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Journal Cover Journal of Biological Chemistry
  [SJR: 3.151]   [H-I: 435]   [197 followers]  Follow
    
   Full-text available via subscription Subscription journal
   ISSN (Print) 0021-9258 - ISSN (Online) 1083-351X
   Published by ASBMB Homepage  [3 journals]
  • At the confluence of ribosomally synthesized peptide modification and
           radical S-adenosylmethionine (SAM) enzymology [Protein Structure and
           Folding]
    • Authors: John A. Latham; Ian Barr, Judith P. Klinman
      Pages: 16397 - 16405
      Abstract: Radical S-adenosylmethionine (RS) enzymology has emerged as a major biochemical strategy for the homolytic cleavage of unactivated C–H bonds. At the same time, the post-translational modification of ribosomally synthesized peptides is a rapidly expanding area of investigation. We discuss the functional cross-section of these two disciplines, highlighting the recently uncovered importance of protein–protein interactions, especially between the peptide substrate and its chaperone, which functions either as a stand-alone protein or as an N-terminal fusion to the respective RS enzyme. The need for further work on this class of enzymes is emphasized, given the poorly understood roles performed by multiple, auxiliary iron–sulfur clusters and the paucity of protein X-ray structural data.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.R117.797399
      Issue No: Vol. 292, No. 40 (2017)
       
  • Janus kinase 3 regulates adherens »∑unctions and epithelial mesenchymal
           transition through {beta}-catenin [Protein Structure and Folding]
    • Authors: Jayshree Mishra; Jugal Kishore Das, Narendra Kumar
      Pages: 16406 - 16419
      Abstract: Compromise in adherens junctions (AJs) is associated with several chronic inflammatory diseases. We reported previously that Janus kinase 3, a non-receptor tyrosine kinase, plays a crucial role in AJ formation through its interaction with β-catenin. In this report, we characterize the structural determinants responsible for Jak3 interactions with β-catenin and determine the functional implications of previously unknown tyrosine residues on β-catenin phosphorylated by Jak3. We demonstrate that Jak3 autophosphorylation was the rate-limiting step during Jak3 trans-phosphorylation of β-catenin, where Jak3 directly phosphorylated three tyrosine residues, viz. Tyr30, Tyr64, and Tyr86 in the N-terminal domain (NTD) of β-catenin. However, prior phosphorylation of β-catenin at Tyr654 was essential for further phosphorylation of β-catenin by Jak3. Interaction studies indicated that phosphorylated Jak3 bound to phosphorylated β-catenin with a dissociation constant of 0.28 μm, and although both the kinase and FERM (Band 4.1, ezrin, radixin, and moesin) domains of Jak3 interacted with β-catenin, the NTD domain of β-catenin facilitated its interactions with Jak3. Physiologically, Jak3-mediated phosphorylation of β-catenin suppressed EGF-mediated epithelial–mesenchymal transition and facilitated epithelial barrier functions by AJ localization of phosphorylated β-catenin through its interactions with α-catenin. Moreover, loss of Jak3-mediated phosphorylation sites in β-catenin abrogated its AJ localization and compromised epithelial barrier functions. Thus, we not only characterize Jak3 interaction with β-catenin but also demonstrate the mechanism of molecular interplay between AJ dynamics and EMT by Jak3-mediated NTD phosphorylation of β-catenin.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.811802
      Issue No: Vol. 292, No. 40 (2017)
       
  • MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast
           proliferation and activation [Gene Regulation]
    • Authors: Chaoqun Huang; Xiao Xiao, Ye Yang, Amorite Mishra, Yurong Liang, Xiangming Zeng, Xiaoyun Yang, Dao Xu, Michael R. Blackburn, Craig A. Henke, Lin Liu
      Pages: 16420 - 16439
      Abstract: Aberrant proliferation and activation of lung fibroblasts contribute to the initiation and progression of idiopathic pulmonary fibrosis (IPF). However, the mechanisms responsible for the proliferation and activation of fibroblasts are not fully understood. The objective of this study was to investigate the role of miR-101 in the proliferation and activation of lung fibroblasts. miR-101 expression was determined in lung tissues from patients with IPF and mice with bleomycin-induced pulmonary fibrosis. The regulation of miR-101 and cellular signaling was investigated in pulmonary fibroblasts in vitro. The role of miR-101 in pulmonary fibrosis in vivo was studied using adenovirus-mediated gene transfer in mice. The expression of miR-101 was down-regulated in fibrotic lungs from patients with IPF and bleomycin-treated mice. The down-regulation of miR-101 occurred via the E26 transformation-specific (ETS) transcription factor. miR-101 suppressed the WNT5a-induced proliferation of lung fibroblasts by inhibiting NFATc2 signaling via targeting Frizzled receptor 4/6 and the TGF-β-induced activation of lung fibroblasts by inhibition of SMAD2/3 signaling via targeting the TGF-β receptor 1. Adenovirus-mediated miR-101 gene transfer in the mouse lung attenuated bleomycin-induced lung fibrosis and improved lung function. Our data suggest that miR-101 is an anti-fibrotic microRNA and a potential therapeutic target for pulmonary fibrosis.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.805747
      Issue No: Vol. 292, No. 40 (2017)
       
  • Ar»∑unolic acid, a peroxisome proliferator-activated receptor {alpha}
           agonist, regresses cardiac fibrosis by inhibiting non-canonical TGF-{beta}
           signaling [Signal Transduction]
    • Authors: Trisha Bansal; Emeli Chatterjee, Jasdeep Singh, Arjun Ray, Bishwajit Kundu, V. Thankamani, Shantanu Sengupta, Sagartirtha Sarkar
      Pages: 16440 - 16462
      Abstract: Cardiac hypertrophy and associated heart fibrosis remain a major cause of death worldwide. Phytochemicals have gained attention as alternative therapeutics for managing cardiovascular diseases. These include the extract from the plant Terminalia arjuna, which is a popular cardioprotectant and may prevent or slow progression of pathological hypertrophy to heart failure. Here, we investigated the mode of action of a principal bioactive T. arjuna compound, arjunolic acid (AA), in ameliorating hemodynamic load-induced cardiac fibrosis and identified its intracellular target. Our data revealed that AA significantly represses collagen expression and improves cardiac function during hypertrophy. We found that AA binds to and stabilizes the ligand-binding domain of peroxisome proliferator-activated receptor α (PPARα) and increases its expression during cardiac hypertrophy. PPARα knockdown during AA treatment in hypertrophy samples, including angiotensin II-treated adult cardiac fibroblasts and renal artery-ligated rat heart, suggests that AA-driven cardioprotection primarily arises from PPARα agonism. Moreover, AA-induced PPARα up-regulation leads to repression of TGF-β signaling, specifically by inhibiting TGF-β-activated kinase1 (TAK1) phosphorylation. We observed that PPARα directly interacts with TAK1, predominantly via PPARα N-terminal transactivation domain (AF-1) thereby masking the TAK1 kinase domain. The AA-induced PPARα-bound TAK1 level thereby shows inverse correlation with the phosphorylation level of TAK1 and subsequent reduction in p38 MAPK and NF-κBp65 activation, ultimately culminating in amelioration of excess collagen synthesis in cardiac hypertrophy. In conclusion, our findings unravel the mechanism of AA action in regressing hypertrophy-associated cardiac fibrosis by assigning a role of AA as a PPARα agonist that inactivates non-canonical TGF-β signaling.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.788299
      Issue No: Vol. 292, No. 40 (2017)
       
