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Journal of Biological Chemistry
Journal Prestige (SJR): 2.672
Citation Impact (citeScore): 4
Number of Followers: 261  
 
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ISSN (Print) 0021-9258 - ISSN (Online) 1083-351X
Published by ASBMB Homepage  [3 journals]
  • Structure of the RAD9-RAD1-HUS1 checkpoint clamp bound to RHINO sheds
           light on the other side of the DNA clamp [DNA and Chromosomes]
    • Authors: Kodai Hara; Nao Iida, Ryota Tamafune, Eiji Ohashi, Hitomi Sakurai, Yoshinobu Ishikawa, Asami Hishiki, Hiroshi Hashimoto
      Pages: 899 - 904
      Abstract: DNA clamp, a highly conserved ring-shaped protein, binds dsDNA within its central pore. Also, DNA clamp interacts with various nuclear proteins on its front, thereby stimulating their enzymatic activities and biological functions. It has been assumed that the DNA clamp is a functionally single-faced ring from bacteria to humans. Here, we report the crystal structure of the heterotrimeric RAD9-RAD1-HUS1 (9-1-1) checkpoint clamp bound to a peptide of RHINO, a recently identified cancer-related protein that interacts with 9-1-1 and promotes activation of the DNA damage checkpoint. This is the first structure of 9-1-1 bound to its partner. The structure reveals that RHINO is unexpectedly bound to the edge and around the back of the 9-1-1 ring through specific interactions with the RAD1 subunit of 9-1-1. Our finding indicates that 9-1-1 is a functionally double-faced DNA clamp.
      PubDate: 2020-01-24T00:05:49-08:00
      DOI: 10.1074/jbc.AC119.011816
      Issue No: Vol. 295, No. 4 (2020)
       
  • The balancing act of R-loop biology: The good, the bad, and the ugly [DNA
           and Chromosomes]
    • Authors: Youssef A. Hegazy; Chrishan M. Fernando, Elizabeth J. Tran
      Pages: 905 - 913
      Abstract: An R-loop is a three-stranded nucleic acid structure that consists of a DNA:RNA hybrid and a displaced strand of DNA. R-loops occur frequently in genomes and have significant physiological importance. They play vital roles in regulating gene expression, DNA replication, and DNA and histone modifications. Several studies have uncovered that R-loops contribute to fundamental biological processes in various organisms. Paradoxically, although they do play essential positive functions required for important biological processes, they can also contribute to DNA damage and genome instability. Recent evidence suggests that R-loops are involved in a number of human diseases, including neurological disorders, cancer, and autoimmune diseases. This review focuses on the molecular basis for R-loop–mediated gene regulation and genomic instability and briefly discusses methods for identifying R-loops in vivo. It also highlights recent studies indicating the role of R-loops in DNA double-strand break repair with an updated view of much-needed future goals in R-loop biology.
      PubDate: 2020-01-24T00:05:49-08:00
      DOI: 10.1074/jbc.REV119.011353
      Issue No: Vol. 295, No. 4 (2020)
       
  • The structural basis of T-cell receptor (TCR) activation: An enduring
           enigma [Signal Transduction]
    • Authors: Roy A. Mariuzza; Pragati Agnihotri, John Orban
      Pages: 914 - 925
      Abstract: T cells are critical for protective immune responses to pathogens and tumors. The T-cell receptor (TCR)–CD3 complex is composed of a diverse αβ TCR heterodimer noncovalently associated with the invariant CD3 dimers CD3ϵγ, CD3ϵδ, and CD3ζζ. The TCR mediates recognition of antigenic peptides bound to MHC molecules (pMHC), whereas the CD3 molecules transduce activation signals to the T cell. Whereas much is known about downstream T-cell signaling pathways, the mechanism whereby TCR engagement by pMHC is first communicated to the CD3 signaling apparatus, a process termed early T-cell activation, remains largely a mystery. In this review, we examine the molecular basis for TCR activation in light of the recently determined cryoEM structure of a complete TCR–CD3 complex. This structure provides an unprecedented opportunity to assess various signaling models that have been proposed for the TCR. We review evidence from single-molecule and structural studies for force-induced conformational changes in the TCR–CD3 complex, for dynamically-driven TCR allostery, and for pMHC-induced structural changes in the transmembrane and cytoplasmic regions of CD3 subunits. We identify major knowledge gaps that must be filled in order to arrive at a comprehensive model of TCR activation that explains, at the molecular level, how pMHC-specific information is transmitted across the T-cell membrane to initiate intracellular signaling. An in-depth understanding of this process will accelerate the rational design of immunotherapeutic agents targeting the TCR–CD3 complex.
      PubDate: 2020-01-24T00:05:49-08:00
      DOI: 10.1074/jbc.REV119.009411
      Issue No: Vol. 295, No. 4 (2020)
       
  • Antibody validation for Western blot: By the user, for the user [Protein
           Structure and Folding]
    • Authors: Lakshmi Pillai-Kastoori; Sam Heaton, Steve D. Shiflett, Annabelle C. Roberts, Alejandra Solache, Amy R. Schutz-Geschwender
      Pages: 926 - 939
      Abstract: Well-characterized antibody reagents play a key role in the reproducibility of research findings, and inconsistent antibody performance leads to variability in Western blotting and other immunoassays. The current lack of clear, accepted standards for antibody validation and reporting of experimental details contributes to this problem. Because the performance of primary antibodies is strongly influenced by assay context, recommendations for validation and usage are unique to each type of immunoassay. Practical strategies are proposed for the validation of primary antibody specificity, selectivity, and reproducibility using Western blot analysis. The antibody should produce reproducible results within and between Western blotting experiments and the observed effect confirmed with a complementary or orthogonal method. Routine implementation of standardized antibody validation and reporting in immunoassays such as Western blotting may promote improved reproducibility across the global life sciences community.
      PubDate: 2020-01-24T00:05:49-08:00
      DOI: 10.1074/jbc.RA119.010472
      Issue No: Vol. 295, No. 4 (2020)
       
