt its conformation to accommodate both ligands. This appears to be driven by the variability of the conformation of the C-lobe binding site, which has ramification for the target specificity and diversity of NCS-1. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 05/2015; DOI:10.1074/jbc.M114.627059
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    ABSTRACT: Gα subunits are central molecular switches in cells. They are activated by G protein-coupled receptors which exchange GDP for GTP, similar to small GTPase activation mechanisms. Gα subunits are turned off by GTP hydrolysis. For the first time we employed time resolved FTIR difference spectroscopy to investigate the molecular reaction mechanisms of Gαi1. FTIR spectroscopy is a powerful tool which monitors reactions label free with high spatio-temporal resolution. In contrast to common multiple turnover assays, FTIR spectroscopy depicts the single turnover GTPase reaction without nucleotide exchange / Mg(2+ binding) bias. Global fit analysis resulted in one apparent rate constant of 0.02 s(-1) at 15 °C. Isotopic labeling was applied to assign the individual phosphate vibrations for α-,β- and γ-GTP (1243, 1224 and 1156 cm(-1), respectively), α- and β-GDP (1214 and 1134 / 1103 cm(-1), respectively) and free phosphate (1078 / 991 cm(-1)). In contrast to Ras·GAP catalysis, the bond breakage of the β-γ-phosphate but not the Pi release is rate limiting in the GTPase reaction. Complementary common GTPase assays were used. Reversed phase HPLC provided multiple turnover rates and tryptophan fluorescence provided nucleotide exchange rates. Experiments were complemented by molecular dynamics simulations. This broad approach provided detailed insights at atomic resolution and allows now to identify key residues of Gαi1 in GTP hydrolysis and nucleotide exchange. Mutants of the intrinsic arginine finger (Gαi1-R178S) affected exclusively the hydrolysis reaction. The effect of nucleotide binding (Gαi1-D272N) and Ras-like / all-α interface coordination (Gαi1-D229N/Gαi1-D231N) on the nucleotide exchange reaction was furthermore elucidated. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 05/2015; DOI:10.1074/jbc.M115.651190
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    ABSTRACT: Lysyl oxidases (LOXs) are a family of copper- dependent oxido-deaminases that can modify the side-chain of lysyl residues in collagen and elastin, thereby leading to the spontaneous formation of non-reducible aldehyde-derived inter-polypeptide chain cross-links. The consequences of LOX inhibition in producing lathyrism are well documented but the consequences on collagen fibril formation are less clear. Here we used β-aminoproprionitrile (BAPN) to inhibit LOX in tendon-like constructs (prepared from human tenocytes), which are an experimental model of cell- mediated collagen fibril formation. The improvement in structure and strength seen with time in control constructs was absent in constructs treated with BAPN. As expected, BAPN inhibited the formation of aldimine- derived cross-links in collagen and the constructs were mechanically weak. However, an unexpected finding was that BAPN- treatment led to structurally abnormal collagen fibrils with irregular profiles and widely dispersed diameters. Of special interest, the abnormal fibril profiles resembled those seen in some Ehlers-Danlos Syndrome (EDS) phenotypes. Importantly, the total collagen content developed normally, and there was no difference in COL1A1 gene expression. Collagen type V , decorin, fibromodulin and tenascin-X proteins were unaffected by the cross-link inhibition, suggesting that LOX regulates fibrillogenesis independently of these molecules. Collectively, the data show the importance of LOX on the mechanical development of early collagenous tissues, and that LOX is essential for correct collagen fibril shape formation. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 05/2015; DOI:10.1074/jbc.M115.641670