  • Phylogenetic sequence analysis and functional studies reveal compensatory
           amino acid substitutions in loop 2 of human ribonucleotide reductase
           [Enzymology]
    • Authors: Andrew J. Knappenberger; Sneha Grandhi, Reena Sheth, Md. Faiz Ahmad, Rajesh Viswanathan, Michael E. Harris
      Pages: 16463 - 16476
      Abstract: Eukaryotic class I ribonucleotide reductases (RRs) generate deoxyribonucleotides for DNA synthesis. Binding of dNTP effectors is coupled to the formation of active dimers and induces conformational changes in a short loop (loop 2) to regulate RR specificity among its nucleoside diphosphate substrates. Moreover, ATP and dATP bind at an additional allosteric site 40 Å away from loop 2 and thereby drive formation of activated or inactive hexamers, respectively. To better understand how dNTP binding influences specificity, activity, and oligomerization of human RR, we aligned>300 eukaryotic RR sequences to examine natural sequence variation in loop 2. We found that most amino acids in eukaryotic loop 2 were nearly invariant in this sample; however, two positions co-varied as nonconservative substitutions (N291G and P294K; human numbering). We also found that the individual N291G and P294K substitutions in human RR additively affect substrate specificity. The P294K substitution significantly impaired effector-induced oligomerization required for enzyme activity, and oligomerization was rescued in the N291G/P294K enzyme. None of the other mutants exhibited altered ATP-mediated hexamerization; however, certain combinations of loop 2 mutations and dNTP effectors perturbed ATP's role as an allosteric activator. Our results demonstrate that the observed compensatory covariation of amino acids in eukaryotic loop 2 is essential for its role in dNTP-induced dimerization. In contrast, defects in substrate specificity are not rescued in the double mutant, implying that functional sequence variation elsewhere in the protein is necessary. These findings yield insight into loop 2's roles in regulating RR specificity, allostery, and oligomerization.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.798769
      Issue No: Vol. 292, No. 40 (2017)
       
  • Cell-cell adhesion in metazoans relies on evolutionarily conserved
           features of the {alpha}-catenin{middle dot}{beta}-catenin-binding
           interface [Protein Structure and Folding]
    • Authors: Xiangqiang Shao; Hyunook Kang, Timothy Loveless, Gyu Rie Lee, Chaok Seok, William I. Weis, Hee-Jung Choi, Jeff Hardin
      Pages: 16477 - 16490
      Abstract: Stable tissue integrity during embryonic development relies on the function of the cadherin·catenin complex (CCC). The Caenorhabditis elegans CCC is a useful paradigm for analyzing in vivo requirements for specific interactions among the core components of the CCC, and it provides a unique opportunity to examine evolutionarily conserved mechanisms that govern the interaction between α- and β-catenin. HMP-1, unlike its mammalian homolog α-catenin, is constitutively monomeric, and its binding affinity for HMP-2/β-catenin is higher than that of α-catenin for β-catenin. A crystal structure shows that the HMP-1·HMP-2 complex forms a five-helical bundle structure distinct from the structure of the mammalian α-catenin·β-catenin complex. Deletion analysis based on the crystal structure shows that the first helix of HMP-1 is necessary for binding HMP-2 avidly in vitro and for efficient recruitment of HMP-1 to adherens junctions in embryos. HMP-2 Ser-47 and Tyr-69 flank its binding interface with HMP-1, and we show that phosphomimetic mutations at these two sites decrease binding affinity of HMP-1 to HMP-2 by 40–100-fold in vitro. In vivo experiments using HMP-2 S47E and Y69E mutants showed that they are unable to rescue hmp-2(zu364) mutants, suggesting that phosphorylation of HMP-2 on Ser-47 and Tyr-69 could be important for regulating CCC formation in C. elegans. Our data provide novel insights into how cadherin-dependent cell–cell adhesion is modulated in metazoans by conserved elements as well as features unique to specific organisms.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.795567
      Issue No: Vol. 292, No. 40 (2017)
       
  • Loss of mucin-type O-glycans impairs the integrity of the glomerular
           filtration barrier in the mouse kidney [Cell Biology]
    • Authors: Kai Song; Jianxin Fu, Jianhua Song, Brett H. Herzog, Kirk Bergstrom, Yuji Kondo, J. Michael McDaniel, Samuel McGee, Robert Silasi-Mansat, Florea Lupu, Hong Chen, Harini Bagavant, Lijun Xia
      Pages: 16491 - 16497
      Abstract: The kidney's filtration activity is essential for removing toxins and waste products from the body. The vascular endothelial cells of the glomerulus are fenestrated, flattened, and surrounded by podocytes, specialized cells that support glomerular endothelial cells. Mucin-type core 1–derived O-glycans (O-glycans) are highly expressed on both glomerular capillary endothelial cells and their supporting podocytes, but their biological role is unclear. Biosynthesis of core 1–derived O-glycans is catalyzed by the glycosyltransferase core 1 β1,3-galactosyltransferase (C1galt1). Here we report that neonatal or adult mice with inducible deletion of C1galt1 (iC1galt1−/−) exhibit spontaneous proteinuria and rapidly progressing glomerulosclerosis. Ultrastructural analysis of the glomerular filtration barrier components revealed that loss of O-glycans results in altered podocyte foot processes. Further analysis indicated that O-glycan is essential for the normal signaling function of podocalyxin, a podocyte foot process–associated glycoprotein. Our results reveal a new function of O-glycosylation in the integrity of the glomerular filtration barrier.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.798512
      Issue No: Vol. 292, No. 40 (2017)
       
  • Complex interplay of kinetic factors governs the synergistic properties of
           HIV-1 entry inhibitors [Microbiology]
    • Authors: Koree W. Ahn; Michael J. Root
      Pages: 16498 - 16510
      Abstract: The homotrimeric HIV-1 envelope glycoprotein (Env) undergoes receptor-triggered structural changes that mediate viral entry through membrane fusion. This process is inhibited by chemokine receptor antagonists (CoRAs) that block Env–receptor interactions and by fusion inhibitors (FIs) that disrupt Env conformational transitions. Synergy between CoRAs and FIs has been attributed to a CoRA-dependent decrease in the rate of viral membrane fusion that extends the lifetime of the intermediate state targeted by FIs. Here, we demonstrated that the magnitude of CoRA/FI synergy unexpectedly depends on FI-binding affinity and the stoichiometry of chemokine receptor binding to trimeric Env. For C-peptide FIs (clinically represented by enfuvirtide), synergy waned as binding strength decreased until inhibitor combinations behaved additively. Curiously, this affinity dependence on synergy was absent for 5-Helix-type FIs. We linked this complex behavior to the CoRA dependence of Env deactivation following FI binding. For both FI classes, reducing chemokine receptor levels on target cells or eliminating competent chemokine receptor-binding sites on Env trimers resulted in a loss of synergistic activity. These data imply that the stoichiometry required for CoRA/FI synergy exceeds that required for HIV-1 entry. Our analysis suggests two distinct roles for chemokine receptor binding, one to trigger formation of the FI-sensitive intermediate state and another to facilitate subsequent conformational transitions. Together, our results could explain the wide variety of previously reported activities for CoRA/FI combinations. These findings also have implications for the combined use of CoRAs and FIs in antiviral therapies and point to a multifaceted role for chemokine receptor binding in promoting HIV-1 entry.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.791731
      Issue No: Vol. 292, No. 40 (2017)
       
  • How entry inhibitors synergize to fight HIV [Microbiology]
    • Authors: Gregory B. Melikyan
      Pages: 16511 - 16512
      Abstract: HIV fusion with the cell membrane can be inhibited by blocking coreceptor binding or by preventing fusion-inducing conformational changes in the Env protein. Logically, inhibitors that act by these two mechanisms should act synergistically, but previous studies have reported conflicting results. A new study by Ahn and Root reconciles these discordant reports by demonstrating that synergy emerges when Env engages multiple coreceptors prior to inducing fusion and when high-affinity inhibitory peptides are used, a condition that may not be satisfied in vivo.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.H117.791731
      Issue No: Vol. 292, No. 40 (2017)
       
