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Journal Cover Journal of Biological Chemistry
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   ISSN (Print) 0021-9258 - ISSN (Online) 1083-351X
   Published by ASBMB Homepage  [3 journals]
  • Membrane protein serendipity [Protein Structure and Folding]
    • Authors: Gunnar von Heijne
      Pages: 3470 - 3476
      Abstract: My scientific career has taken me from chemistry, via theoretical physics and bioinformatics, to molecular biology and even structural biology. Along the way, serendipity led me to work on problems such as the identification of signal peptides that direct protein trafficking, membrane protein biogenesis, and cotranslational protein folding. I've had some great collaborations that came about because of a stray conversation or from following up on an interesting paper. And I've had the good fortune to be asked to sit on the Nobel Committee for Chemistry, where I am constantly reminded of the amazing pace and often intricate history of scientific discovery. Could I have planned this' No way! I just went with the flow …
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.X118.001958
      Issue No: Vol. 293, No. 10 (2018)
  • Restricted processing of CD16a/Fc {gamma} receptor IIIa N-glycans from
           primary human NK cells impacts structure and function [Genomics and
    • Authors: Kashyap R. Patel; Jacob T. Roberts, Ganesh P. Subedi, Adam W. Barb
      Pages: 3477 - 3489
      Abstract: CD16a/Fc γ receptor IIIa is the most abundant antibody Fc receptor expressed on human natural killer (NK) cells and activates a protective cytotoxic response following engagement with antibody clustered on the surface of a pathogen or diseased tissue. Therapeutic monoclonal antibodies (mAbs) with greater Fc-mediated affinity for CD16a show superior therapeutic outcome; however, one significant factor that promotes antibody–CD16a interactions, the asparagine-linked carbohydrates (N-glycans), remains undefined. Here, we purified CD16a from the primary NK cells of three donors and identified a large proportion of hybrid (22%) and oligomannose N-glycans (23%). These proportions indicated restricted N-glycan processing and were unlike those of the recombinant CD16a forms, which have predominantly complex-type N-glycans (82%). Tethering recombinant CD16a to the membrane by including the transmembrane and intracellular domains and via coexpression with the Fc ϵ receptor γ–chain in HEK293F cells was expected to produce N-glycoforms similar to NK cell–derived CD16a but yielded N-glycoforms different from NK cell–derived CD16a and recombinant soluble CD16a. Of note, these differences in CD16a N-glycan composition affected antibody binding: CD16a with oligomannose N-glycans bound IgG1 Fc with 12-fold greater affinity than did CD16a having primarily complex-type and highly branched N-glycans. The changes in binding activity mirrored changes in NMR spectra of the two CD16a glycoforms, indicating that CD16a glycan composition also affects the glycoprotein's structure. These results indicated that CD16a from primary human NK cells is compositionally, and likely also functionally, distinct from commonly used recombinant forms. Furthermore, our study provides critical evidence that cell lineage determines CD16a N-glycan composition and antibody-binding affinity.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.RA117.001207
      Issue No: Vol. 293, No. 10 (2018)
  • Antibody receptors steal the sweet spotlight [Immunology]
    • Authors: Kelsey D. Oliva; Jill M. Cavanaugh, Brian A. Cobb
      Pages: 3490 - 3491
      Abstract: Immunoglobulin G (IgG) antibodies function, in part, through ligation of cell-surface Fc receptors such as FcγRIIIA (also known as CD16A). IgG glycosylation is known to impact antibody function, but the role of FcγRIIIA glycans, if any, is unclear. Patel et al. now reveal that these glycans do impact protein conformation and IgG affinity and display cell-specific glycosylation patterns, leading to a potential model in which the affinity and possibly function of Fc receptors is dictated by the cell type and its surface glycome.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.H118.001955
      Issue No: Vol. 293, No. 10 (2018)
  • Computational antimicrobial peptide design and evaluation against
    • Authors: Deepesh Nagarajan; Tushar Nagarajan, Natasha Roy, Omkar Kulkarni, Sathyabaarathi Ravichandran, Madhulika Mishra, Dipshikha Chakravortty, Nagasuma Chandra
      Pages: 3492 - 3509
      Abstract: There is a pressing need for new therapeutics to combat multidrug- and carbapenem-resistant bacterial pathogens. This challenge prompted us to use a long short-term memory (LSTM) language model to understand the underlying grammar, i.e. the arrangement and frequencies of amino acid residues, in known antimicrobial peptide sequences. According to the output of our LSTM network, we synthesized 10 peptides and tested them against known bacterial pathogens. All of these peptides displayed broad-spectrum antimicrobial activity, validating our LSTM-based peptide design approach. Our two most effective antimicrobial peptides displayed activity against multidrug-resistant clinical isolates of Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and coagulase-negative staphylococci strains. High activity against extended-spectrum β-lactamase, methicillin-resistant S. aureus, and carbapenem-resistant strains was also observed. Our peptides selectively interacted with and disrupted bacterial cell membranes and caused secondary gene-regulatory effects. Initial structural characterization revealed that our most effective peptide appeared to be well folded. We conclude that our LSTM-based peptide design approach appears to have correctly deciphered the underlying grammar of antimicrobial peptide sequences, as demonstrated by the experimentally observed efficacy of our designed peptides.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.M117.805499
      Issue No: Vol. 293, No. 10 (2018)
  • Cholesterol binding to a conserved site modulates the conformation,
           pharmacology, and transport kinetics of the human serotonin transporter
           [Membrane Biology]
    • Authors: Louise Laursen; Kasper Severinsen, Kristina Birch Kristensen, Xavier Periole, Malene Overby, Heidi Kaastrup Muller, Birgit Schiott, Steffen Sinning
      Pages: 3510 - 3523