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    ABSTRACT: Angiogenesis is a highly regulated process orchestrated by the VEGF system. Heparin/heparan sulfate (HS) proteoglycans and neuropilin-1 (NRP-1) have been identified as co-receptors, yet the mechanisms of action have not been fully defined. In the present study we characterized molecular interactions between receptors and co-receptors, using surface plasmon resonance (SPR) and in vitro binding assays. Additionally, we demonstrate these binding events are relevant to VEGF activity witihin endothelial cells. We defined interactions and structural requirements for heparin/HS interactions with VEGF receptor-1 (VEGFR-1), NRP-1, and VEGF165 in complex with VEGFR-2 and NRP-1. We demonstrate that these structural requirements are distinct for each interaction. We further show that VEGF165, VEGFR-2 and monomeric NRP-1 bind weakly to heparin alone, yet show synergistic binding to heparin when presented together in various combinations. This synergistic binding appears to translate to alterations in VEGF signaling in endothelial cells. We found that soluble NRP-1 increases VEGF binding and activation of VEGFR-2 and Erk1/2 in endothelial cells, and that these effects require sulfated HS. These data suggest that the presence of HS/heparin and NRP-1 may dictate the specific receptor type activated by VEGF and ultimately determine the biological output of the system. The ability of co-receptors to fine-tune VEGF responsiveness suggests the possibility that VEGF-mediated angiogenesis can be selectively stimulated or inhibited by targeting HS/heparin and NRP-1. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 05/2015; DOI:10.1074/jbc.M114.627372
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    ABSTRACT: Urokinase plasminogen activator (uPA) converts plasminogen to plasmin, resulting in a proteolytic cascade that has been implicated in tissue destruction during inflammation. Periodontitis is a highly prevalent chronic inflammatory disease characterized by destruction of the tissue and bone that support the teeth. We demonstrate that stimulation of macrophages with the arginine- and lysine-specific cysteine protease complex (RgpA-Kgp complex), produced by the keystone pathogen Porphyromonas gingivalis, dramatically increased their ability to degrade matrix in a uPA-dependent manner. We show the RgpA-Kgp complex cleaves the inactive zymogens, pro-uPA (at consensus sites K(158)-I(159) and K(135)-K(136)) and plasminogen, yielding active uPA and plasmin, respectively. These findings are consistent with activation of the uPA proteolytic cascade by P.gingivalis being required for the pathogen to induce alveolar bone loss in a model of periodontitis and reveal a new host-pathogen interaction in which P.gingivalis activates a critical host proteolytic pathway to promote tissue destruction and pathogen virulence. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 05/2015; DOI:10.1074/jbc.M115.645572
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    ABSTRACT: People with Rett Syndrome (RTT) and mouse models show autonomic dysfunction involving brainstem locus coeruleus (LC). Neurons in the LC of Mecp2-null mice are overly excited, likely to result from defect in neuronal intrinsic membrane properties and deficiency in GABA synaptic inhibition. In addition to the synaptic GABA receptors, there is a group of GABAA receptors (GABAARs), which is located extrasynaptically, and mediates tonic inhibition. Here we show evidence for augmentation of the extrasynaptic GABAARs in Mecp2-null mice. In brain slices, exposure of LC neurons to GABAAR agonists increased tonic currents that were blocked by GABAAR antagonists. With 10 μM GABA, the bicuculline-sensitive tonic currents were ~ 4 folds larger in Mecp2-null LC neurons than the wild-type (WT). Single-cell PCR analysis showed that δ subunit, the principal subunit of extrasynaptic GABAARs, was present in LC neurons. Expression levels of the δ were ~ 50% higher in Mecp2-null neurons than in the WT. Also increased in expression in Mecp2-null mice was another extrasynaptic GABAAR subunit α 6 by ~ 4 folds. The δ subunit-selective agonists THIP and DS2 activated the tonic GABAA currents in LC neurons and reduced neuronal excitability to a greater degree in Mecp2-null mice than in the WT. Consistent with these findings, in-vivo application of THIP alleviated breathing abnormalities of conscious Mecp2-null mice. These results suggest that extrasynaptic GABAARs seem to be augmented with Mecp2 disruption, which may be a compensatory response to the deficiency in GABA-ergic synaptic inhibition and allow a control of neuronal excitability and breathing abnormalities. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 05/2015; DOI:10.1074/jbc.M115.650465