  • Disease-causing mutations in the serpin antithrombin reveal a key domain
           critical for inhibiting protease activities [Protein Structure and
           Folding]
    • Authors: Sonia Aguila; Gonzalo Izaguirre, Irene Martinez–Martinez, Vicente Vicente, Steven T. Olson, Javier Corral
      Pages: 16513 - 16520
      Abstract: Antithrombin mainly inhibits factor Xa and thrombin. The reactive center loop (RCL) is crucial for its interactions with its protease targets and is fully inserted into the A-sheet after its cleavage, causing translocation of the covalently linked protease to the opposite end of the A-sheet. Antithrombin variants with altered RCL hinge residues behave as substrates rather than inhibitors, resulting in stoichiometries of inhibition greater than one. Other antithrombin residues have been suggested to interfere with RCL insertion or the stability of the antithrombin–protease complex, but available crystal structures or mutagenesis studies have failed to identify such residues. Here, we characterized two mutations, S365L and I207T, present in individuals with type II antithrombin deficiency and identified a new antithrombin functional domain. S365L did not form stable complexes with thrombin or factor Xa, and the I207T/I207A variants inhibited both proteases with elevated stoichiometries of inhibition. Close proximity of Ile-207 and Ser-365 to the inserted RCL suggested that the preferred reaction of these mutants as protease substrates reflects an effect on the rate of the RCL insertion and protease translocation. However, both residues lie within the final docking site for the protease in the antithrombin–protease complex, supporting the idea that the enhanced substrate reactions may result from an increased dissociation of the final complexes. Our findings demonstrate that the distal end of the antithrombin A-sheet is crucial for the last steps of protease inhibition either by affecting the rate of RCL insertion or through critical interactions with proteases at the end of the A-sheet.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.787325
      Issue No: Vol. 292, No. 40 (2017)
       
  • A novel antithrombin domain dictates the journey's end of a proteinase
           [Protein Structure and Folding]
    • Authors: Ingrid M. Verhamme
      Pages: 16521 - 16522
      Abstract: Antithrombin (AT) is an anticoagulant serpin that irreversibly inactivates the clotting proteinases factor Xa and thrombin by forming covalent complexes with them. Mutations in its critical domains, such as those that impair the conformational rearrangement required for proteinase inactivation, increase the risk of venous thrombosis. Águila et al. characterize for the first time the destabilizing effects of mutations in the region of AT that makes contact with the proteinase in the final acyl-enzyme complex. Their work adds new insight into the unique structural intricacies of the inhibitory mechanism.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.H117.787325
      Issue No: Vol. 292, No. 40 (2017)
       
  • Adaptor protein CD2AP and L-type lectin LMAN2 regulate exosome cargo
           protein trafficking through the Golgi complex. [Additions and Corrections]
           
    • Authors: Sang-Ho Kwon; Sekyung Oh, Marisa Nacke, Keith E. Mostov, Joshua H. Lipschutz
      Pages: 16523 - 16523
      Abstract: VOLUME 291 (2016) PAGES 25462–25475PAGES 25466 and 25469:Figs. 3D and 5C in the initially published version of this article were mistakenly repeated using the images from Figs. 3C and 5B, respectively. These figures have now been corrected with the original experimental images. The rat anti-HA antibody used in these figures was HRP-conjugated (anti-HA-HRP) and was added to the nitrocellulose membrane together with a rabbit anti-β-catenin antibody. The HA (GPRC5B-HA) and the β-catenin chemiluminescent signals were therefore obtained from a single membrane per experiment. The ratio of GPRC5B-HA signal intensity in exosomes to that in total cell lysates from three independent experiments was calculated and normalized to the value calculated for the wild-type cells. This correction does not affect the results or conclusions of the work.jbc;292/40/16523/FU1F1FU1jbc;292/40/16523/FU2F2FU2
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.A116.729202
      Issue No: Vol. 292, No. 40 (2017)
       
  • O-GlcNAcase is an RNA polymerase II elongation factor coupled to pausing
           factors SPT5 and TIF1{beta}. [Additions and Corrections]
    • Authors: Melissa Resto; Bong-Hyun Kim, Alfonso G. Fernandez, Brian J. Abraham, Keji Zhao, Brian A. Lewis
      Pages: 16524 - 16525
      Abstract: VOLUME 291 (2016) PAGES 22703–22713PAGES 22705, 22706, and 22708:There were several errors in this article. Figs. 1C and 2A did not indicate the borders between different sections of the same gel. In Fig. 4, one of the figure panel labels was missing. These errors have now been corrected and do not affect the results or conclusions of this work.jbc;292/40/16524/FU1F1FU1jbc;292/40/16524/FU2F2FU2jbc;292/40/16524/FU3F3FU3
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.A116.751420
      Issue No: Vol. 292, No. 40 (2017)
       
  • Molecular identification and functional characterization of the human
           colonic thiamine pyrophosphate transporter. [Additions and Corrections]
    • Authors: Svetlana M. Nabokina; Katsuhisa Inoue, Veedamali S. Subramanian, Judith E. Valle, Hiroaki Yuasa, Hamid M. Said
      Pages: 16526 - 16526
      Abstract: VOLUME 289 (2014) PAGES 4405–4416A grant was omitted from the grant support footnote. The following information should be added: This work was supported in part by National Institutes of Health Grant AA 18071.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.A113.528257
      Issue No: Vol. 292, No. 40 (2017)
       
  • Leucine-rich repeat-containing G protein-coupled receptor 4 facilitates
           vesicular stomatitis virus infection by binding vesicular stomatitis virus
           glycoprotein [Immunology]
    • Authors: Na Zhang; Hongjun Huang, Binghe Tan, Yinglei Wei, Qingqing Xiong, Yan Yan, Lili Hou, Nannan Wu, Stefan Siwko, Andrea Cimarelli, Jianrong Xu, Honghui Han, Min Qian, Mingyao Liu, Bing Du
      Pages: 16527 - 16538
      Abstract: Vesicular stomatitis virus (VSV) and rabies and Chandipura viruses belong to the Rhabdovirus family. VSV is a common laboratory virus to study viral evolution and host immune responses to viral infection, and recombinant VSV-based vectors have been widely used for viral oncolysis, vaccination, and gene therapy. Although the tropism of VSV is broad, and its envelope glycoprotein G is often used for pseudotyping other viruses, the host cellular components involved in VSV infection remain unclear. Here, we demonstrate that the host protein leucine-rich repeat-containing G protein–coupled receptor 4 (Lgr4) is essential for VSV and VSV-G pseudotyped lentivirus (VSVG-LV) to infect susceptible cells. Accordingly, Lgr4-deficient mice had dramatically decreased VSV levels in the olfactory bulb. Furthermore, Lgr4 knockdown in RAW 264.7 cells also significantly suppressed VSV infection, and Lgr4 overexpression in RAW 264.7 cells enhanced VSV infection. Interestingly, only VSV infection relied on Lgr4, whereas infections with Newcastle disease virus, influenza A virus (A/WSN/33), and herpes simplex virus were unaffected by Lgr4 status. Of note, assays of virus entry, cell ELISA, immunoprecipitation, and surface plasmon resonance indicated that VSV bound susceptible cells via the Lgr4 extracellular domain. Pretreating cells with an Lgr4 antibody, soluble LGR4 extracellular domain, or R-spondin 1 blocked VSV infection by competitively inhibiting VSV binding to Lgr4. Taken together, the identification of Lgr4 as a VSV-specific host factor provides important insights into understanding VSV entry and its pathogenesis and lays the foundation for VSV-based gene therapy and viral oncolytic therapeutics.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.802090
      Issue No: Vol. 292, No. 40 (2017)
       
  • UbMES and UbFluor: Novel probes for ring-between-ring (RBR) E3 ubiquitin
           ligase PARKIN [Protein Synthesis and Degradation]
    • Authors: Sungjin Park; Peter K. Foote, David T. Krist, Sarah E. Rice, Alexander V. Statsyuk
      Pages: 16539 - 16553
      Abstract: Ring-between-ring (RBR) E3 ligases have been implicated in autoimmune disorders and neurodegenerative diseases. The functions of many RBR E3s are poorly defined, and their regulation is complex, involving post-translational modifications and allosteric regulation with other protein partners. The functional complexity of RBRs, coupled with the complexity of the native ubiquitination reaction that requires ATP and E1 and E2 enzymes, makes it difficult to study these ligases for basic research and therapeutic purposes. To address this challenge, we developed novel chemical probes, ubiquitin C-terminal fluorescein thioesters UbMES and UbFluor, to qualitatively and quantitatively assess the activity of the RBR E3 ligase PARKIN in a simple experimental setup and in real time using fluorescence polarization. First, we confirmed that PARKIN does not require an E2 enzyme for substrate ubiquitination, lysine selection, and polyubiquitin chain formation. Second, we confirmed that UbFluor quantitatively detects naturally occurring activation states of PARKIN caused by Ser65 phosphorylation (pPARKIN) and phosphorylated ubiquitin (pUb). Third, we showed that both pUb and the ubiquitin-accepting substrate contribute to maximal pPARKIN ubiquitin conjugation turnover. pUb enhances the transthiolation step, whereas the substrate clears the pPARKIN∼Ub thioester intermediate. Finally, we established that UbFluor can quantify activation or inhibition of PARKIN by structural mutations. These results demonstrate the feasibility of using UbFluor for quantitative studies of the biochemistry of RBR E3s and for high-throughput screening of small-molecule activators or inhibitors of PARKIN and other RBR E3 ligases.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M116.773200
      Issue No: Vol. 292, No. 40 (2017)
       