  • Overexpression of mitochondrial histidyl-tRNA synthetase restores
           mitochondrial dysfunction caused by a deafness-associated tRNAHis mutation
           [Molecular Bases of Disease]
    • Authors: Shasha Gong; Xiaoqiong Wang, Feilong Meng, Limei Cui, Qiuzi Yi, Qiong Zhao, Xiaohui Cang, Zhiyi Cai, Jun Qin Mo, Yong Liang, Min-Xin Guan
      Pages: 940 - 954
      Abstract: The deafness-associated m.12201T>C mutation affects the A5-U68 base-pairing within the acceptor stem of mitochondrial tRNAHis. The primary defect in this mutation is an alteration in tRNAHis aminoacylation. Here, we further investigate the molecular mechanism of the deafness-associated tRNAHis 12201T>C mutation and test whether the overexpression of the human mitochondrial histidyl-tRNA synthetase gene (HARS2) in cytoplasmic hybrid (cybrid) cells carrying the m.12201T>C mutation reverses mitochondrial dysfunctions. Using molecular dynamics simulations, we demonstrate that the m.12201T>C mutation perturbs the tRNAHis structure and function, supported by decreased melting temperature, conformational changes, and instability of mutated tRNA. We show that the m.12201T>C mutation-induced alteration of aminoacylation tRNAHis causes mitochondrial translational defects and respiratory deficiency. We found that the transfer of HARS2 into the cybrids carrying the m.12201T>C mutation raises the levels of aminoacylated tRNAHis from 56.3 to 75.0% but does not change the aminoacylation of other tRNAs. Strikingly, HARS2 overexpression increased the steady-state levels of tRNAHis and of noncognate tRNAs, including tRNAAla, tRNAGln, tRNAGlu, tRNALeu(UUR), tRNALys, and tRNAMet, in cells bearing the m.12201T>C mutation. This improved tRNA metabolism elevated the efficiency of mitochondrial translation, activities of oxidative phosphorylation complexes, and respiration capacity. Furthermore, HARS2 overexpression markedly increased mitochondrial ATP levels and membrane potential and reduced production of reactive oxygen species in cells carrying the m.12201T>C mutation. These results indicate that HARS2 overexpression corrects the mitochondrial dysfunction caused by the tRNAHis mutation. These findings provide critical insights into the pathophysiology of mitochondrial disease and represent a step toward improved therapeutic interventions for mitochondrial disorders.
      PubDate: 2020-01-24T00:05:49-08:00
      DOI: 10.1074/jbc.RA119.010998
      Issue No: Vol. 295, No. 4 (2020)
       
  • The protein tyrosine phosphatase RPTP{zeta}/phosphacan is critical for
           perineuronal net structure [Neurobiology]
    • Authors: Geoffrey J. Eill; Ashis Sinha, Markus Morawski, Mariano S. Viapiano, Russell T. Matthews
      Pages: 955 - 968
      Abstract: Perineuronal nets (PNNs) are conspicuous neuron-specific substructures within the extracellular matrix of the central nervous system that have generated an explosion of interest over the last decade. These reticulated structures appear to surround synapses on the cell bodies of a subset of the neurons in the central nervous system and play key roles in both developmental and adult-brain plasticity. Despite the interest in these structures and compelling demonstrations of their importance in regulating plasticity, their precise functional mechanisms remain elusive. The limited mechanistic understanding of PNNs is primarily because of an incomplete knowledge of their molecular composition and structure and a failure to identify PNN-specific targets. Thus, it has been challenging to precisely manipulate PNNs to rigorously investigate their function. Here, using mouse models and neuronal cultures, we demonstrate a role of receptor protein tyrosine phosphatase zeta (RPTPζ) in PNN structure. We found that in the absence of RPTPζ, the reticular structure of PNNs is lost and phenocopies the PNN structural abnormalities observed in tenascin-R knockout brains. Furthermore, we biochemically analyzed the contribution of RPTPζ to PNN formation and structure, which enabled us to generate a more detailed model for PNNs. We provide evidence for two distinct kinds of interactions of PNN components with the neuronal surface, one dependent on RPTPζ and the other requiring the glycosaminoglycan hyaluronan. We propose that these findings offer important insight into PNN structure and lay important groundwork for future strategies to specifically disrupt PNNs to precisely dissect their function.
      PubDate: 2020-01-24T00:05:49-08:00
      DOI: 10.1074/jbc.RA119.010830
      Issue No: Vol. 295, No. 4 (2020)
       