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    ABSTRACT: Human protein arginine methyltransferase 9 (PRMT9) symmetrically dimethylates arginine residues on splicing factor SF3B2 (SAP145) and has been functionally linked to the regulation of alternative splicing of pre-mRNA. Site-directed mutagenesis studies on this enzyme and its substrate had revealed essential unique residues in the double E loop and the importance of the C-terminal duplicated methyltransferase domain. In contrast to what had been observed with other PRMTs and their physiological substrates, a peptide containing the methylatable Arg-508 of SF3B2 was not recognized by PRMT9 in vitro. While amino acid substitutions of residues surrounding Arg-508 had no great effect on PRMT9 recognition of SF3B2, moving the arginine residue within this sequence abolished methylation. PRMT9 and PRMT5 are the only known mammalian enzymes capable of forming symmetric dimethylarginine (SDMA) residues as type II PRMTs. We demonstrate here that the specificity of these enzymes for their substrates is distinct and not redundant. The loss of PRMT5 activity in mouse embryo fibroblasts results in almost complete loss of SDMA, suggesting that PRMT5 is the primary SDMA-forming enzyme in these cells. PRMT9, with its duplicated methyltransferase domain and conserved sequence in the double E loop, appears to have a unique structure and specificity among PRMTs for methylating SF3B2 and potentially other polypeptides. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 05/2015; DOI:10.1074/jbc.M115.659433
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    ABSTRACT: Low levels of reactive oxygen species (ROS) act as important signaling molecules but in excess they can damage biomolecules. ROS regulation is therefore of key importance. Several polyphenols in general and flavonoids in particular have the potential to generate hydroxyl radicals, the most hazardous among all ROS. However, the generation of a hydroxyl radical and subsequent ROS formation can be prevented by methylation of the hydroxyl group of the flavonoids. O‑methylation is performed by O‑methyltransferases, members of the S‑adenosylmethionine (SAM)-dependent O‑methyltransferase superfamily involved in the secondary metabolism of many species across all kingdoms. In the filamentous fungus Podospora anserina, a well-established aging model, the O-methyltransferase (PaMTH1) was reported to accumulate in total and mitochondrial protein extracts during aging. In vitro functional studies revealed flavonoids and in particular myricetin as its potential substrate. The molecular architecture of PaMTH1 and the mechanism of the methyl transfer reaction remains unknown. Here, we report the crystal structures of PaMTH1 apoenzyme, PaMTH1-SAM (co-factor) and PaMTH1-SAH (by-product) co-complexes refined to 2.0, 1.9 and 1.9 Å, respectively. PaMTH1 forms a tight dimer through swapping of the N‑termini. Each monomer adopts the Rossmann-fold typical for many SAM-binding methyltransferases. Structural comparisons between different O‑methyltransferases reveal a strikingly similar co-factor binding pocket but differences in the substrate binding pocket indicating specific molecular determinants required for substrate selection. Furthermore, using NMR, mass spectrometry and site-directed active-site mutagenesis, we show that PaMTH1 catalyzes the transfer of the methyl group from SAM to one hydroxyl group of the myricetin in a cation-dependent manner. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 05/2015; DOI:10.1074/jbc.M115.660829
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    ABSTRACT: Thrombin acts on the endothelium by activating protease-activated receptors (PARs). The endothelial thrombin-PAR system becomes deregulated during pathological conditions resulting in loss of barrier function and a pro-inflammatory and pro-angiogenic endothelial phenotype. We reported recently that the ion transporter Na+/Ca2+ exchanger (NCX) operating in the Ca2+-influx (reverse) mode promoted ERK1/2 activation and angiogenesis in vascular endothelial growth factor-stimulated primary human vascular endothelial cells (HUVEC). Here we investigated whether Ca2+ influx through NCX was involved in ERK1/2 activation, angiogenesis and endothelial barrier dysfunction in response to thrombin. Reverse-mode NCX inhibitors and RNAi-mediated