      Abstract: The serotonin transporter (SERT) is important for reuptake of the neurotransmitter serotonin from the synaptic cleft and is also the target of most antidepressants. It has previously been shown that cholesterol in the membrane bilayer affects the conformation of SERT. Although recent crystal structures have identified several potential cholesterol-binding sites, it is unclear whether any of these potential cholesterol sites are occupied by cholesterol and functionally relevant. In the present study, we focus on the conserved cholesterol site 1 (CHOL1) located in a hydrophobic groove between TM1a, TM5, and TM7. By molecular dynamics simulations, we demonstrate a strong binding of cholesterol to CHOL1 in a membrane bilayer environment. In biochemical experiments, we find that cholesterol depletion induces a more inward-facing conformation favoring substrate analog binding. Consistent with this, we find that mutations in CHOL1 with a negative impact on cholesterol binding induce a more inward-facing conformation, and, vice versa, mutations with a positive impact on cholesterol binding induce a more outward-facing conformation. This shift in transporter conformation dictated by the ability to bind cholesterol in CHOL1 affects the apparent substrate affinity, maximum transport velocity, and turnover rates. Taken together, we show that occupation of CHOL1 by cholesterol is of major importance in the transporter conformational equilibrium, which in turn dictates ligand potency and serotonin transport activity. Based on our findings, we propose a mechanistic model that incorporates the role of cholesterol binding to CHOL1 in the function of SERT.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.M117.809046
      Issue No: Vol. 293, No. 10 (2018)
  • Glucose regulates MafA transcription factor abundance and insulin gene
           expression by inhibiting AMP-activated protein kinase in pancreatic
           {beta}-cells [Gene Regulation]
    • Authors: Ryo Iwaoka; Kohsuke Kataoka
      Pages: 3524 - 3534
      Abstract: Insulin mRNA expression in pancreatic islet β-cells is up-regulated by extracellular glucose concentration, but the underlying mechanism remains incompletely understood. MafA is a transcriptional activator specifically enriched in β-cells that binds to the insulin gene promoter. Its expression is transcriptionally and posttranscriptionally regulated by glucose. Moreover, AMP-activated protein kinase (AMPK), a regulator of cellular energy homeostasis, is inhibited by high glucose, and this inhibition is essential for the up-regulation of insulin gene expression and glucose-stimulated insulin secretion (GSIS). Here we mutagenized the insulin promoter and found that the MafA-binding element C1/RIPE3b is required for glucose- or AMPK-induced alterations in insulin gene promoter activity. Under high-glucose conditions, pharmacological activation of AMPK in isolated mouse islets or MIN6 cells by metformin or 5-aminoimidazole-4-carboxamide riboside decreased MafA protein levels and mRNA expression of insulin and GSIS-related genes (i.e. glut2 and sur1). Overexpression of constitutively active AMPK also reduced MafA and insulin expression. Conversely, pharmacological AMPK inhibition by dorsomorphin (compound C) or expression of a dominant-negative form of AMPK increased MafA and insulin expression under low-glucose conditions. However, AMPK activation or inhibition did not change the expression levels of the β-cell-enriched transcription factors Pdx1 and Beta2/NeuroD1. AMPK activation accelerated MafA protein degradation, which is not dependent on the proteasome. We also noted that MafA overexpression prevents metformin-induced decreases in insulin and GSIS-related gene expression. These findings indicate that high glucose concentrations inhibit AMPK, thereby increasing MafA protein levels and activating the insulin promoter.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.M117.817932
      Issue No: Vol. 293, No. 10 (2018)
  • Characterization of the interactions of potent allosteric inhibitors with
           glutaminase C, a key enzyme in cancer cell glutamine metabolism [Signal
    • Authors: Qingqiu Huang; Clint Stalnecker, Chengliang Zhang, Lee A. McDermott, Prema Iyer, Jason O'Neill, Shawn Reimer, Richard A. Cerione, William P. Katt
      Pages: 3535 - 3545
      Abstract: Altered glycolytic flux in cancer cells (the “Warburg effect”) causes their proliferation to rely upon elevated glutamine metabolism (“glutamine addiction”). This requirement is met by the overexpression of glutaminase C (GAC), which catalyzes the first step in glutamine metabolism and therefore represents a potential therapeutic target. The small molecule CB-839 was reported to be more potent than other allosteric GAC inhibitors, including the parent compound bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl (BPTES), and is in clinical trials. Recently, we described the synthesis of BPTES analogs having distinct saturated heterocyclic cores as a replacement for the flexible chain moiety, with improved microsomal stability relative to CB-839 and BPTES. Here, we show that one of these new compounds, UPGL00004, like CB-839, more potently inhibits the enzymatic activity of GAC, compared with BPTES. We also compare the abilities of UPGL00004, CB-839, and BPTES to directly bind to recombinant GAC and demonstrate that UPGL00004 has a similar binding affinity as CB-839 for GAC. We also show that UPGL00004 potently inhibits the growth of triple-negative breast cancer cells, as well as tumor growth when combined with the anti-vascular endothelial growth factor antibody bevacizumab. Finally, we compare the X-ray crystal structures for UPGL00004 and CB-839 bound to GAC, verifying that UPGL00004 occupies the same binding site as CB-839 or BPTES and that all three inhibitors regulate the enzymatic activity of GAC via a similar allosteric mechanism. These results provide insights regarding the potency of these inhibitors that will be useful in designing novel small-molecules that target a key enzyme in cancer cell metabolism.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.M117.810101
      Issue No: Vol. 293, No. 10 (2018)
  • Hydrogen sulfide inhibits Kir2 and Kir3 channels by decreasing sensitivity
           to the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) [Cell
    • Authors: Junghoon Ha; Yu Xu, Takeharu Kawano, Tyler Hendon, Lia Baki, Sumanta Garai, Andreas Papapetropoulos, Ganesh A. Thakur, Leigh D. Plant, Diomedes E. Logothetis
      Pages: 3546 - 3561