  • Distinct alterations of gut morphology and microbiota characterize
           accelerated diabetes onset in nonobese diabetic mice [Molecular Bases of
           Disease]
    • Authors: Marie–Christine Simon; Anna Lena Reinbeck, Corinna Wessel, Julia Heindirk, Tomas Jelenik, Kirti Kaul, Juan Arreguin–Cano, Alexander Strom, Michael Blaut, Fredrik Backhed, Volker Burkart, Michael Roden
      Pages: 969 - 980
      Abstract: The rising prevalence of type 1 diabetes (T1D) over the past decades has been linked to lifestyle changes, but the underlying mechanisms are largely unknown. Recent findings point to gut-associated mechanisms in the control of T1D pathogenesis. In nonobese diabetic (NOD) mice, a model of T1D, diabetes development accelerates after deletion of the Toll-like receptor 4 (TLR4). We hypothesized that altered intestinal functions contribute to metabolic alterations, which favor accelerated diabetes development in TLR4-deficient (TLR4−/−) NOD mice. In 70–90-day-old normoglycemic (prediabetic) female NOD TLR4+/+ and NOD TLR4−/− mice, gut morphology and microbiome composition were analyzed. Parameters of lipid metabolism, glucose homeostasis, and mitochondrial respiratory activity were measured in vivo and ex vivo. Compared with NOD TLR4+/+ mice, NOD TLR4−/− animals showed lower muscle mass of the small intestine, higher abundance of Bacteroidetes, and lower Firmicutes in the large intestine, along with lower levels of circulating short-chain fatty acids (SCFA). These changes are associated with higher body weight, hyperlipidemia, and severe insulin and glucose intolerance, all occurring before the onset of diabetes. These mice also exhibited insulin resistance–related abnormalities of energy metabolism, such as lower total respiratory exchange rates and higher hepatic oxidative capacity. Distinct alterations of gut morphology and microbiota composition associated with reduction of circulating SCFA may contribute to metabolic disorders promoting the progression of insulin-deficient diabetes/T1D development.
      PubDate: 2020-01-24T00:05:49-08:00
      DOI: 10.1074/jbc.RA119.010816
      Issue No: Vol. 295, No. 4 (2020)
       
  • Cultivation at high osmotic pressure confers ubiquinone 8-independent
           protection of respiration on Escherichia coli [Microbiology]
    • Authors: Laura Tempelhagen; Anita Ayer, Doreen E. Culham, Roland Stocker, Janet M. Wood
      Pages: 981 - 993
      Abstract: Ubiquinone 8 (coenzyme Q8 or Q8) mediates electron transfer within the aerobic respiratory chain, mitigates oxidative stress, and contributes to gene expression in Escherichia coli. In addition, Q8 was proposed to confer bacterial osmotolerance by accumulating during growth at high osmotic pressure and altering membrane stability. The osmolyte trehalose and membrane lipid cardiolipin accumulate in E. coli cells cultivated at high osmotic pressure. Here, Q8 deficiency impaired E. coli growth at low osmotic pressure and rendered growth osmotically sensitive. The Q8 deficiency impeded cellular O2 uptake and also inhibited the activities of two proton symporters, the osmosensing transporter ProP and the lactose transporter LacY. Q8 supplementation decreased membrane fluidity in liposomes, but did not affect ProP activity in proteoliposomes, which is respiration-independent. Liposomes and proteoliposomes prepared with E. coli lipids were used for these experiments. Similar oxygen uptake rates were observed for bacteria cultivated at low and high osmotic pressures. In contrast, respiration was dramatically inhibited when bacteria grown at the same low osmotic pressure were shifted to high osmotic pressure. Thus, respiration was restored during prolonged growth of E. coli at high osmotic pressure. Of note, bacteria cultivated at low and high osmotic pressures had similar Q8 concentrations. The protection of respiration was neither diminished by cardiolipin deficiency nor conferred by trehalose overproduction during growth at low osmotic pressure, but rather might be achieved by Q8-independent respiratory chain remodeling. We conclude that osmotolerance is conferred through Q8-independent protection of respiration, not by altering physical properties of the membrane.
      PubDate: 2020-01-24T00:05:49-08:00
      DOI: 10.1074/jbc.RA119.011549
      Issue No: Vol. 295, No. 4 (2020)
       
  • Disruption of hepatic small heterodimer partner induces dissociation of
           steatosis and inflammation in experimental nonalcoholic steatohepatitis
           [Gene Regulation]
    • Authors: Nancy Magee; An Zou, Priyanka Ghosh, Forkan Ahamed, Don Delker, Yuxia Zhang
      Pages: 994 - 1008
      Abstract: Nonalcoholic steatohepatitis (NASH) is a leading cause of chronic liver disease worldwide and is characterized by steatosis, inflammation, and fibrosis. The molecular mechanisms underlying NASH development remain obscure. The nuclear receptor small heterodimer partner (Shp) plays a complex role in lipid metabolism and inflammation. Here, we sought to determine SHP's role in regulating steatosis and inflammation in NASH. Shp deletion in murine hepatocytes (ShpHep−/−) resulted in massive infiltration of macrophages and CD4+ T cells in the liver. ShpHep−/− mice developed reduced steatosis, but surprisingly increased hepatic inflammation and fibrosis after being fed a high-fat, -cholesterol, and -fructose (HFCF) diet. RNA-Seq analysis revealed that pathways involved in inflammation and fibrosis are significantly activated in the liver of ShpHep−/− mice fed a chow diet. After having been fed the HFCF diet, WT mice displayed up-regulated peroxisome proliferator-activated receptor γ (Pparg) signaling in the liver; however, this response was completely abolished in the ShpHep−/− mice. In contrast, livers of ShpHep−/− mice had consistent NF-κB activation. To further characterize the role of Shp specifically in the transition of steatosis to NASH, mice were fed the HFCF diet for 4 weeks, followed by Shp deletion. Surprisingly, Shp deletion after steatosis development exacerbated hepatic inflammation and fibrosis without affecting liver steatosis. Together, our results indicate that, depending on NASH stage, hepatic Shp plays an opposing role in steatosis and inflammation. Mechanistically, Shp deletion in hepatocytes activated NF-κB and impaired Pparg activation, leading to the dissociation of steatosis, inflammation, and fibrosis in NASH development.
      PubDate: 2020-01-24T00:05:49-08:00
      DOI: 10.1074/jbc.RA119.010233
      Issue No: Vol. 295, No. 4 (2020)
       