  • BNGR-A25L and -A27 are two functional G protein-coupled receptors for CAPA
           periviscerokinin neuropeptides in the silkworm Bombyx mori [Signal
           Transduction]
    • Authors: Zhangfei Shen; Yu Chen, Lingjuan Hong, Zhenteng Cui, Huipeng Yang, Xiaobai He, Ying Shi, Liangen Shi, Feng Han, Naiming Zhou
      Pages: 16554 - 16570
      Abstract: CAPA peptides, such as periviscerokinin (PVK), are insect neuropeptides involved in many signaling pathways controlling, for example, metabolism, behavior, and reproduction. They are present in a large number of insects and, together with their cognate receptors, are important for research into approaches for improving insect control. However, the CAPA receptors in the silkworm (Bombyx mori) insect model are unknown. Here, we cloned cDNAs of two putative CAPA peptide receptor genes, BNGR-A27 and -A25, from the brain of B. mori larvae. We found that the predicted BNGR-A27 ORF encodes 450 amino acids and that one BNGR-A25 splice variant encodes a full-length isoform (BNGR-A25L) of 418 amino acid residues and another a short isoform (BNGR-A25S) of 341 amino acids with a truncated C-terminal tail. Functional assays indicated that both BNGR-A25L and -A27 are activated by the PVK neuropeptides Bom-CAPA-PVK-1 and -PVK-2, leading to a significant increase in cAMP-response element–controlled luciferase activity and Ca2+ mobilization in a Gq inhibitor–sensitive manner. In contrast, BNGR-A25S was not significantly activated in response to the PVK peptides. Moreover, Bom-CAPA-PVK-1 directly bound to BNGR-A25L and -A27, but not BNGR-A25S. Of note, CAPA-PVK–mediated ERK1/2 phosphorylation and receptor internalization confirmed that BNGR-A25L and -A27 are two canonical receptors for Bombyx CAPA-PVKs. However, BNGR-A25S alone is a nonfunctional receptor but serves as a dominant-negative protein for BNGR-A25L. These results provide evidence that BNGR-A25L and -A27 are two functional Gq-coupled receptors for Bombyx CAPA-PVKs, enabling the further elucidation of the endocrinological roles of Bom-CAPA-PVKs and their receptors in insect biology.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.803445
      Issue No: Vol. 292, No. 40 (2017)
       
  • A small-molecule modulator of cardiac myosin acts on multiple stages of
           the myosin chemomechanical cycle [Molecular Biophysics]
    • Authors: Raja F. Kawas; Robert L. Anderson, Sadie R. Bartholomew Ingle, Yonghong Song, Arvinder S. Sran, Hector M. Rodriguez
      Pages: 16571 - 16577
      Abstract: Mavacamten, formerly known as MYK-461 is a recently discovered novel small-molecule modulator of cardiac myosin that targets the underlying sarcomere hypercontractility of hypertrophic cardiomyopathy, one of the most prevalent heritable cardiovascular disorders. Studies on isolated cells and muscle fibers as well as intact animals have shown that mavacamten inhibits sarcomere force production, thereby reducing cardiac contractility. Initial mechanistic studies have suggested that mavacamten primarily reduces the steady-state ATPase activity by inhibiting the rate of phosphate release of β-cardiac myosin-S1, but the molecular mechanism of action of mavacamten has not been described. Here we used steady-state and presteady-state kinetic analyses to investigate the mechanism of action of mavacamten. Transient kinetic analyses revealed that mavacamten modulates multiple steps of the myosin chemomechanical cycle. In addition to decreasing the rate-limiting step of the cycle (phosphate release), mavacamten reduced the number of myosin-S1 heads that can interact with the actin thin filament during transition from the weakly to the strongly bound state without affecting the intrinsic rate. Mavacamten also decreased the rate of myosin binding to actin in the ADP-bound state and the ADP-release rate from myosin-S1 alone. We, therefore, conclude that mavacamten acts on multiple stages of the myosin chemomechanical cycle. Although the primary mechanism of mavacamten-mediated inhibition of cardiac myosin is the decrease of phosphate release from β-cardiac myosin-S1, a secondary mechanism decreases the number of actin-binding heads transitioning from the weakly to the strongly bound state, which occurs before phosphate release and may provide an additional method to modulate myosin function.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.776815
      Issue No: Vol. 292, No. 40 (2017)
       
  • Two plant-derived aporphinoid alkaloids exert their antifungal activity by
           disrupting mitochondrial iron-sulfur cluster biosynthesis [Microbiology]
    • Authors: Siddharth K. Tripathi; Tao Xu, Qin Feng, Bharathi Avula, Xiaomin Shi, Xuewen Pan, Melanie M. Mask, Scott R. Baerson, Melissa R. Jacob, Ranga Rao Ravu, Shabana I. Khan, Xing-Cong Li, Ikhlas A. Khan, Alice M. Clark, Ameeta K. Agarwal
      Pages: 16578 - 16593
      Abstract: Eupolauridine and liriodenine are plant-derived aporphinoid alkaloids that exhibit potent inhibitory activity against the opportunistic fungal pathogens Candida albicans and Cryptococcus neoformans. However, the molecular mechanism of this antifungal activity is unknown. In this study, we show that eupolauridine 9591 (E9591), a synthetic analog of eupolauridine, and liriodenine methiodide (LMT), a methiodide salt of liriodenine, mediate their antifungal activities by disrupting mitochondrial iron-sulfur (Fe-S) cluster synthesis. Several lines of evidence supported this conclusion. First, both E9591 and LMT elicited a transcriptional response indicative of iron imbalance, causing the induction of genes that are required for iron uptake and for the maintenance of cellular iron homeostasis. Second, a genome-wide fitness profile analysis showed that yeast mutants with deletions in iron homeostasis-related genes were hypersensitive to E9591 and LMT. Third, treatment of wild-type yeast cells with E9591 or LMT generated cellular defects that mimicked deficiencies in mitochondrial Fe-S cluster synthesis including an increase in mitochondrial iron levels, a decrease in the activities of Fe-S cluster enzymes, a decrease in respiratory function, and an increase in oxidative stress. Collectively, our results demonstrate that E9591 and LMT perturb mitochondrial Fe-S cluster biosynthesis; thus, these two compounds target a cellular pathway that is distinct from the pathways commonly targeted by clinically used antifungal drugs. Therefore, the identification of this pathway as a target for antifungal compounds has potential applications in the development of new antifungal therapies.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.781773
      Issue No: Vol. 292, No. 40 (2017)
       