      Abstract: Inwardly rectifying potassium (Kir) channels establish and regulate the resting membrane potential of excitable cells in the heart, brain, and other peripheral tissues. Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key direct activator of ion channels, including Kir channels. The gasotransmitter carbon monoxide has been shown to regulate Kir channel activity by altering channel–PIP2 interactions. Here, we tested in two cellular models the effects and mechanism of action of another gasotransmitter, hydrogen sulfide (H2S), thought to play a key role in cellular responses under ischemic conditions. Direct administration of sodium hydrogen sulfide as an exogenous H2S source and expression of cystathionine γ-lyase, a key enzyme that produces endogenous H2S in specific brain tissues, resulted in comparable current inhibition of several Kir2 and Kir3 channels. This effect resulted from changes in channel-gating kinetics rather than in conductance or cell-surface localization. The extent of H2S regulation depended on the strength of the channel–PIP2 interactions. H2S regulation was attenuated when channel–PIP2 interactions were strengthened and was increased when channel–PIP2 interactions were weakened by depleting PIP2 levels. These H2S effects required specific cytoplasmic cysteine residues in Kir3.2 channels. Mutation of these residues abolished H2S inhibition, and reintroduction of specific cysteine residues back into the background of the cytoplasmic cysteine-lacking mutant rescued H2S inhibition. Molecular dynamics simulation experiments provided mechanistic insights into how potential sulfhydration of specific cysteine residues could lead to changes in channel–PIP2 interactions and channel gating.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.RA117.001679
      Issue No: Vol. 293, No. 10 (2018)
  • Tumor stressors induce two mechanisms of intracellular
           P-glycoprotein-mediated resistance that are overcome by lysosomal-targeted
           thiosemicarbazones [Molecular Bases of Disease]
    • Authors: Lina Al-Akra; Dong-Hun Bae, Sumit Sahni, Michael L. H. Huang, Kyung Chan Park, Darius J. R. Lane, Patric J. Jansson, Des R. Richardson
      Pages: 3562 - 3587
      Abstract: Multidrug resistance (MDR) is a major obstacle in cancer treatment due to the ability of tumor cells to efflux chemotherapeutics via drug transporters (e.g. P-glycoprotein (Pgp; ABCB1)). Although the mechanism of Pgp-mediated drug efflux is known at the plasma membrane, the functional role of intracellular Pgp is unclear. Moreover, there has been intense focus on the tumor micro-environment as a target for cancer treatment. This investigation aimed to dissect the effects of tumor micro-environmental stress on subcellular Pgp expression, localization, and its role in MDR. These studies demonstrated that tumor micro-environment stressors (i.e. nutrient starvation, low glucose levels, reactive oxygen species, and hypoxia) induce Pgp-mediated drug resistance. This occurred by two mechanisms, where stressors induced 1) rapid Pgp internalization and redistribution via intracellular trafficking (within 1 h) and 2) hypoxia-inducible factor-1α expression after longer incubations (4–24 h), which up-regulated Pgp and was accompanied by lysosomal biogenesis. These two mechanisms increased lysosomal Pgp and facilitated lysosomal accumulation of the Pgp substrate, doxorubicin, resulting in resistance. This was consistent with lysosomal Pgp being capable of transporting substrates into lysosomes. Hence, tumor micro-environmental stressors result in: 1) Pgp redistribution to lysosomes; 2) increased Pgp expression; 3) lysosomal biogenesis; and 4) potentiation of Pgp substrate transport into lysosomes. In contrast to doxorubicin, when stress stimuli increased lysosomal accumulation of the cytotoxic Pgp substrate, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), this resulted in the agent overcoming resistance. Overall, this investigation describes a novel approach to overcoming resistance in the stressful tumor micro-environment.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.M116.772699
      Issue No: Vol. 293, No. 10 (2018)
  • Anti-microRNA-222 (anti-miR-222) and -181B suppresses growth of
           tamoxifen-resistant xenografts in mouse by targeting TIMP3 protein and
           modulating mitogenic signal. [Withdrawals/Retractions]
    • Authors: Yuanzhi Lu; Satavisha Roy, Gerard Nuovo, Bhuvaneswari Ramaswamy, Tyler Miller, Charles Shapiro, Samson T. Jacob, Sarmila Majumder
      Pages: 3588 - 3588
      Abstract: VOLUME 286 (2011) PAGES 42292–42302This article has been withdrawn by the authors. The Journal raised questions regarding Figs. 5C, 5D, 6A, 6B, 6C, 6D, 7A, 7C, 7D, 8E, and S3. The authors were able to locate some, but not all, of the original data and were able to locate some repeat experiments performed at the time of the original work, which the authors state support the conclusions of the paper. The authors state that the results of this paper are confirmed by the results of complementary experiments presented in the manuscript and that the principal observation of this paper was further confirmed in a 2014 paper (PMID Medline), in which tamoxifen resistance in breast cancer was connected to miR-221/222, by suppression of TIMP3. The authors stand by the reproducibility of the experimental data and the conclusions of the paper.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.W118.002430
      Issue No: Vol. 293, No. 10 (2018)
  • PTPROt inactivates the oncogenic fusion protein BCR/ABL and suppresses
           transformation of K562 cells. [Withdrawals/Retractions]
    • Authors: Tasneem Motiwala; Sarmila Majumder, Kalpana Ghoshal, Huban Kutay, Jharna Datta, Satavisha Roy, David M. Lucas, Samson T. Jacob
      Pages: 3589 - 3589
      Abstract: VOLUME 284 (2009) PAGES 455–464This article has been withdrawn by the authors. In June 2017, the Journal raised questions about Figs. 1A and 5D. The original data and originally submitted figures were not available for evaluation. The authors were able to locate data for a repeat experiment conducted at the time of the original work for Fig. 1A and offered to prepare a new figure. The authors also offered to repeat the experiment reflected in Fig. 5D and to publish an amended figure. The Journal declined both offers, a decision with which the authors disagree. The authors stand by the reproducibility of the experimental data and the conclusions of the paper. The paper, with confirmatory data supporting the results, can be obtained by contacting the authors.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.W118.002431
      Issue No: Vol. 293, No. 10 (2018)
  • Treatment of PC12 cells with nerve growth factor induces proteasomal
           degradation of T-cadherin that requires tyrosine phosphorylation of its