  • The terminal sialic acid of stage-specific embryonic antigen-4 has a
           crucial role in binding to a cancer-targeting antibody [Glycobiology and
           Extracellular Matrices]
    • Authors: Caroline Soliman; Jia Xin Chua, Mireille Vankemmelbeke, Richard S. McIntosh, Andrew J. Guy, Ian Spendlove, Lindy G. Durrant, Paul A. Ramsland
      Pages: 1009 - 1020
      Abstract: Cancer remains a leading cause of morbidity and mortality worldwide, requiring ongoing development of targeted therapeutics such as monoclonal antibodies. Carbohydrates on embryonic cells are often highly expressed in cancer and are therefore attractive targets for antibodies. Stage-specific embryonic antigen-4 (SSEA-4) is one such glycolipid target expressed in many cancers, including breast and ovarian carcinomas. Here, we defined the structural basis for recognition of SSEA-4 by a novel monospecific chimeric antibody (ch28/11). Five X-ray structures of ch28/11 Fab complexes with the SSEA-4 glycan headgroup, determined at 1.5–2.7 Å resolutions, displayed highly similar three-dimensional structures indicating a stable binding mode. The structures also revealed that by adopting a horseshoe-shaped conformation in a deep groove, the glycan headgroup likely sits flat against the membrane to allow the antibody to interact with SSEA-4 on cancer cells. Moreover, we found that the terminal sialic acid of SSEA-4 plays a dominant role in dictating the exquisite specificity of the ch28/11 antibody. This observation was further supported by molecular dynamics simulations of the ch28/11-glycan complex, which show that SSEA-4 is stabilized by its terminal sialic acid, unlike SSEA-3, which lacks this sialic acid modification. These high-resolution views of how a glycolipid interacts with an antibody may help to advance a new class of cancer-targeting immunotherapy.
      PubDate: 2020-01-24T00:05:49-08:00
      DOI: 10.1074/jbc.RA119.011518
      Issue No: Vol. 295, No. 4 (2020)
       
  • Infection-induced signals generated at the plasma membrane epigenetically
           regulate Wnt signaling in vitro and in vivo [Molecular Bases of Disease]
    • Authors: Ishfaq Ahmed; Badal Chandra Roy, Laxmi Uma Maheswar Rao Jakkula, Dharmalingam Subramaniam, Prasad Dandawate, Shrikant Anant, Venkatesh Sampath, Shahid Umar
      Pages: 1021 - 1035
      Abstract: Wnt signaling regulates immunomodulatory functions during infection and inflammation. Employing NCCIT and HCT116 cells, having high endogenous Wnt signaling, we observed elevated levels of low-density lipoprotein receptor–related protein 5/6 (LRP5/6) and Frizzled class receptor 10 (FZD10) and increases in β-catenin, doublecortin-like kinase 1 (DCLK1), CD44 molecule (CD44), and aldehyde dehydrogenase 1 family member A1 (ALDH1A1). siRNA-induced knockdown of these receptors antagonized TOPflash reporter activity and spheroid growth in vitro and elevated Wnt-inhibitory factor 1 (WIF1) activity. Elevated mRNA and protein levels of LRP5/6 and FZD10 paralleled expression of WNT2b and WNT4 in colonic crypts at days 6 and 12 post-infection with Citrobacter rodentium (CR) and tended to decline at days 20–34. The CR mutant escV or the tankyrase inhibitor XAV939 attenuated these responses. A three-dimensional organoid assay in colonic crypts isolated from CR-infected mice revealed elevated levels of LRP5/6 and FZD10 and β-catenin co-localization with enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2). Co-immunoprecipitation in the membrane fraction revealed that axin associates with LRP5/6 in CR-infected crypts, and this association was correlated with increased β-catenin. Colon tumors from either CR-infected ApcPMin/+ or azoxymethane/dextran sodium sulfate (AOM/DSS)-treated mice had high LRP5/6 or FZD10 levels, and chronic Notch blockade through the γ-secretase inhibitor dibenzazepine down-regulated LRP5/6 and FZD10 expression. In CR-responsive CT-26 cells, siRNA-induced LRP5/6 or FZD10 knockdown antagonized TOPflash reporter activity. Elevated miR-153-3p levels correlated with LRP5/6 and FZD10, and miR-153-3p sequestration via a plasmid-based miR inhibitor system attenuated Wnt signaling. We conclude that infection-induced signals from the plasma membrane epigenetically regulate Wnt signaling.
      PubDate: 2020-01-24T00:05:49-08:00
      DOI: 10.1074/jbc.RA119.010285
      Issue No: Vol. 295, No. 4 (2020)
       