  • The transmembrane domain of the p75 neurotrophin receptor stimulates
           phosphorylation of the TrkB tyrosine kinase receptor [Cell Biology]
    • Authors: Khalil Saadipour; Michael MacLean, Sean Pirkle, Solav Ali, Maria-Luisa Lopez-Redondo, David L. Stokes, Moses V. Chao
      Pages: 16594 - 16604
      Abstract: The function of protein products generated from intramembraneous cleavage by the γ-secretase complex is not well defined. The γ-secretase complex is responsible for the cleavage of several transmembrane proteins, most notably the amyloid precursor protein that results in Aβ, a transmembrane (TM) peptide. Another protein that undergoes very similar γ-secretase cleavage is the p75 neurotrophin receptor. However, the fate of the cleaved p75 TM domain is unknown. p75 neurotrophin receptor is highly expressed during early neuronal development and regulates survival and process formation of neurons. Here, we report that the p75 TM can stimulate the phosphorylation of TrkB (tyrosine kinase receptor B). In vitro phosphorylation experiments indicated that a peptide representing p75 TM increases TrkB phosphorylation in a dose- and time-dependent manner. Moreover, mutagenesis analyses revealed that a valine residue at position 264 in the rat p75 neurotrophin receptor is necessary for the ability of p75 TM to induce TrkB phosphorylation. Because this residue is just before the γ-secretase cleavage site, we then investigated whether the p75(αγ) peptide, which is a product of both α- and γ-cleavage events, could also induce TrkB phosphorylation. Experiments using TM domains from other receptors, EGFR and FGFR1, failed to stimulate TrkB phosphorylation. Co-immunoprecipitation and biochemical fractionation data suggested that p75 TM stimulates TrkB phosphorylation at the cell membrane. Altogether, our results suggest that TrkB activation by p75(αγ) peptide may be enhanced in situations where the levels of the p75 receptor are increased, such as during brain injury, Alzheimer's disease, and epilepsy.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.788729
      Issue No: Vol. 292, No. 40 (2017)
       
  • Hypothyroidism induced by loss of the manganese efflux transporter
           SLC30A10 may be explained by reduced thyroxine production [Metabolism]
    • Authors: Chunyi Liu; Steven Hutchens, Thomas Jursa, William Shawlot, Elena V. Polishchuk, Roman S. Polishchuk, Beth K. Dray, Andrea C. Gore, Michael Aschner, Donald R. Smith, Somshuvra Mukhopadhyay
      Pages: 16605 - 16615
      Abstract: SLC30A10 and SLC39A14 are manganese efflux and influx transporters, respectively. Loss-of-function mutations in genes encoding either transporter induce hereditary manganese toxicity. Patients have elevated manganese in the blood and brain and develop neurotoxicity. Liver manganese is increased in patients lacking SLC30A10 but not SLC39A14. These organ-specific changes in manganese were recently recapitulated in knockout mice. Surprisingly, Slc30a10 knockouts also had elevated thyroid manganese and developed hypothyroidism. To determine the mechanisms of manganese-induced hypothyroidism and understand how SLC30A10 and SLC39A14 cooperatively mediate manganese detoxification, here we produced Slc39a14 single and Slc30a10/Slc39a14 double knockout mice and compared their phenotypes with that of Slc30a10 single knockouts. Compared with wild-type controls, Slc39a14 single and Slc30a10/Slc39a14 double knockouts had higher manganese levels in the blood and brain but not in the liver. In contrast, Slc30a10 single knockouts had elevated manganese levels in the liver as well as in the blood and brain. Furthermore, SLC30A10 and SLC39A14 localized to the canalicular and basolateral domains of polarized hepatic cells, respectively. Thus, transport activities of both SLC39A14 and SLC30A10 are required for hepatic manganese excretion. Compared with Slc30a10 single knockouts, Slc39a14 single and Slc30a10/Slc39a14 double knockouts had lower thyroid manganese levels and normal thyroid function. Moreover, intrathyroid thyroxine levels of Slc30a10 single knockouts were lower than those of controls. Thus, the hypothyroidism phenotype of Slc30a10 single knockouts is induced by elevated thyroid manganese, which blocks thyroxine production. These findings provide new insights into the mechanisms of manganese detoxification and manganese-induced thyroid dysfunction.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.804989
      Issue No: Vol. 292, No. 40 (2017)
       
  • Both brown adipose tissue and skeletal muscle thermogenesis processes are
           activated during mild to severe cold adaptation in mice [Bioenergetics]
    • Authors: Naresh C. Bal; Sushant Singh, Felipe C. G. Reis, Santosh K. Maurya, Sunil Pani, Leslie A. Rowland, Muthu Periasamy
      Pages: 16616 - 16625
      Abstract: Thermogenesis is an important homeostatic mechanism essential for survival and normal physiological functions in mammals. Both brown adipose tissue (BAT) (i.e. uncoupling protein 1 (UCP1)-based) and skeletal muscle (i.e. sarcolipin (SLN)-based) thermogenesis processes play important roles in temperature homeostasis, but their relative contributions differ from small to large mammals. In this study, we investigated the functional interplay between skeletal muscle- and BAT-based thermogenesis under mild versus severe cold adaptation by employing UCP1−/− and SLN−/− mice. Interestingly, adaptation of SLN−/− mice to mild cold conditions (16 °C) significantly increased UCP1 expression, suggesting increased reliance on BAT-based thermogenesis. This was also evident from structural alterations in BAT morphology, including mitochondrial architecture, increased expression of electron transport chain proteins, and depletion of fat droplets. Similarly, UCP1−/− mice adapted to mild cold up-regulated muscle-based thermogenesis, indicated by increases in muscle succinate dehydrogenase activity, SLN expression, mitochondrial content, and neovascularization, compared with WT mice. These results further confirm that SLN-based thermogenesis is a key player in muscle non-shivering thermogenesis (NST) and can compensate for loss of BAT activity. We also present evidence that the increased reliance on BAT-based NST depends on increased autonomic input, as indicated by abundant levels of tyrosine hydroxylase and neuropeptide Y. Our findings demonstrate that both BAT and muscle-based NST are equally recruited during mild and severe cold adaptation and that loss of heat production from one thermogenic pathway leads to increased recruitment of the other, indicating a functional interplay between these two thermogenic processes.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.790451
      Issue No: Vol. 292, No. 40 (2017)
       
  • P2X7 receptor antagonism prevents IL-1{beta} release from salivary
           epithelial cells and reduces inflammation in a mouse model of autoimmune
           exocrinopathy [Immunology]
    • Authors: Mahmoud G. Khalafalla; Lucas T. Woods, Jean M. Camden, Aslam A. Khan, Kirsten H. Limesand, Michael J. Petris, Laurie Erb, Gary A. Weisman
      Pages: 16626 - 16637
      Abstract: Salivary gland inflammation is a hallmark of Sjögren's syndrome (SS), a common autoimmune disease characterized by lymphocytic infiltration of the salivary gland and loss of saliva secretion, predominantly in women. The P2X7 receptor (P2X7R) is an ATP-gated nonselective cation channel that induces inflammatory responses in cells and tissues, including salivary gland epithelium. In immune cells, P2X7R activation induces the production of proinflammatory cytokines, including IL-1β and IL-18, by inducing the oligomerization of the multiprotein complex NLRP3-type inflammasome. Here, our results show that in primary mouse submandibular gland (SMG) epithelial cells, P2X7R activation also induces the assembly of the NLRP3 inflammasome and the maturation and release of IL-1β, a response that is absent in SMG cells isolated from mice deficient in P2X7Rs (P2X7R−/−). P2X7R-mediated IL-1β release in SMG epithelial cells is dependent on transmembrane Na+ and/or K+ flux and the activation of heat shock protein 90 (HSP90), a protein required for the activation and stabilization of the NLRP3 inflammasome. Also, using the reactive oxygen species (ROS) scavengers N-acetyl cysteine and Mito-TEMPO, we determined that mitochondrial reactive oxygen species are required for P2X7R-mediated IL-1β release. Lastly, in vivo administration of the P2X7R antagonist A438079 in the CD28−/−, IFNγ−/−, NOD.H-2h4 mouse model of salivary gland exocrinopathy ameliorated salivary gland inflammation and enhanced carbachol-induced saliva secretion. These findings demonstrate that P2X7R antagonism in vivo represents a promising therapeutic strategy to limit salivary gland inflammation and improve secretory function.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.790741
      Issue No: Vol. 292, No. 40 (2017)
       