           cadherin domain. [Withdrawals/Retractions]
    • Authors: Shoumei Bai; Jharna Datta, Samson T. Jacob, Kalpana Ghoshal
      Pages: 3590 - 3590
      Abstract: VOLUME 282 (2007) PAGES 27171–27180This article has been withdrawn by the authors. In June 2017, the Journal raised questions about Figs. 1B, 2C, 3A, 3C, 3E, 4C, 5A, 5C, 6B, 7A, and 7D. The original data and originally submitted figures were not available for evaluation. The authors were able to locate some data for repeat experiments conducted at the time of the original work, which they state support the conclusions of the paper, and offered to prepare new figures based on that data. However, the Journal declined that offer, a decision with which the authors disagree. The authors state that any errors in the construction of figures in the paper do not alter the scientific conclusions of the work, and they stand by the reproducibility of the experimental data and the conclusions of the paper.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.W118.002432
      Issue No: Vol. 293, No. 10 (2018)
  • Role of DNA methyltransferases in regulation of human ribosomal RNA gene
           transcription. [Withdrawals/Retractions]
    • Authors: Sarmila Majumder; Kalpana Ghoshal, Jharna Datta, David Spencer Smith, Shoumei Bai, Samson T. Jacob
      Pages: 3591 - 3591
      Abstract: VOLUME 281 (2006) PAGES 22062–22072This article has been withdrawn by the authors. In June 2017, the Journal questioned whether there were duplications in Figs. 1E and 6C and questioned the absence of data in one field of Fig. 5B. The original data and originally submitted figures were not available for evaluation. The authors are not convinced there are any duplications in Figs. 1E and 6C. Regarding Fig. 5B, the authors agree that the data for “Vector” was inadvertently omitted from the final figure. The authors were able to locate replicate data performed at the time of the original work, which they state shows an absence of expression in the Vector panel. The authors offered to publish a correction based on this repeat experiment and, alternatively, offered to repeat the experiment. However, the Journal declined both these offers, a decision with which the authors disagree. The authors stand by the reproducibility of the experimental data and the conclusions of the paper. The paper, with confirmatory data supporting the results, can be obtained by contacting the authors.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.W118.002433
      Issue No: Vol. 293, No. 10 (2018)
  • Identification of T-cadherin as a novel target of DNA methyltransferase 3B
           and its role in the suppression of nerve growth factor-mediated neurite
           outgrowth in PC12 cells. [Withdrawals/Retractions]
    • Authors: Shoumei Bai; Kalpana Ghoshal, Samson T. Jacob
      Pages: 3592 - 3592
      Abstract: VOLUME 281 (2006) PAGES 13604–13611This article has been withdrawn by the authors. In June 2017, the Journal raised questions concerning Figs. 2B, 3B, and 5A. The original data and originally submitted figures were not available for evaluation. Regarding Fig. 2B, the authors were able to locate a repeated experiment performed at the time of the original work, which they state confirm the results. Regarding Figs. 3B and 5A, the authors are not convinced that any duplication occurred, and they were able to provide to the Journal data from multiple partial repeat experiments performed at the time of the original work, which they state confirm the results. The authors offered to publish substitute figures based on the repeated experiments and, alternatively, offered to repeat the experiments. However, the Journal declined both of these offers, a decision with which the authors disagree. The authors stand by the reproducibility of the experimental data and the conclusions of the paper. The paper, with confirmatory data supporting the results, can be obtained by contacting the authors.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.W118.002434
      Issue No: Vol. 293, No. 10 (2018)
  • Dynamic cycling of t-SNARE acylation regulates platelet exocytosis
           [Molecular Bases of Disease]
    • Authors: Jinchao Zhang; Yunjie Huang, Jing Chen, Haining Zhu, Sidney W. Whiteheart
      Pages: 3593 - 3606
      Abstract: Platelets regulate vascular integrity by secreting a host of molecules that promote hemostasis and its sequelae. Given the importance of platelet exocytosis, it is critical to understand how it is controlled. The t-SNAREs, SNAP-23 and syntaxin-11, lack classical transmembrane domains (TMDs), yet both are associated with platelet membranes and redistributed into cholesterol-dependent lipid rafts when platelets are activated. Using metabolic labeling and hydroxylamine (HA)/HCl treatment, we showed that both contain thioester-linked acyl groups. Mass spectrometry mapping further showed that syntaxin-11 was modified on cysteine 275, 279, 280, 282, 283, and 285, and SNAP-23 was modified on cysteine 79, 80, 83, 85, and 87. Interestingly, metabolic labeling studies showed incorporation of [3H]palmitate into the t-SNAREs increased although the protein levels were unchanged, suggesting that acylation turns over on the two t-SNAREs in resting platelets. Exogenously added fatty acids did compete with [3H]palmitate for t-SNARE labeling. To determine the effects of acylation, we measured aggregation, ADP/ATP release, as well as P-selectin exposure in platelets treated with the acyltransferase inhibitor cerulenin or the thioesterase inhibitor palmostatin B. We found that cerulenin pretreatment inhibited t-SNARE acylation and platelet function in a dose- and time-dependent manner whereas palmostatin B had no detectable effect. Interestingly, pretreatment with palmostatin B blocked the inhibitory effects of cerulenin, suggesting that maintaining the acylation state is important for platelet function. Thus, our work shows that t-SNARE acylation is actively cycling in platelets and suggests that the enzymes regulating protein acylation could be potential targets to control platelet exocytosis in vivo.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.RA117.000140
      Issue No: Vol. 293, No. 10 (2018)
  • Oligomer formation and G-quadruplex binding by purified murine Rif1
           protein, a key organizer of higher-order chromatin architecture [DNA and
    • Authors: Kenji Moriyama; Naoko Yoshizawa-Sugata, Hisao Masai
      Pages: 3607 - 3624