  • The chloroplast metalloproteases VAR2 and EGY1 act synergistically to
           regulate chloroplast development in Arabidopsis [Developmental Biology]
    • Authors: Yafei Qi; Xiaomin Wang, Pei Lei, Huimin Li, Liru Yan, Jun Zhao, Jingjing Meng, Jingxia Shao, Lijun An, Fei Yu, Xiayan Liu
      Pages: 1036 - 1046
      Abstract: Chloroplast development and photosynthesis require the proper assembly and turnover of photosynthetic protein complexes. Chloroplasts harbor a repertoire of proteases to facilitate proteostasis and development. We have previously used an Arabidopsis leaf variegation mutant, yellow variegated2 (var2), defective in thylakoid FtsH protease complexes, as a tool to dissect the genetic regulation of chloroplast development. Here, we report a new genetic enhancer mutant of var2, enhancer of variegation3–1 (evr3–1). We confirm that EVR3 encodes a chloroplast metalloprotease, reported previously as ethylene-dependent gravitropism-deficient and yellow-green1 (EGY1)/ammonium overly sensitive1 (AMOS1). We observed that mutations in EVR3/EGY1/AMOS1 cause more severe leaf variegation in var2–5 and synthetic lethality in var2–4. Using a modified blue-native PAGE system, we reveal abnormal accumulations of photosystem I, photosystem II, and light-harvesting antenna complexes in EVR3/EGY1/AMOS1 mutants. Moreover, we discover distinct roles of VAR2 and EVR3/EGY1/AMOS1 in the turnover of photosystem II reaction center under high light stress. In summary, our findings indicate that two chloroplast metalloproteases, VAR2/AtFtsH2 and EVR3/EGY1/AMOS1, function coordinately to regulate chloroplast development and reveal new roles of EVR3/EGY1/AMOS1 in regulating chloroplast proteostasis in Arabidopsis.
      PubDate: 2020-01-24T00:05:49-08:00
      DOI: 10.1074/jbc.RA119.011853
      Issue No: Vol. 295, No. 4 (2020)
       
  • Structural insights into the catalytic mechanism of lovastatin hydrolase
           [Protein Structure and Folding]
    • Authors: Yajing Liang; Xuefeng Lu
      Pages: 1047 - 1055
      Abstract: The lovastatin hydrolase PcEST from the fungus Penicillium chrysogenum exhibits enormous potential for industrial-scale applications in single-step production of monacolin J, the key precursor for synthesis of the cholesterol-lowering drug simvastatin. This enzyme specifically and efficiently catalyzes the conversion of lovastatin to monacolin J but cannot hydrolyze simvastatin. Understanding the catalytic mechanism and the structure–function relationship of PcEST is therefore important for further lovastatin hydrolase screening, engineering, and commercial applications. Here, we solved four X-ray crystal structures, including apo PcEST (2.3 Å), PcEST in complex with monacolin J (2.48 Å), PcEST complexed with the substrate analog simvastatin (2.4 Å), and an inactivated PcEST variant (S57A) with the lovastatin substrate (2.3 Å). Structure-based biochemical analyses and mutagenesis assays revealed that the Ser57 (nucleophile)–Tyr170 (general base)–Lys60 (general acid) catalytic triad, the hydrogen-bond network (Trp344 and Tyr127) around the active site, and the specific substrate-binding tunnel together determine efficient and specific lovastatin hydrolysis by PcEST. Moreover, steric effects on nucleophilic attack caused by the 2′,2-dimethybutyryl group of simvastatin resulted in no activity of PcEST on simvastatin. On the basis of structural comparisons, we propose several indicators to define lovastatin esterases. Furthermore, using structure-guided enzyme engineering, we developed a PcEST variant, D106A, having improved solubility and thermostability, suggesting a promising application of this variant in industrial processes. To our knowledge, this is the first report describing the mechanism and structure–function relationship of lovastatin hydrolase and providing insights that may guide rapid screening and engineering of additional lovastatin esterase variants.
      PubDate: 2020-01-24T00:05:49-08:00
      DOI: 10.1074/jbc.RA119.011936
      Issue No: Vol. 295, No. 4 (2020)
       
  • SMARCAD1-mediated recruitment of the DNA mismatch repair protein
           MutL{alpha} to MutS{alpha} on damaged chromatin induces apoptosis in human
           cells [DNA and Chromosomes]
    • Authors: Yukimasa Takeishi; Ryosuke Fujikane, Mihoko Rikitake, Yuko Obayashi, Mutsuo Sekiguchi, Masumi Hidaka
      Pages: 1056 - 1065
      Abstract: The mismatch repair (MMR) complex is composed of MutSα (MSH2-MSH6) and MutLα (MLH1-PMS2) and specifically recognizes mismatched bases during DNA replication. O6-Methylguanine is produced by treatment with alkylating agents, such as N-methyl-N-nitrosourea (MNU), and during DNA replication forms a DNA mismatch (i.e. an O6-methylguanine/thymine pair) and induces a G/C to A/T transition mutation. To prevent this outcome, cells carrying this DNA mismatch are eliminated by MMR-dependent apoptosis, but the underlying molecular mechanism is unclear. In this study, we provide evidence that the chromatin-regulatory and ATP-dependent nucleosome-remodeling protein SMARCAD1 is involved in the induction of MMR-dependent apoptosis in human cells. Unlike control cells, SMARCAD1-knockout cells (ΔSMARCAD1) were MNU-resistant, and the appearance of a sub-G1 population and caspase-9 activation were significantly suppressed in the ΔSMARCAD1 cells. Furthermore, the MNU-induced mutation frequencies were increased in these cells. Immunoprecipitation analyses revealed that the recruitment of MutLα to chromatin-bound MutSα, observed in SMARCAD1-proficient cells, is suppressed in ΔSMARCAD1 cells. Of note, the effect of SMARCAD1 on the recruitment of MutLα exclusively depended on the ATPase activity of the protein. On the basis of these findings, we propose that SMARCAD1 induces apoptosis via its chromatin-remodeling activity, which helps recruit MutLα to MutSα on damaged chromatin.
      PubDate: 2020-01-24T00:05:49-08:00
      DOI: 10.1074/jbc.RA119.008854
      Issue No: Vol. 295, No. 4 (2020)
       