  • Arginine mutations in antibody complementarity-determining regions display
           context-dependent affinity/specificity trade-offs [Protein Structure and
           Folding]
    • Authors: Kathryn E. Tiller; Lijuan Li, Sandeep Kumar, Mark C. Julian, Shekhar Garde, Peter M. Tessier
      Pages: 16638 - 16652
      Abstract: Antibodies commonly accumulate charged mutations in their complementarity-determining regions (CDRs) during affinity maturation to enhance electrostatic interactions. However, charged mutations can mediate non-specific interactions, and it is unclear to what extent CDRs can accumulate charged residues to increase antibody affinity without compromising specificity. This is especially concerning for positively charged CDR mutations that are linked to antibody polyspecificity. To better understand antibody affinity/specificity trade-offs, we have selected single-chain antibody fragments specific for the negatively charged and hydrophobic Alzheimer's amyloid β peptide using weak and stringent selections for antibody specificity. Antibody variants isolated using weak selections for specificity were enriched in arginine CDR mutations and displayed low specificity. Alanine-scanning mutagenesis revealed that the affinities of these antibodies were strongly dependent on their arginine mutations. Antibody variants isolated using stringent selections for specificity were also enriched in arginine CDR mutations, but these antibodies possessed significant improvements in specificity. Importantly, the affinities of the most specific antibodies were much less dependent on their arginine mutations, suggesting that over-reliance on arginine for affinity leads to reduced specificity. Structural modeling and molecular simulations reveal unique hydrophobic environments near the arginine CDR mutations. The more specific antibodies contained arginine mutations in the most hydrophobic portions of the CDRs, whereas the less specific antibodies contained arginine mutations in more hydrophilic regions. These findings demonstrate that arginine mutations in antibody CDRs display context-dependent impacts on specificity and that affinity/specificity trade-offs are governed by the relative contribution of arginine CDR residues to the overall antibody affinity.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.783837
      Issue No: Vol. 292, No. 40 (2017)
       
  • Ablating the protein TBC1D1 impairs contraction-induced sarcolemmal
           glucose transporter 4 redistribution but not insulin-mediated responses in
           rats [Metabolism]
    • Authors: Jamie Whitfield; Sabina Paglialunga, Brennan K. Smith, Paula M. Miotto, Genevieve Simnett, Holly L. Robson, Swati S. Jain, Eric A. F. Herbst, Eric M. Desjardins, David J. Dyck, Lawrence L. Spriet, Gregory R. Steinberg, Graham P. Holloway
      Pages: 16653 - 16664
      Abstract: TBC1 domain family member 1 (TBC1D1), a Rab GTPase-activating protein and paralogue of Akt substrate of 160 kDa (AS160), has been implicated in both insulin- and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase-mediated glucose transporter type 4 (GLUT4) translocation. However, the role of TBC1D1 in contracting muscle remains ambiguous. We therefore explored the metabolic consequence of ablating TBC1D1 in both resting and contracting skeletal muscles, utilizing a rat TBC1D1 KO model. Although insulin administration rapidly increased (p < 0.05) plasma membrane GLUT4 content in both red and white gastrocnemius muscles, the TBC1D1 ablation did not alter this response nor did it affect whole-body insulin tolerance, suggesting that TBC1D1 is not required for insulin-induced GLUT4 trafficking events. Consistent with findings in other models of altered TBC1D1 protein levels, whole-animal and ex vivo skeletal muscle fat oxidation was increased in the TBC1D1 KO rats. Although there was no change in mitochondrial content in the KO rats, maximal ADP-stimulated respiration was higher in permeabilized muscle fibers, which may contribute to the increased reliance on fatty acids in resting KO animals. Despite this increase in mitochondrial oxidative capacity, run time to exhaustion at various intensities was impaired in the KO rats. Moreover, contraction-induced increases in sarcolemmal GLUT4 content and glucose uptake were lower in the white gastrocnemius of the KO animals. Altogether, our results highlight a critical role for TBC1D1 in exercise tolerance and contraction-mediated translocation of GLUT4 to the plasma membrane in skeletal muscle.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.806786
      Issue No: Vol. 292, No. 40 (2017)
       
  • Conformational characterization of nerve growth factor-{beta} reveals that
           its regulatory pro-part domain stabilizes three loop regions in its mature
           part [Neurobiology]
    • Authors: Esben Trabȷerg; Fredrik Kartberg, Soren Christensen, Kasper D. Rand
      Pages: 16665 - 16676
      Abstract: Nerve growth factor-β (NGF) is essential for the correct development of the nervous system. NGF exists in both a mature form and a pro-form (proNGF). The two forms have opposing effects on neurons: NGF induces proliferation, whereas proNGF induces apoptosis via binding to a receptor complex of the common neurotrophin receptor (p75NTR) and sortilin. The overexpression of both proNGF and sortilin has been associated with several neurodegenerative diseases. Insights into the conformational differences between proNGF and NGF are central to a better understanding of the opposing mechanisms of action of NGF and proNGF on neurons. However, whereas the structure of NGF has been determined by X-ray crystallography, the structural details for proNGF remain elusive. Here, using a sensitive MS-based analytical method to measure the hydrogen/deuterium exchange of proteins in solution, we analyzed the conformational properties of proNGF and NGF. We detected the presence of a localized higher-order structure motif in the pro-part of proNGF. Furthermore, by comparing the hydrogen/deuterium exchange in the mature part of NGF and proNGF, we found that the presence of the pro-part in proNGF causes a structural stabilization of three loop regions in the mature part, possibly through a direct molecular interaction. Moreover, using tandem MS analyses, we identified two N-linked and two O-linked glycosylations in the pro-part of proNGF. These results advance our knowledge of the conformational properties of proNGF and NGF and help provide a rationale for the diverse biological effects of NGF and proNGF at the molecular level.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.803320
      Issue No: Vol. 292, No. 40 (2017)
       
  • Single-domain antibodies pinpoint potential targets within Shigella
           invasion plasmid antigen D of the needle tip complex for inhibition of
           type III secretion [Protein Structure and Folding]
    • Authors: Michael L. Barta; Jonathan P. Shearer, Olivia Arizmendi, Jacqueline M. Tremblay, Nurjahan Mehzabeen, Qi Zheng, Kevin P. Battaile, Scott Lovell, Saul Tzipori, William D. Picking, Charles B. Shoemaker, Wendy L. Picking
      Pages: 16677 - 16687
      Abstract: Numerous Gram-negative pathogens infect eukaryotes and use the type III secretion system (T3SS) to deliver effector proteins into host cells. One important T3SS feature is an extracellular needle with an associated tip complex responsible for assembly of a pore-forming translocon in the host cell membrane. Shigella spp. cause shigellosis, also called bacillary dysentery, and invade colonic epithelial cells via the T3SS. The tip complex of Shigella flexneri contains invasion plasmid antigen D (IpaD), which initially regulates secretion and provides a physical platform for the translocon pore. The tip complex represents a promising therapeutic target for many important T3SS-containing pathogens. Here, in an effort to further elucidate its function, we created a panel of single-VH domain antibodies (VHHs) that recognize distinct epitopes within IpaD. These VHHs recognized the in situ tip complex and modulated the infectious properties of Shigella. Moreover, structural elucidation of several IpaD–VHH complexes provided critical insights into tip complex formation and function. Of note, one VHH heterodimer could reduce Shigella hemolytic activity by>80%. Our observations along with previous findings support the hypothesis that the hydrophobic translocator (IpaB in Shigella) likely binds to a region within the tip protein that is structurally conserved across all T3SS-possessing pathogens, suggesting potential therapeutic avenues for managing infections by these pathogens.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.802231
      Issue No: Vol. 292, No. 40 (2017)
       