      Abstract: Rap1-interacting protein 1 (Rif1) regulates telomere length in budding yeast. We previously reported that, in metazoans and fission yeast, Rif1 also plays pivotal roles in controlling genome-wide DNA replication timing. We proposed that Rif1 may assemble chromatin compartments that contain specific replication-timing domains by promoting chromatin loop formation. Rif1 also is involved in DNA lesion repair, restart after replication fork collapse, anti-apoptosis activities, replicative senescence, and transcriptional regulation. Although multiple physiological functions of Rif1 have been characterized, biochemical and structural information on mammalian Rif1 is limited, mainly because of difficulties in purifying the full-length protein. Here, we expressed and purified the 2418-amino-acid-long, full-length murine Rif1 as well as its partially truncated variants in human 293T cells. Hydrodynamic analyses indicated that Rif1 forms elongated or extended homo-oligomers in solution, consistent with the presence of a HEAT-type helical repeat segment known to adopt an elongated shape. We also observed that the purified murine Rif1 bound G-quadruplex (G4) DNA with high specificity and affinity, as was previously shown for Rif1 from fission yeast. Both the N-terminal (HEAT-repeat) and C-terminal segments were involved in oligomer formation and specifically bound G4 DNA, and the central intrinsically disordered polypeptide segment increased the affinity for G4. Of note, pulldown assays revealed that Rif1 simultaneously binds multiple G4 molecules. Our findings support a model in which Rif1 modulates chromatin loop structures through binding to multiple G4 assemblies and by holding chromatin fibers together.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.RA117.000446
      Issue No: Vol. 293, No. 10 (2018)
  • An ornithine {omega}-aminotransferase required for growth in the absence
           of exogenous proline in the archaeon Thermococcus kodakarensis
    • Authors: Ren-Chao Zheng; Shin-ichi Hachisuka, Hiroya Tomita, Tadayuki Imanaka, Yu-Guo Zheng, Makoto Nishiyama, Haruyuki Atomi
      Pages: 3625 - 3636
      Abstract: Aminotransferases are pyridoxal 5′-phosphate–dependent enzymes that catalyze reversible transamination reactions between amino acids and α-keto acids, and are important for the cellular metabolism of nitrogen. Many bacterial and eukaryotic ω-aminotransferases that use l-ornithine (Orn), l-lysine (Lys), or γ-aminobutyrate (GABA) have been identified and characterized, but the corresponding enzymes from archaea are unknown. Here, we examined the activity and function of TK2101, a gene annotated as a GABA aminotransferase, from the hyperthermophilic archaeon Thermococcus kodakarensis. We overexpressed the TK2101 gene in T. kodakarensis and purified and characterized the recombinant protein and found that it displays only low levels of GABA aminotransferase activity. Instead, we observed a relatively high ω-aminotransferase activity with l-Orn and l-Lys as amino donors. The most preferred amino acceptor was 2-oxoglutarate. To examine the physiological role of TK2101, we created a TK2101 gene–disruption strain (ΔTK2101), which was auxotrophic for proline. Growth comparison with the parent strain KU216 and the biochemical characteristics of the protein strongly suggested that TK2101 encodes an Orn aminotransferase involved in the biosynthesis of l-Pro. Phylogenetic comparisons of the TK2101 sequence with related sequences retrieved from the databases revealed the presence of several distinct protein groups, some of which having no experimentally studied member. We conclude that TK2101 is part of a novel group of Orn aminotransferases that are widely distributed at least in the genus Thermococcus, but perhaps also throughout the Archaea.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.RA117.001222
      Issue No: Vol. 293, No. 10 (2018)
  • TRPM2 channel-mediated regulation of autophagy maintains mitochondrial
           function and promotes gastric cancer cell survival via the JNK-signaling
           pathway [Membrane Biology]
    • Authors: Shekoufeh Almasi; Barry E. Kennedy, Mariam El–Aghil, Andra M. Sterea, Shashi Guȷar, Santiago Partida–Sanchez, Yassine El Hiani
      Pages: 3637 - 3650
      Abstract: A lack of effective treatment is one of the main factors contributing to gastric cancer–related death. Discovering effective targets and understanding their underlying anti-cancer mechanism are key to achieving the best response to treatment and to limiting side effects. Although recent studies have shown that the cation channel transient receptor potential melastatin-2 (TRPM2) is crucial for cancer cell survival, the exact mechanism remains unclear, limiting its therapeutic potential. Here, using molecular and functional assays, we investigated the role of TRPM2 in survival of gastric cancer cells. Our results indicated that TRPM2 knockdown in AGS and MKN-45 cells decreases cell proliferation and enhances apoptosis. We also observed that the TRPM2 knockdown impairs mitochondrial metabolism, indicated by a decrease in basal and maximal mitochondrial oxygen consumption rates and ATP production. These mitochondrial defects coincided with a decrease in autophagy and mitophagy, indicated by reduced levels of autophagy- and mitophagy-associated proteins (i.e. ATGs, LC3A/B II, and BNIP3). Moreover, we found that TRPM2 modulates autophagy through a c-Jun N-terminal kinase (JNK)-dependent and mechanistic target of rapamycin-independent pathway. We conclude that in the absence of TRPM2, down-regulation of the JNK-signaling pathway impairs autophagy, ultimately causing the accumulation of damaged mitochondria and death of gastric cancer cells. Of note, by inhibiting cell proliferation and promoting apoptosis, the TRPM2 down-regulation enhanced the efficacy of paclitaxel and doxorubicin in gastric cancer cells. Collectively, we provide compelling evidence that TRPM2 inhibition may benefit therapeutic approaches for managing gastric cancer.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.M117.817635
      Issue No: Vol. 293, No. 10 (2018)
  • Mycolyltransferase from Mycobacterium tuberculosis in covalent complex
           with tetrahydrolipstatin provides insights into antigen 85 catalysis
           [Computational Biology]
    • Authors: Christopher M. Goins; Steven Dajnowicz, Micholas D. Smith, Jerry M. Parks, Donald R. Ronning
      Pages: 3651 - 3662