  • C-Mannosylation of Toxoplasma gondii proteins promotes attachment to host
           cells and parasite virulence [Microbiology]
    • Authors: Andreia Albuquerque–Wendt; Damien Jacot, Nicolas Dos Santos Pacheco, Carla Seegers, Patricia Zarnovican, Falk F. R. Buettner, Hans Bakker, Dominique Soldati–Favre, Francoise H. Routier
      Pages: 1066 - 1076
      Abstract: C-Mannosylation is a common modification of thrombospondin type 1 repeats present in metazoans and recently identified also in apicomplexan parasites. This glycosylation is mediated by enzymes of the DPY19 family that transfer α-mannoses to tryptophan residues in the sequence WX2WX2C, which is part of the structurally essential tryptophan ladder. Here, deletion of the dpy19 gene in the parasite Toxoplasma gondii abolished C-mannosyltransferase activity and reduced levels of the micronemal protein MIC2. The loss of C-mannosyltransferase activity was associated with weakened parasite adhesion to host cells and with reduced parasite motility, host cell invasion, and parasite egress. Interestingly, the C-mannosyltransferase–deficient Δdpy19 parasites were strongly attenuated in virulence and induced protective immunity in mice. This parasite attenuation could not simply be explained by the decreased MIC2 level and strongly suggests that absence of C-mannosyltransferase activity leads to an insufficient level of additional proteins. In summary, our results indicate that T. gondii C-mannosyltransferase DPY19 is not essential for parasite survival, but is important for adhesion, motility, and virulence.
      PubDate: 2020-01-24T00:05:49-08:00
      DOI: 10.1074/jbc.RA119.010590
      Issue No: Vol. 295, No. 4 (2020)
       
  • A 49-residue sequence motif in the C terminus of Nav1.9 regulates
           trafficking of the channel to the plasma membrane [Neurobiology]
    • Authors: Daria V. Sizova; Jianying Huang, Elizabeth J. Akin, Mark Estacion, Carolina Gomis-Perez, Stephen G. Waxman, Sulayman D. Dib-Hajj
      Pages: 1077 - 1090
      Abstract: Genetic and functional studies have confirmed an important role for the voltage-gated sodium channel Nav1.9 in human pain disorders. However, low functional expression of Nav1.9 in heterologous systems (e.g. in human embryonic kidney 293 (HEK293) cells) has hampered studies of its biophysical and pharmacological properties and the development of high-throughput assays for drug development targeting this channel. The mechanistic basis for the low level of Nav1.9 currents in heterologous expression systems is not understood. Here, we implemented a multidisciplinary approach to investigate the mechanisms that govern functional Nav1.9 expression. Recombinant expression of a series of Nav1.9-Nav1.7 C-terminal chimeras in HEK293 cells identified a 49-amino-acid-long motif in the C terminus of the two channels that regulates expression levels of these chimeras. We confirmed the critical role of this motif in the context of a full-length channel chimera, Nav1.9-Ct49aaNav1.7, which displayed significantly increased current density in HEK293 cells while largely retaining the characteristic Nav1.9-gating properties. High-resolution live microscopy indicated that the newly identified C-terminal motif dramatically increases the number of channels on the plasma membrane of HEK293 cells. Molecular modeling results suggested that this motif is exposed on the cytoplasmic face of the folded C terminus, where it might interact with other channel partners. These findings reveal that a 49-residue-long motif in Nav1.9 regulates channel trafficking to the plasma membrane.
      PubDate: 2020-01-24T00:05:49-08:00
      DOI: 10.1074/jbc.RA119.011424
      Issue No: Vol. 295, No. 4 (2020)
       
  • Phosphatidylinositol 3,4-bisphosphate synthesis and turnover are spatially
           segregated in the endocytic pathway [Lipids]
    • Authors: Haibin Wang; Dinah Loerke, Caroline Bruns, Rainer Muller, Philipp–Alexander Koch, Dmytro Puchkov, Carsten Schultz, Volker Haucke
      Pages: 1091 - 1104
      Abstract: Phosphoinositides play crucial roles in intracellular membrane dynamics and cell signaling, with phosphatidylinositol (PI) 3-phosphates being the predominant phosphoinositide lipids at endosomes and lysosomes, whereas PI 4-phosphates, such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), are enriched at the cell surface including sites of endocytosis. How PI 4-phosphates and PI 3-phosphates are dynamically interconverted within the endocytic pathway and how this is controlled in space and time remains poorly understood. Here, combining live imaging, genome engineering, and acute chemical and genetic manipulations, we found that local synthesis of PI(3,4)P2 by phosphatidylinositol 3-kinase C2α at plasma membrane clathrin-coated pits is spatially segregated from its hydrolysis by the PI(3,4)P2-specific inositol polyphosphate 4-phosphatase 4A (INPP4A). We observed that INPP4A is dispensable for clathrin-mediated endocytosis and is undetectable in endocytic clathrin-coated pits. Instead, we found that INPP4A partially localizes to endosomes and that loss of INPP4A in HAP1 cancer cells perturbs signaling via AKT kinase and mTOR complex 1. These results reveal a function for INPP4-mediated PI(3,4)P2 hydrolysis in local regulation of growth factor and nutrient signals at endosomes in cancer cells. They further suggest a model whereby synthesis and turnover of PI(3,4)P2 are spatially segregated within the endocytic pathway to couple endocytic membrane traffic to growth factor and nutrient signaling.
      PubDate: 2020-01-24T00:05:49-08:00
      DOI: 10.1074/jbc.RA119.011774
      Issue No: Vol. 295, No. 4 (2020)
       