  • Region-specific protein misfolding cyclic amplification reproduces brain
           tropism of prion strains [Protein Structure and Folding]
    • Authors: Nicolas Privat; Etienne Levavasseur, Serfildan Yildirim, Samia Hannaoui, Jean–Philippe Brandel, Jean–Louis Laplanche, Vincent Beringue, Danielle Seilhean, Stephane Haik
      Pages: 16688 - 16696
      Abstract: Human prion diseases such as Creutzfeldt-Jakob disease are transmissible brain proteinopathies, characterized by the accumulation of a misfolded isoform of the host cellular prion protein (PrP) in the brain. According to the prion model, prions are defined as proteinaceous infectious particles composed solely of this abnormal isoform of PrP (PrPSc). Even in the absence of genetic material, various prion strains can be propagated in experimental models. They can be distinguished by the pattern of disease they produce and especially by the localization of PrPSc deposits within the brain and the spongiform lesions they induce. The mechanisms involved in this strain-specific targeting of distinct brain regions still are a fundamental, unresolved question in prion research. To address this question, we exploited a prion conversion in vitro assay, protein misfolding cyclic amplification (PMCA), by using experimental scrapie and human prion strains as seeds and specific brain regions from mice and humans as substrates. We show here that region-specific PMCA in part reproduces the specific brain targeting observed in experimental, acquired, and sporadic Creutzfeldt-Jakob diseases. Furthermore, we provide evidence that, in addition to cellular prion protein, other region- and species-specific molecular factors influence the strain-dependent prion conversion process. This important step toward understanding prion strain propagation in the human brain may impact research on the molecular factors involved in protein misfolding and the development of ultrasensitive methods for diagnosing prion disease.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.793646
      Issue No: Vol. 292, No. 40 (2017)
       
  • Reactive oxygen species trigger Parkin/PINK1 pathway-dependent mitophagy
           by inducing mitochondrial recruitment of Parkin [Cell Biology]
    • Authors: Bin Xiao; Jian-Yuan Goh, Lin Xiao, Hongxu Xian, Kah-Leong Lim, Yih-Cherng Liou
      Pages: 16697 - 16708
      Abstract: Defective mitophagy linked to dysfunction in the proteins Parkin and PTEN-induced putative kinase 1 (PINK1) is implicated in the pathogenesis of Parkinson's disease. Although the mechanism by which Parkin mediates mitophagy in a PINK1-dependent manner is becoming clearer, the triggers for this mitophagy pathway remain elusive. Reactive oxygen species (ROS) have been suggested as such triggers, but this proposal remains controversial because ROS scavengers fail to retard mitophagy. Here we demonstrate that the role of ROS in mitophagy has been underappreciated as a result of the inefficiency of ROS scavengers to control ROS bursts after high-dose treatment with carbonyl cyanide m-chlorophenylhydrazone. Supporting this, combinatorial treatment with N-acetyl-l-cysteine and catalase substantially inhibited the ROS upsurge and PINK1-dependent Parkin translocation to mitochondria in response to carbonyl cyanide m-chlorophenylhydrazone treatment. In addition to the chemical mitophagy inducer, overexpression of voltage-dependent anion channel 1 (VDAC1) induced Parkin translocation to mitochondria, presumably by stimulating ROS generation. Similarly, combined N-acetyl-l-cysteine and catalase treatment also suppressed VDAC1-induced redistribution of Parkin. Alongside these observations, we also found that the elevated protein level of PINK1 was not necessary for Parkin translocation to mitochondria. Thus, our data suggest that ROS may act as a trigger for the induction of Parkin/PINK1-dependent mitophagy. In addition, our study casts doubt on the importance of protein quantity of PINK1 in the recruitment of Parkin to mitochondria.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.787739
      Issue No: Vol. 292, No. 40 (2017)
       
  • The unique C terminus of the calcineurin isoform CNA{beta}1 confers
           non-canonical regulation of enzyme activity by Ca2+ and calmodulin
           [Enzymology]
    • Authors: Rachel Bond; Nina Ly, Martha S. Cyert
      Pages: 16709 - 16721
      Abstract: Calcineurin, the conserved Ca2+/calmodulin-regulated phosphatase and target of immunosuppressants, plays important roles in the circulatory, nervous, and immune systems. Calcineurin activity strictly depends on Ca2+ and Ca2+-bound calmodulin (Ca2+/CaM) to relieve autoinhibition of the catalytic subunit (CNA) by its C terminus. The C terminus contains two regulatory domains, the autoinhibitory domain (AID) and calmodulin-binding domain (CBD), which block the catalytic center and a conserved substrate-binding groove, respectively. However, this mechanism cannot apply to CNAβ1, an atypical CNA isoform generated by alternative 3′-end processing, whose divergent C terminus shares the CBD common to all isoforms, but lacks the AID. We present the first biochemical characterization of CNAβ1, which is ubiquitously expressed and conserved in vertebrates. We identify a distinct C-terminal autoinhibitory four-residue sequence in CNAβ1, 462LAVP465, which competitively inhibits substrate dephosphorylation. In vitro and cell-based assays revealed that the CNAβ1-containing holoenzyme, CNβ1, is autoinhibited at a single site by either of two inhibitory regions, CBD and LAVP, which block substrate access to the substrate-binding groove. We found that the autoinhibitory segment (AIS), located within the CBD, is progressively removed by Ca2+ and Ca2+/CaM, whereas LAVP remains engaged. This regulatory strategy conferred higher basal and Ca2+-dependent activity to CNβ1, decreasing its dependence on CaM, but also limited maximal enzyme activity through persistence of LAVP-mediated autoinhibiton during Ca2+/CaM stimulation. These regulatory properties may underlie observed differences between the biological activities of CNβ1 and canonical CNβ2. Our insights lay the groundwork for further studies of CNβ1, whose physiological substrates are currently unknown.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.795146
      Issue No: Vol. 292, No. 40 (2017)
       
  • Heartland virus NSs protein disrupts host defenses by blocking the TBK1
           kinase-IRF3 transcription factor interaction and signaling required for
           interferon induction [Molecular Bases of Disease]
    • Authors: Yun-Jia Ning; Kuan Feng, Yuan-Qin Min, Fei Deng, Zhihong Hu, Hualin Wang
      Pages: 16722 - 16733
      Abstract: Heartland virus (HRTV) is a pathogenic phlebovirus related to the severe fever with thrombocytopenia syndrome virus (SFTSV), another phlebovirus causing life-threatening disease in humans. Previous findings have suggested that SFTSV can antagonize the host interferon (IFN) system via viral nonstructural protein (NSs)-mediated sequestration of antiviral signaling proteins into NSs-induced inclusion bodies. However, whether and how HRTV counteracts the host innate immunity is unknown. Here, we report that HRTV NSs (HNSs) also antagonizes IFN and cytokine induction and bolsters viral replication, although no noticeable inclusion body formation was observed in HNSs-expressing cells. Furthermore, HNSs inhibited the virus-triggered activation of IFN-β promoter by specifically targeting the IFN-stimulated response element but not the NF-κB response element. Consistently, HNSs blocked the phosphorylation and nuclear translocation of IFN regulatory factor 3 (IRF3, an IFN-stimulated response element-activating transcription factor). Reporter gene assays next showed that HNSs blockades the antiviral signaling mediated by RIG-I-like receptors likely at the level of TANK-binding kinase 1 (TBK1). Indeed, HNSs strongly interacts with TBK1 as indicated by confocal microscopy and pulldown analyses, and we also noted that the scaffold dimerization domain of TBK1 is required for the TBK1-HNSs interaction. Finally, pulldown assays demonstrated that HNSs expression dose-dependently diminishes a TBK1-IRF3 interaction, further explaining the mechanism for HNSs function. Collectively, these data suggest that HNSs, an antagonist of host innate immunity, interacts with TBK1 and thereby hinders the association of TBK1 with its substrate IRF3, thus blocking IRF3 activation and transcriptional induction of the cellular antiviral responses.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.805127
      Issue No: Vol. 292, No. 40 (2017)
       
  • The ATAD2 bromodomain binds different acetylation marks on the histone H4
           in similar fuzzy complexes [Computational Biology]
    • Authors: Cassiano Langini; Amedeo Caflisch, Andreas Vitalis
      Pages: 16734 - 16745
      Abstract: Bromodomains are protein modules adopting conserved helix bundle folds. Some bromodomain-containing proteins, such as ATPase family AAA domain-containing protein 2 (ATAD2), isoform A, have attracted much interest because they are overexpressed in many types of cancer. Bromodomains bind to acetylated lysine residues on histone tails and thereby facilitate the reading of the histone code. Epigenetic regulators in general have been implicated as indicators, mediators, or causes of a large number of diseases and disorders. To interfere with or modulate these processes, it is therefore of fundamental interest to understand the molecular mechanisms by which epigenetic regulation occurs. Here, we present results from molecular dynamics simulations of a doubly acetylated histone H4 peptide bound to the bromodomain of ATAD2 (hereafter referred to as ATAD2A). These simulations revealed how the flexibility of ATAD2A's major loop, the so-called ZA loop, creates an adaptable interface that preserves the disorder of both peptide and loop in the bound state. We further demonstrate that the binding involves an almost identical average pattern of interactions irrespective of which acetyl mark is inserted into the pocket. In conjunction with a likely mechanism of electrostatically driven recruitment, our simulation results highlight how the bromodomain is built toward promiscuous binding with low specificity. In conclusion, the simulations indicate that disorder and electrostatic steering function jointly to recruit ATAD2A to the histone core and that these fuzzy interactions may promote cooperativity between nearby epigenetic marks.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.786350
      Issue No: Vol. 292, No. 40 (2017)
       