      Abstract: Mycobacterium tuberculosis antigen 85 (Ag85) enzymes catalyze the transfer of mycolic acid (MA) from trehalose monomycolate to produce the mycolyl arabinogalactan (mAG) or trehalose dimycolate (TDM). These lipids define the protective mycomembrane of mycobacteria. The current model of substrate binding within the active sites of Ag85s for the production of TDM is not sterically and geometrically feasible; additionally, this model does not account for the production of mAG. Furthermore, this model does not address how Ag85s limit the hydrolysis of the acyl-enzyme intermediate while catalyzing acyl transfer. To inform an updated model, we obtained an Ag85 acyl-enzyme intermediate structure that resembles the mycolated form. Here, we present a 1.45-Å X-ray crystal structure of M. tuberculosis Ag85C covalently modified by tetrahydrolipstatin (THL), an esterase inhibitor that suppresses M. tuberculosis growth and mimics structural attributes of MAs. The mode of covalent inhibition differs from that observed in the reversible inhibition of the human fatty-acid synthase by THL. Similarities between the Ag85-THL structure and previously determined Ag85C structures suggest that the enzyme undergoes structural changes upon acylation, and positioning of the peptidyl arm of THL limits hydrolysis of the acyl-enzyme adduct. Molecular dynamics simulations of the modeled mycolated-enzyme form corroborate the structural analysis. From these findings, we propose an alternative arrangement of substrates that rectifies issues with the previous model and suggest a direct role for the β-hydroxy of MA in the second half-reaction of Ag85 catalysis. This information affords the visualization of a complete mycolyltransferase catalytic cycle.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.RA117.001681
      Issue No: Vol. 293, No. 10 (2018)
  • LSD1 demethylase and the methyl-binding protein PHF20L1 prevent SET7
           methyltransferase-dependent proteolysis of the stem-cell protein SOX2
           [Gene Regulation]
    • Authors: Chunxiao Zhang; Nam Hoang, Feng Leng, Lovely Saxena, Logan Lee, Salvador Alejo, Dandan Qi, Anthony Khal, Hong Sun, Fei Lu, Hui Zhang
      Pages: 3663 - 3674
      Abstract: The pluripotency-controlling stem-cell protein SRY-box 2 (SOX2) plays a pivotal role in maintaining the self-renewal and pluripotency of embryonic stem cells and also of teratocarcinoma or embryonic carcinoma cells. SOX2 is monomethylated at lysine 119 (Lys-119) in mouse embryonic stem cells by the SET7 methyltransferase, and this methylation triggers ubiquitin-dependent SOX2 proteolysis. However, the molecular regulators and mechanisms controlling SET7-induced SOX2 proteolysis are unknown. Here, we report that in human ovarian teratocarcinoma PA-1 cells, methylation-dependent SOX2 proteolysis is dynamically regulated by the LSD1 lysine demethylase and a methyl-binding protein, PHD finger protein 20–like 1 (PHF20L1). We found that LSD1 not only removes the methyl group from monomethylated Lys-117 (equivalent to Lys-119 in mouse SOX2), but it also demethylates monomethylated Lys-42 in SOX2, a reaction that SET7 also regulated and that also triggered SOX2 proteolysis. Our studies further revealed that PHF20L1 binds both monomethylated Lys-42 and Lys-117 in SOX2 and thereby prevents SOX2 proteolysis. Down-regulation of either LSD1 or PHF20L1 promoted SOX2 proteolysis, which was prevented by SET7 inactivation in both PA-1 and mouse embryonic stem cells. Our studies also disclosed that LSD1 and PHF20L1 normally regulate the growth of pluripotent mouse embryonic stem cells and PA-1 cells by preventing methylation-dependent SOX2 proteolysis. In conclusion, our findings reveal an important mechanism by which the stability of the pluripotency-controlling stem-cell protein SOX2 is dynamically regulated by the activities of SET7, LSD1, and PHF20L1 in pluripotent stem cells.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.RA117.000342
      Issue No: Vol. 293, No. 10 (2018)
  • Mapping allosteric linkage to channel gating by extracellular domains in
           the human epithelial sodium channel [Protein Structure and Folding]
    • Authors: Mahmoud Shobair; Konstantin I. Popov, Yan L. Dang, Hong He, M. Jackson Stutts, Nikolay V. Dokholyan
      Pages: 3675 - 3684
      Abstract: The epithelial sodium channel (ENaC) mediates sodium absorption in lung, kidney, and colon epithelia. Channels in the ENaC/degenerin family possess an extracellular region that senses physicochemical changes in the extracellular milieu and allosterically regulates the channel opening. Proteolytic cleavage activates the ENaC opening, by the removal of specific segments in the finger domains of the α- and γ ENaC-subunits. Cleavage causes perturbations in the extracellular region that propagate to the channel gate. However, it is not known how the channel structure mediates the propagation of activation signals through the extracellular sensing domains. Here, to identify the structure–function determinants that mediate allosteric ENaC activation, we performed MD simulations, thiol modification of residues substituted by cysteine, and voltage-clamp electrophysiology recordings. Our simulations of an ENaC heterotetramer, α1βα2γ, in the proteolytically cleaved and uncleaved states revealed structural pathways in the α-subunit that are responsible for ENaC proteolytic activation. To validate these findings, we performed site-directed mutagenesis to introduce cysteine substitutions in the extracellular domains of the α-, β-, and γ ENaC-subunits. Insertion of a cysteine at the α-subunit Glu557 site, predicted to stabilize a closed state of ENaC, inhibited ENaC basal activity and retarded the kinetics of proteolytic activation by 2-fold. Our results suggest that the lower palm domain of αENaC is essential for ENaC activation. In conclusion, our integrated computational and experimental approach suggests key structure–function determinants for ENaC proteolytic activation and points toward a mechanistic model for the allosteric communication in the extracellular domains of the ENaC/degenerin family channels.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.RA117.000604
      Issue No: Vol. 293, No. 10 (2018)
  • Unraveling the molecular mechanism of interactions of the Rho GTPases
    • Authors: E. Sila Ozdemir; Hyunbum Jang, Attila Gursoy, Ozlem Keskin, Zhigang Li, David B. Sacks, Ruth Nussinov
      Pages: 3685 - 3699