  • Biochemical and structural analyses reveal that the tumor suppressor
           neurofibromin (NF1) forms a high-affinity dimer [Molecular Biophysics]
    • Authors: Mukul Sherekar; Sae-Won Han, Rodolfo Ghirlando, Simon Messing, Matthew Drew, Dana Rabara, Timothy Waybright, Puneet Juneja, Hugh O'Neill, Christopher B. Stanley, Debsindhu Bhowmik, Arvind Ramanathan, Sriram Subramaniam, Dwight V. Nissley, William Gillette, Frank McCormick, Dominic Esposito
      Pages: 1105 - 1119
      Abstract: Neurofibromin is a tumor suppressor encoded by the NF1 gene, which is mutated in Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to aberrant signaling through the RAS–mitogen-activated protein kinase pathway due to disruption of the neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of most of the neurofibromin protein; to date, biochemical and structural data exist only for its GAP domain and a region containing a Sec-PH motif. To better understand the role of this large protein, here we carried out a series of biochemical and biophysical experiments, including size-exclusion chromatography–multiangle light scattering (SEC-MALS), small-angle X-ray and neutron scattering, and analytical ultracentrifugation, indicating that full-length neurofibromin forms a high-affinity dimer. We observed that neurofibromin dimerization also occurs in human cells and likely has biological and clinical implications. Analysis of purified full-length and truncated neurofibromin variants by negative-stain EM revealed the overall architecture of the dimer and predicted the potential interactions that contribute to the dimer interface. We could reconstitute structures resembling high-affinity full-length dimers by mixing N- and C-terminal protein domains in vitro. The reconstituted neurofibromin was capable of GTPase activation in vitro, and co-expression of the two domains in human cells effectively recapitulated the activity of full-length neurofibromin. Taken together, these results suggest how neurofibromin dimers might form and be stabilized within the cell.
      PubDate: 2020-01-24T00:05:50-08:00
      DOI: 10.1074/jbc.RA119.010934
      Issue No: Vol. 295, No. 4 (2020)
       
  • The zebrafish NLRP3 inflammasome has functional roles in ASC-dependent
           interleukin-1{beta} maturation and gasdermin E-mediated pyroptosis [Cell
           Biology]
    • Authors: Jiang-Yuan Li; Yue-Yi Wang, Tong Shao, Dong-Dong Fan, Ai-Fu Lin, Li-Xin Xiang, Jian-Zhong Shao
      Pages: 1120 - 1141
      Abstract: The NLR family pyrin domain containing 3 (NLRP3) inflammasome is one of the best-characterized inflammasomes in humans and other mammals. However, knowledge about the NLRP3 inflammasome in nonmammalian species remains limited. Here, we report the molecular and functional identification of an NLRP3 homolog (DrNLRP3) in a zebrafish (Danio rerio) model. We found that DrNLRP3's overall structural architecture was shared with mammalian NLRP3s. It initiates a classical inflammasome assembly for zebrafish inflammatory caspase (DrCaspase-A/-B) activation and interleukin 1β (DrIL-1β) maturation in an apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)-dependent manner, in which DrNLRP3 organizes DrASC into a filament that recruits DrCaspase-A/-B by homotypic pyrin domain (PYD)–PYD interactions. DrCaspase-A/-B activation in the DrNLRP3 inflammasome occurred in two steps, with DrCaspase-A being activated first and DrCaspase-B second. DrNLRP3 also directly activated full-length DrCaspase-B and elicited cell pyroptosis in a gasdermin E (GSDME)-dependent but ASC-independent manner. These two events were tightly coordinated by DrNLRP3 to ensure efficient IL-1β secretion for the initiation of host innate immunity. By knocking down DrNLRP3 in zebrafish embryos and generating a DrASC-knockout (DrASC−/−) fish clone, we characterized the function of the DrNLRP3 inflammasome in anti-bacterial immunity in vivo. The results of our study disclosed the origin of the NLRP3 inflammasome in teleost fish, providing a cross-species understanding of the evolutionary history of inflammasomes. Our findings also indicate that the NLRP3 inflammasome may coordinate inflammatory cytokine processing and secretion through a GSDME-mediated pyroptotic pathway, uncovering a previously unrecognized regulatory function of NLRP3 in both inflammation and cell pyroptosis.
      PubDate: 2020-01-24T00:05:50-08:00
      DOI: 10.1074/jbc.RA119.011751
      Issue No: Vol. 295, No. 4 (2020)
       