NCX1 knockdown attenuated ERK1/2 phosphorylation in response to thrombin or an agonist of PAR-1, the main endothelial thrombin receptor. Conversely, promoting reverse-mode NCX by suppressing Na+-K+-ATPase activity enhanced ERK1/2 activation. Reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced HUVEC angiogenesis, quantified as proliferation and tubular differentiation. Reverse-mode NCX inhibitors or NCX1 knock-down preserved barrier integrity upon thrombin stimulation in vitro. Moreover, the reverse-mode NCX inhibitor SEA0400 suppressed Evans blue albumin extravasation to the lung and kidneys and attenuated oedema formation and ERK1/2 activation in the lungs of mice challenged with a peptide activator of PAR-1. Mechanistically, thrombin-induced ERK1/2 activation required NADPH oxidase 2-mediated reactive oxygen species (ROS) production and reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced ROS production. We propose that reverse-mode NCX is a novel mechanism contributing to thrombin-induced angiogenesis and hyperpermeability by mediating ERK1/2 activation in a ROS-dependent manner. Targeting reverse-mode NCX could be beneficial in pathological conditions involving unregulated thrombin signalling. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 05/2015; DOI:10.1074/jbc.M114.628156
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    ABSTRACT: T follicular helper (Tfh) cells support differentiation of B cells to plasma cells and high affinity antibody production in germinal centers (GC) and Tfh differentiation requires the function of B cell lymphoma 6 (Bcl6). We have now discovered that early growth response gene (Egr) 2 and 3 directly regulate the expression of Bcl6 in Tfh cells which is required for their function in regulation of GC formation. In the absence of Egr2 and 3, the expression of Bcl6 in Tfh cells is defective leading to impaired differentiation of Tfh cells resulting in a failure to form GCs following virus infection and defects in production of anti-viral antibodies. Enforced expression of Bcl6 in Egr2/3 deficient CD4 T cells partially restored Tfh differentiation and GC formation in response to virus infection. Our findings demonstrate a novel function of Egr2/3 which is important for Tfh cell development and Tfh cell mediated B cell immune responses. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 05/2015; DOI:10.1074/jbc.M114.634816
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    ABSTRACT: Syndecans, a family of transmembrane heparansulfate proteoglycans, are known to interact through their transmembrane domains to form non-covalently linked homodimers, a process essential for their individual functions. Because all syndecan transmembrane domains are highly conserved, and thus might mediate interactions between different members of the syndecan family, we investigated syndecan interactions in detail. All recombinant syndecan-2 and -4 protein variants containing the transmembrane domain formed not only sodium dodecyl sulfate (SDS)-resistant homodimers but also SDS-resistant heterodimers. Biochemical and structural data revealed that recombinant syndecan-2 and -4 formed inter-molecular interactions in vitro and GxxxG motif in transmembrane domain mediated this interaction. When exogenously expressed in rat embryonic fibroblasts, syndecan-2 interacted with syndecan-4 and vice versa. Furthermore, bimolecular fluorescence complementation-based assay demonstrated specific hetero-molecular interactions between syndecan-2 and -4, supporting hetero-oligomer formation of syndecans in vivo. Interestingly, hetero-oligomerization significantly reduced syndecan-4-mediated cellular processes such as protein kinase Cα activation and protein kinase Cα-mediated cell adhesion, as well as syndecan-2-mediated tumorigenic activities in colon cancer cells such as migration and anchorage-independent growth. Taken together, these data provide evidence that hetero-oligomerization produces distinct syndecan functions and offer insights into the underlying signaling mechanisms of syndecans. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 05/2015; DOI:10.1074/jbc.M114.611798