      Abstract: IQ motif–containing GTPase-activating proteins (IQGAPs) are scaffolding proteins playing central roles in cell–cell adhesion, polarity, and motility. The Rho GTPases Cdc42 and Rac1, in their GTP-bound active forms, interact with all three human IQGAPs. The IQGAP–Cdc42 interaction promotes metastasis by enhancing actin polymerization. However, despite their high sequence identity, Cdc42 and Rac1 differ in their interactions with IQGAP. Two Cdc42 molecules can bind to the Ex-domain and the RasGAP site of the GTPase-activating protein (GAP)-related domain (GRD) of IQGAP and promote IQGAP dimerization. Only one Rac1 molecule might bind to the RasGAP site of GRD and may not facilitate the dimerization, and the exact mechanism of Cdc42 and Rac1 binding to IQGAP is unclear. Using all-atom molecular dynamics simulations, site-directed mutagenesis, and Western blotting, we unraveled the detailed mechanisms of Cdc42 and Rac1 interactions with IQGAP2. We observed that Cdc42 binding to the Ex-domain of GRD of IQGAP2 (GRD2) releases the Ex-domain at the C-terminal region of GRD2, facilitating IQGAP2 dimerization. Cdc42 binding to the Ex-domain promoted allosteric changes in the RasGAP site, providing a binding site for the second Cdc42 in the RasGAP site. Of note, the Cdc42 “insert loop” was important for the interaction of the first Cdc42 with the Ex-domain. By contrast, differences in Rac1 insert-loop sequence and structure precluded its interaction with the Ex-domain. Rac1 could bind only to the RasGAP site of apo-GRD2 and could not facilitate IQGAP2 dimerization. Our detailed mechanistic insights help decipher how Cdc42 can stimulate actin polymerization in metastasis.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.RA117.001596
      Issue No: Vol. 293, No. 10 (2018)
  • Controlled dimerization of insulin-like growth factor-1 and insulin
           receptors reveals shared and distinct activities of holo and hybrid
           receptors [Cell Biology]
    • Authors: Jingci Chen; Alison M. Nagle, Yu-Fen Wang, David N. Boone, Adrian V. Lee
      Pages: 3700 - 3709
      Abstract: Breast cancer development and progression are influenced by insulin-like growth factor receptor 1 (IGF1R) and insulin receptor (InsR) signaling, which drive cancer phenotypes such as cell growth, proliferation, and migration. IGF1R and InsR form IGF1R/InsR hybrid receptors (HybRs) consisting of one molecule of IGF1R and one molecule of InsR. The specific signaling and functions of HybR are largely unknown, as HybR is activated by both IGF1 and insulin, and no cellular system expresses HybR in the absence of holo-IGF1R or holo-InsR. Here we studied the role of HybR by constructing inducible chimeric receptors and compared HybR signaling with that of holo-IGF1R and holo-InsR. We cloned chemically inducible chimeric IGF1R and InsR constructs consisting of the extracellular domains of the p75 nerve growth factor receptor fused to the intracellular β subunit of IGF1R or InsR and a dimerization domain. Dimerization with the drugs AP20187 or AP21967 allowed specific and independent activation of holo-IGF1R, holo-InsR, or HybR, resulting in activation of the PI3K pathway. Holo-IGF1R and HybR both promoted cell proliferation and glucose uptake, whereas holo-InsR only promoted glucose uptake, and only holo-IGF1R showed anti-apoptotic effects. We also found that the three receptors differentially regulated gene expression: holo-IGF1R and HybR up-regulated EGR3; holo-InsR specifically down-regulated JUN and BCL2L1; holo-InsR down-regulated but HybR up-regulated HK2; and HybR specifically up-regulated FHL2, ITGA6, and PCK2. Our findings suggest that, when expressed and activated in mammary epithelial cells, HybR acts in a manner similar to IGF1R and support further investigation of the role of HybR in breast cancer.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.M117.789503
      Issue No: Vol. 293, No. 10 (2018)
  • An N-terminal motif unique to primate tau enables differential
           protein-protein interactions [Cell Biology]
    • Authors: Kristie Stefanoska; Alexander Volkerling, Josefine Bertz, Anne Poljak, Yazi D. Ke, Lars M. Ittner, Arne Ittner
      Pages: 3710 - 3719
      Abstract: Compared with other mammalian species, humans are particularly susceptible to tau-mediated neurodegenerative disorders. Differential interactions of the tau protein with other proteins are critical for mediating tau's physiological functions as well as tau-associated pathological processes. Primate tau harbors an 11-amino acid-long motif in its N-terminal region (residues 18–28), which is not present in non-primate species and whose function is unknown. Here, we used deletion mutagenesis to remove this sequence region from the longest human tau isoform, followed by glutathione S-transferase (GST) pulldown assays paired with isobaric tags for relative and absolute quantitation (iTRAQ) multiplex labeling, a quantitative method to measure protein abundance by mass spectrometry. Using this method, we found that the primate-specific N-terminal tau motif differentially mediates interactions with neuronal proteins. Among these binding partners are proteins involved in synaptic transmission (synapsin-1 and synaptotagmin-1) and signaling proteins of the 14-3-3 family. Furthermore, we identified an interaction of tau with a member of the annexin family (annexin A5) that was linked to the 11-residue motif. These results suggest that primate Tau has evolved specific residues that differentially regulate protein–protein interactions compared with tau proteins from other non-primate mammalian species. Our findings provide in vitro insights into tau's interactions with other proteins that may be relevant to human disease.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.RA118.001784
      Issue No: Vol. 293, No. 10 (2018)
  • Evidence of a sequestered imine intermediate during reduction of nitrile
    • Authors: Jihye Jung; Bernd Nidetzky
      Pages: 3720 - 3733
      Abstract: In the biosynthesis of the tRNA-inserted nucleoside queuosine, the nitrile reductase QueF catalyzes conversion of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine (preQ1), a biologically unique four-electron reduction of a nitrile to an amine. The QueF mechanism involves a covalent thioimide adduct between the enzyme and preQ0 that undergoes reduction to preQ1 in two NADPH-dependent steps, presumably via an imine intermediate. Protecting a labile imine from interception by water is fundamental to QueF catalysis for proper enzyme function. In the QueF from Escherichia coli, the conserved Glu89 and Phe228 residues together with a mobile structural element composing the catalytic Cys190 form a substrate-binding pocket that secludes the bound preQ0 completely from solvent. We show here that residue substitutions (E89A, E89L, and F228A) targeted at opening up the binding pocket weakened preQ0 binding at the preadduct stage by up to +10 kJ/mol and profoundly affected catalysis. Unlike wildtype enzyme, the QueF variants, including L191A and I192A, were no longer selective for preQ1 formation. The E89A, E89L, and F228A variants performed primarily (≥90%) a two-electron reduction of preQ0, releasing hydrolyzed imine (7-formyl-7-deazaguanine) as the product. The preQ0 reduction by L191A and I192A gave preQ1 and 7-formyl-7-deazaguanine at a 4:1 and 1:1 ratio, respectively. The proportion of 7-formyl-7-deazaguanine in total product increased with increasing substrate concentration, suggesting a role for preQ0 in a competitor-induced release of the imine intermediate. Collectively, these results provide direct evidence for the intermediacy of an imine in the QueF-catalyzed reaction. They reveal determinants of QueF structure required for imine sequestration and hence for a complete nitrile-to-amine conversion by this class of enzymes.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.M117.804583