  • Correction: Defining {alpha}-synuclein species responsible for Parkinson's
           disease phenotypes in mice. [Additions and Corrections]
    • Authors: Jessica M. Froula; Marta Castellana–Cruz, Nadia M. Anabtawi, Jose D. Camino, Serene W. Chen, Drake R. Thrasher, Jennifer Freire, Allen A. Yazdi, Sheila Fleming, Christopher M. Dobson, Janet R. Kumita, Nunilo Cremades, Laura A. Volpicelli–Daley
      Pages: 1142 - 1142
      Abstract: VOLUME 294 (2019) PAGES 10392–10406In the published article, there was an inadvertent error in Fig. 5. The panel for the retrobeads (red) and p-α-synuclein inclusions (green) in the cortex at 1 week post-injection was the same as 2 weeks post-injection. The 1-week post-injection panel was incorrect and has been corrected. There are no phosphorylated α-synuclein inclusions or retrotracer beads present in the cortex (or amygdala or SNc) 1 week post-injection.jbc;295/4/1142/F5F1F5Figure 5.
      PubDate: 2020-01-24T00:05:50-08:00
      DOI: 10.1074/jbc.AAC119.012485
      Issue No: Vol. 295, No. 4 (2020)
       
  • Activation of sphingosine 1-phosphate receptor 2 attenuates
           chemotherapy-induced neuropathy [Neurobiology]
    • Authors: Wei Wang; Ping Xiang, Wee Siong Chew, Federico Torta, Aishwarya Bandla, Violeta Lopez, Wei Lun Seow, Brenda Wan Shing Lam, Jing Kai Chang, Peiyan Wong, Kanokporn Chayaburakul, Wei-Yi Ong, Markus R. Wenk, Raghav Sundar, Deron R. Herr
      Pages: 1143 - 1152
      Abstract: Platinum-based therapeutics are used to manage many forms of cancer, but frequently result in peripheral neuropathy. Currently, the only option available to attenuate chemotherapy-induced neuropathy is to limit or discontinue this treatment. Sphingosine 1-phosphate (S1P) is a lipid-based signaling molecule involved in neuroinflammatory processes by interacting with its five cognate receptors: S1P1–5. In this study, using a combination of drug pharmacodynamic analysis in human study participants, disease modeling in rodents, and cell-based assays, we examined whether S1P signaling may represent a potential target in the treatment of chemotherapy-induced neuropathy. To this end, we first investigated the effects of platinum-based drugs on plasma S1P levels in human cancer patients. Our analysis revealed that oxaliplatin treatment specifically increases one S1P species, d16:1 S1P, in these patients. Although d16:1 S1P is an S1P2 agonist, it has lower potency than the most abundant S1P species (d18:1 S1P). Therefore, as d16:1 S1P concentration increases, it is likely to disproportionately activate proinflammatory S1P1 signaling, shifting the balance away from S1P2. We further show that a selective S1P2 agonist, CYM-5478, reduces allodynia in a rat model of cisplatin-induced neuropathy and attenuates the associated inflammatory processes in the dorsal root ganglia, likely by activating stress-response proteins, including ATF3 and HO-1. Cumulatively, the findings of our study suggest that the development of a specific S1P2 agonist may represent a promising therapeutic approach for the management of chemotherapy-induced neuropathy.
      PubDate: 2020-01-24T00:05:50-08:00
      DOI: 10.1074/jbc.RA119.011699
      Issue No: Vol. 295, No. 4 (2020)
       
  • The influenza NS1 protein modulates RIG-I activation via a strain-specific
           direct interaction with the second CARD of RIG-I [Microbiology]
    • Authors: Alexander S. Jureka; Alex B. Kleinpeter, Jennifer L. Tipper, Kevin S. Harrod, Chad M. Petit
      Pages: 1153 - 1164
      Abstract: A critical role of influenza A virus nonstructural protein 1 (NS1) is to antagonize the host cellular antiviral response. NS1 accomplishes this role through numerous interactions with host proteins, including the cytoplasmic pathogen recognition receptor, retinoic acid–inducible gene I (RIG-I). Although the consequences of this interaction have been studied, the complete mechanism by which NS1 antagonizes RIG-I signaling remains unclear. We demonstrated previously that the NS1 RNA-binding domain (NS1RBD) interacts directly with the second caspase activation and recruitment domain (CARD) of RIG-I. We also identified that a single strain-specific polymorphism in the NS1RBD (R21Q) completely abrogates this interaction. Here we investigate the functional consequences of an R21Q mutation on NS1's ability to antagonize RIG-I signaling. We observed that an influenza virus harboring the R21Q mutation in NS1 results in significant up-regulation of RIG-I signaling. In support of this, we determined that an R21Q mutation in NS1 results in a marked deficit in NS1's ability to antagonize TRIM25-mediated ubiquitination of the RIG-I CARDs, a critical step in RIG-I activation. We also observed that WT NS1 is capable of binding directly to the tandem RIG-I CARDs, whereas the R21Q mutation in NS1 significantly inhibits this interaction. Furthermore, we determined that the R21Q mutation does not impede the interaction between NS1 and TRIM25 or NS1RBD's ability to bind RNA. The data presented here offer significant insights into NS1 antagonism of RIG-I and illustrate the importance of understanding the role of strain-specific polymorphisms in the context of this specific NS1 function.
      PubDate: 2020-01-24T00:05:50-08:00
      DOI: 10.1074/jbc.RA119.011410
      Issue No: Vol. 295, No. 4 (2020)
       
 
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