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    ABSTRACT: The twin arginine translocase (Tat) transports folded proteins of widely varying size across ionically tight membranes with only 2-3 components of machinery and the protonmotive force. Tat operates by a cycle in which the receptor complex combines with the pore-forming component to assemble a new translocase for each substrate. Recent data on component and substrate organization in the receptor complex and on the structure of the pore complex inform models for translocase assembly and translocation. A translocation mechanism involving local transient bilayer rupture is discussed. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 05/2015; DOI:10.1074/jbc.R114.626820
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    ABSTRACT: Exposure to lead (Pb) from environmental sources remains an overlooked and serious public health risk. Starting in childhood Pb in the skeleton can disrupt epiphyseal plate function, constrain the growth of long bones, and prevent attainment of a high peak bone mass, all of which will increase susceptibility to osteoporosis later in life. We hypothesize that the effects of Pb on bone mass, in part, come from depression of Wnt/β-catenin signaling, a critical anabolic pathway for osteoblastic bone formation. In this study we show that depression of Wnt signaling by Pb is due to increased sclerostin levels in vitro and in vivo. Downstream activation of the β- catenin pathway using a pharmacological inhibitor of GSK-3β ameliorates the Pb inhibition of Wnt signaling activity in the TOPGAL reporter mouse. The effect of Pb was determined to be dependent on sclerostin expression through use of the SOST gene knockout mouse, which are resistant to Pb- induced trabecular bone loss and maintain their mechanical bone strength. Moreover, isolated bone marrow cells from the sclerostin null mice show improved bone formation potential even after exposure to Pb. Also, our data implicate the TGFβ canonical signaling pathway is the mechanism by which Pb controls sclerostin production. Taken together these results support our hypothesis that the osteoporotic-like phenotype observed after Pb exposure is, in part, regulated through modulation of the Wnt/β-catenin pathway. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 05/2015; DOI:10.1074/jbc.M114.629204
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    ABSTRACT: Viral flice interacting protein (vFLIP), encoded by the oncogenic Kaposi's sarcoma associated herpes virus (KSHV), constitutively activates the canonical nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. This is achieved through subversion of the IκB kinase (IKK) complex (or signalosome) which involves a physical interaction between vFLIP and the modulatory subunit IKKγ. Although this interaction has been examined both in vivo and in vitro, the mechanism by which vFLIP activates the kinase remains to be determined. Since IKKγ functions as a scaffold, recruiting both vFLIP and the IKKα/β subunits, it has been proposed that binding of vFLIP could trigger a structural rearrangement in IKKγ conducive to activation. To investigate this hypothesis we engineered a series of mutants along the length of the IKKγ molecule that could be individually modified with nitroxide spin labels. Subsequent distance measurements using electron paramagnetic resonance (EPR) spectroscopy combined with molecular modelling and molecular dynamics (MD) simulations revealed that IKKγ is a parallel coiled coil whose response to binding of vFLIP or IKKβ is localised twisting/stiffening and not large-scale rearrangements. The coiled coil comprises N- and C-terminal regions with distinct registers accommodated by a twist: this structural motif is exploited by vFLIP, allowing it to bind and subsequently activate the NF-κB Pathway. In vivo assays confirm that NF-κB activation by vFLIP only requires the N-terminal region up to the transition between the registers which is located directly C-terminal of the vFLIP binding site. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 05/2015; DOI:10.1074/jbc.M114.622928