  • Nuclear Gene 33/Mig6 regulates the DNA damage response through an ATM
           serine/threonine kinase-dependent mechanism [Molecular Bases of Disease]
    • Authors: Cen Li; Soyoung Park, Xiaowen Zhang, Leonard M. Eisenberg, Hong Zhao, Zbigniew Darzynkiewicz, Dazhong Xu
      Pages: 16746 - 16759
      Abstract: Gene 33 (Mig6, ERRFI1) is an adaptor protein with multiple cellular functions. We recently linked Gene 33 to the DNA damage response (DDR) induced by hexavalent chromium (Cr(VI)), but the molecular mechanism remains unknown. Here we show that ectopic expression of Gene 33 triggers DDR in an ATM serine/threonine kinase (ATM)–dependent fashion and through pathways dependent or not dependent on ABL proto-oncogene 1 non-receptor tyrosine kinase (c-Abl). We observed the clear presence of Gene 33 in the nucleus and chromatin fractions of the cell. We also found that the nuclear localization of Gene 33 is regulated by its 14-3-3–binding domain and that the chromatin localization of Gene 33 is partially dependent on its ErbB-binding domain. Our data further indicated that Gene 33 may regulate the targeting of c-Abl to chromatin. Moreover, we observed a clear association of Gene 33 with histone H2AX and that ectopic expression of Gene 33 promotes the interaction between ATM and histone H2AX without triggering DNA damage. In summary, our results reveal nuclear functions of Gene 33 that regulate DDR. The nuclear localization of Gene 33 also provides a spatial explanation of the previously reported regulation of apoptosis by Gene 33 via the c-Abl/p73 pathway. On the basis of these findings and our previous studies, we propose that Gene 33 is a proximal regulator of DDR that promotes DNA repair.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.803338
      Issue No: Vol. 292, No. 40 (2017)
       
  • Chimeric rabbit/human Fab antibodies against the hepatitis Be-antigen and
           their potential applications in assays, characterization, and therapy
           [Molecular Bases of Disease]
    • Authors: Xiaolei Zhuang; Norman R. Watts, Ira W. Palmer, Joshua D. Kaufman, Altaira D. Dearborn, Joni L. Trenbeath, Elif Eren, Alasdair C. Steven, Christoph Rader, Paul T. Wingfield
      Pages: 16760 - 16772
      Abstract: Hepatitis B virus (HBV) infection afflicts millions worldwide, causing cirrhosis and liver cancer. HBV e-antigen (HBeAg), a clinical marker for disease severity, is a soluble variant of the viral capsid protein. HBeAg is not required for viral replication but is implicated in establishing immune tolerance and chronic infection. The structure of recombinant e-antigen (rHBeAg) was recently determined, yet to date, the exact nature and quantitation of HBeAg still remain uncertain. Here, to further characterize HBeAg, we used phage display to produce a panel of chimeric rabbit/human monoclonal antibody fragments (both Fab and scFv) against rHBeAg. Several of the Fab/scFv, expressed in Escherichia coli, had unprecedentedly high binding affinities (Kd ∼10−12 m) and high specificity. We used Fab/scFv in the context of an enzyme-linked immunosorbent assay (ELISA) for HBeAg quantification, which we compared with commercially available kits and verified with seroconversion panels, the WHO HBeAg standard, rHBeAg, and patient plasma samples. We found that the specificity and sensitivity are superior to those of existing commercial assays. To identify potential fine differences between rHBeAg and HBeAg, we used these Fabs in microscale immunoaffinity chromatography to purify HBeAg from individual patient plasmas. Western blotting and MS results indicated that rHBeAg and HBeAg are essentially structurally identical, although HBeAg from different patients exhibits minor carboxyl-terminal heterogeneity. We discuss several potential applications for the humanized Fab/scFv.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.802272
      Issue No: Vol. 292, No. 40 (2017)
       
  • Conformational state interactions provide clues to the pharmacochaperone
           potential of serotonin transporter partial substrates [Membrane Biology]
    • Authors: Shreyas Bhat; Peter S. Hasenhuetl, Ameya Kasture, Ali El-Kasaby, Michael H. Baumann, Bruce E. Blough, Sonja Sucic, Walter Sandtner, Michael Freissmuth
      Pages: 16773 - 16786
      Abstract: Point mutations in SLC6 transporters cause misfolding, which can be remedied by pharmacochaperones. The serotonin transporter (SERT/SLC6A4) has a rich pharmacology including inhibitors, releasers (amphetamines, which promote the exchange mode), and more recently, discovered partial substrates. We hypothesized that partial substrates trapped the transporter in one or several states of the transport cycle. This conformational trapping may also be conducive to folding. We selected naphthylpropane-2-amines of the phenethylamine library (PAL) including the partial substrate PAL1045 and its congeners PAL287 and PAL1046. We analyzed their impact on the transport cycle of SERT by biochemical approaches and by electrophysiological recordings; substrate-induced peak currents and steady-state currents monitored the translocation of substrate and co-substrate Na+ across the lipid bilayer and the transport cycle, respectively. These experiments showed that PAL1045 and its congeners bound with different affinities (ranging from nm to μm) to various conformational intermediates of SERT during the transport cycle. Consistent with the working hypothesis, PAL1045 was the most efficacious compound in restoring surface expression and transport activity to the folding-deficient mutant SERT-601PG602-AA. These experiments provide a proof-of-principle for a rational search for pharmacochaperones, which may be useful to restore function to clinically relevant folding-deficient transporter mutants.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.794081
      Issue No: Vol. 292, No. 40 (2017)
       
  • The binding of activated G{alpha}q to phospholipase C-{beta} exhibits
           anomalous affinity [Membrane Biology]
    • Authors: Punya Navaratnarajah; Anne Gershenson, Elliott M. Ross
      Pages: 16787 - 16801
      Abstract: Upon activation by the Gq family of Gα subunits, Gβγ subunits, and some Rho family GTPases, phospholipase C-β (PLC-β) isoforms hydrolyze phosphatidylinositol 4,5-bisphosphate to the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. PLC-β isoforms also function as GTPase-activating proteins, potentiating Gq deactivation. To elucidate the mechanism of this mutual regulation, we measured the thermodynamics and kinetics of PLC-β3 binding to Gαq. FRET and fluorescence correlation spectroscopy, two physically distinct methods, both yielded Kd values of about 200 nm for PLC-β3–Gαq binding. This Kd is 50–100 times greater than the EC50 for Gαq-mediated PLC-β3 activation and for the Gαq GTPase-activating protein activity of PLC-β. The measured Kd was not altered either by the presence of phospholipid vesicles, phosphatidylinositol 4,5-bisphosphate and Ca2+, or by the identity of the fluorescent labels. FRET-based kinetic measurements were also consistent with a Kd of 200 nm. We determined that PLC-β3 hysteresis, whereby PLC-β3 remains active for some time following either Gαq–PLC-β3 dissociation or PLC-β3–potentiated Gαq deactivation, is not sufficient to explain the observed discrepancy between EC50 and Kd. These results indicate that the mechanism by which Gαq and PLC-β3 mutually regulate each other is far more complex than a simple, two-state allosteric model and instead is probably kinetically determined.
      PubDate: 2017-10-06T00:06:27-07:00
      DOI: 10.1074/jbc.M117.809673
      Issue No: Vol. 292, No. 40 (2017)
       
 
 
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