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    ABSTRACT: Phosducin (Pdc), a highly conserved phosphoprotein involved in the regulation of retinal phototransduction cascade, transcriptional control, and modulation of blood pressure, is controlled in the phosphorylation-dependent manner including the binding to the 14-3-3 protein. However, the molecular mechanism of this regulation is largely unknown. Here, the solution structure of Pdc and its interaction with the 14-3-3 protein were investigated using small angle X-ray scattering, time-resolved fluorescence spectroscopy and hydrogen-deuterium exchange coupled to mass spectrometry. The 14-3-3 protein dimer interacts with Pdc using surfaces both inside and outside its central channel. The N-terminal domain of Pdc, where both phosphorylation sites and the 14-3-3 binding motifs are located, is intrinsically disordered protein which reduces its flexibility in several regions without undergoing dramatic disorder-to-order transition upon binding to 14-3-3. Our data also indicate that the C-terminal domain of Pdc interacts with the outside surface of the 14-3-3 dimer through region involved in Gtβγ binding. In conclusion, we show that the 14-3-3 protein interacts with and sterically occludes both the N- and C-terminal Gtβγ binding interfaces of phosphorylated Pdc, thus providing a mechanistic explanation for the 14-3-3-dependent inhibition of Pdc function. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 05/2015; DOI:10.1074/jbc.M115.636563
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    ABSTRACT: Metabotropic (slow) and ionotropic (fast) neurotransmission are integrated by intracellular signal transduction mechanisms involving protein phosphorylation/ dephosphorylation to achieve experience-dependent alterations in brain circuitry. Extracellular signal-regulated kinase (ERK) is an important effector of both slow and fast forms of neurotransmission and has been implicated in normal brain function and CNS diseases. Here, we characterize phosphorylation of the ERK-activating protein kinase MEK1 by Cdk5, ERK, and Cdk1 in vitro, in intact mouse brain tissue, and in the context of an animal behavioral paradigm of stress. Cdk5 only phosphorylates Thr292, while ERK and Cdk1 phosphorylate both Thr292 and Thr286 MEK1. These sites interact in a kinase-specific manner and inhibit the ability of MEK1 to activate ERK. Thr292 and Thr286 MEK1 are phosphorylated in most mouse brain regions to stoichiometries of approximately 5% or less. Phosphorylation of Thr292 MEK1 is regulated by cAMP-dependent signaling in mouse striatum in a manner consistent with negative feedback inhibition in response to ERK activation. Protein phosphatase 1 and 2A contribute to the maintenance of the basal phosphorylation state of both Thr292 and Thr286 MEK1, as well as that of ERK. Activation of the NMDA-class of ionotropic glutamate receptors reduces inhibitory MEK1 phosphorylation, while forced swim, a paradigm of acute stress, attenuates Thr292 MEK1 phosphorylation. Together the data indicate that these inhibitory MEK1 sites phosphorylated by Cdk5 and ERK1 serve as mechanistic points of convergence for the regulation of ERK signaling by both slow and fast neurotransmission. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 05/2015; DOI:10.1074/jbc.M115.654897
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    ABSTRACT: The Escherichia coli Fumarate-Nitrate Reduction regulator (FNR) protein is the paradigm for bacterial O2-sensing transcription factors. However, unlike E. coli, some bacterial species possess multiple FNR proteins that presumably have evolved to fulfill distinct roles. Here, three FNR proteins (ANR, PP_3233 and PP_3287) from a single bacterial species, Pseudomonas putida KT2440, have been analyzed. Under anaerobic conditions, all three proteins had spectral properties resembling those of [4Fe-4S] proteins. The reactivity of the ANR [4Fe-4S] cluster with O2 was similar to that of E. coli FNR and during conversion to the apo-protein, via a [2Fe-2S] intermediate, cluster sulfur was retained. Like ANR, reconstituted PP_3233 and PP_3287 were converted to [2Fe-2S] forms when exposed to O2, but their [4Fe-4S] clusters reacted more slowly. Transcription from an FNR-dependent promoter with a consensus FNR-binding site in P. putida and E. coli strains expressing only one FNR protein was consistent with the in vitro responses to O2. Taken together the experimental results suggest that the local environments of the iron-sulfur clusters in the different P. putida FNR proteins influence their reactivity with O2, such that ANR resembles E. coli FNR and is highly-responsive to low concentrations of O2, whereas PP_3233 and PP_3287 have evolved to be less sensitive to O2. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 05/2015; DOI:10.1074/jbc.M115.654079