      Issue No: Vol. 293, No. 10 (2018)
  • Profilin reduces aggregation and phase separation of huntingtin N-terminal
           fragments by preferentially binding to soluble monomers and oligomers
           [Protein Structure and Folding]
    • Authors: Ammon E. Posey; Kiersten M. Ruff, Tyler S. Harmon, Scott L. Crick, Aimin Li, Marc I. Diamond, Rohit V. Pappu
      Pages: 3734 - 3746
      Abstract: Huntingtin N-terminal fragments (Htt-NTFs) with expanded polyglutamine tracts form a range of neurotoxic aggregates that are associated with Huntington's disease. Here, we show that aggregation of Htt-NTFs, irrespective of polyglutamine length, yields at least three phases (designated M, S, and F) that are delineated by sharp concentration thresholds and distinct aggregate sizes and morphologies. We found that monomers and oligomers make up the soluble M phase, ∼25-nm spheres dominate in the soluble S phase, and long, linear fibrils make up the insoluble F phase. Previous studies showed that profilin, an abundant cellular protein, reduces Htt-NTF aggregation and toxicity in cells. We confirm that profilin achieves its cellular effects through direct binding to the C-terminal proline-rich region of Htt-NTFs. We show that profilin preferentially binds to Htt-NTF M-phase species and destabilizes aggregation and phase separation by shifting the concentration boundaries for phase separation to higher values through a process known as polyphasic linkage. Our experiments, aided by coarse-grained computer simulations and theoretical analysis, suggest that preferential binding of profilin to the M-phase species of Htt-NTFs is enhanced through a combination of specific interactions between profilin and polyproline segments and auxiliary interactions between profilin and polyglutamine tracts. Polyphasic linkage may be a general strategy that cells utilize to regulate phase behavior of aggregation-prone proteins. Accordingly, detailed knowledge of phase behavior and an understanding of how ligands modulate phase boundaries may pave the way for developing new therapeutics against a variety of aggregation-prone proteins.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.RA117.000357
      Issue No: Vol. 293, No. 10 (2018)
  • A human xenobiotic nuclear receptor contributes to nonresponsiveness of
           Mycobacterium tuberculosis to the antituberculosis drug rifampicin
    • Authors: Ella Bhagyaraj; Drishti Tiwari, Nancy Ahuja, Ravikanth Nanduri, Ankita Saini, Rashi Kalra, Sumit Kumar, Ashok Kumar Janmeja, Pawan Gupta
      Pages: 3747 - 3757
      Abstract: Mycobacterium tuberculosis is the causative agent of tuberculosis (TB). It acquires phenotypic drug resistance inside macrophages, and this resistance mainly arises from host-induced stress. However, whether cellular drug-efflux mechanisms in macrophages contribute to nonresponsiveness of M. tuberculosis to anti-TB drugs is unclear. Here, we report that xenobiotic nuclear receptors mediate TB drug nonresponsiveness by modulating drug-efflux transporters in macrophages. This was evident from expression analysis of drug-efflux transporters in macrophages isolated from TB patients. Among patients harboring rifampicin-susceptible M. tuberculosis, we observed increased intracellular survival of M. tuberculosis upon rifampicin treatment of macrophages isolated from patients not responding to anti-TB drugs compared with macrophages from patients who did respond. Of note, M. tuberculosis infection and rifampicin exposure synergistically modulated macrophage drug-efflux transporters in vitro. We also found that the xenobiotic nuclear receptor pregnane X receptor (PXR) modulates macrophage drug-efflux transporter expression and activity, which compromised the anti-TB efficacy of rifampicin. We further validated this finding in a TB mouse model in which use of the PXR antagonist ketoconazole rescued rifampicin anti-TB activity. We conclude that PXR activation in macrophages compromises the efficacy of the anti-TB drug rifampicin. Alternative therapeutic strategies, such as use of the rifampicin derivatives rifapentine and rifabutin, which do not activate PXR, or of a PXR antagonist, may be effective for tackling drug nonresponsiveness of M. tuberculosis that arises from drug-efflux systems of the host.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.M117.818377
      Issue No: Vol. 293, No. 10 (2018)
  • Fibroblast growth factor 2 induces proliferation and fibrosis via
           SNAI1-mediated activation of CDK2 and ZEB1 in corneal endothelium [Signal
    • Authors: Jeong Goo Lee; Eric Jung, Martin Heur
      Pages: 3758 - 3769
      Abstract: Investigating stimulation of endogenous wound healing in corneal endothelial cells (CECs) may help address the global shortage of donor corneas by decreasing the number of transplants performed for blindness because of endothelial dysfunction. We previously reported that IL-1β stimulation leads to fibroblast growth factor (FGF2) expression, enhancing migration and proliferation of mammalian CECs. However, FGF2 also promotes the endothelial-mesenchymal transition, which can lead to retrocorneal membrane formation and blindness. This prompted us to investigate downstream FGF2 signaling targets that could be manipulated to prevent retrocorneal membrane formation. FGF2 stimulation altered cell morphology and induced expression of mesenchymal transition marker genes such as snail family transcriptional repressor 1 (SNAI1), SNAI2, zinc finger E-box–binding homeobox 1 (ZEB1), and ZEB2. This, in turn, induced expression of fibronectin, vimentin, and type I collagen, and suppressed E-cadherin in CECs in vitro and ex vivo. siRNA-mediated SNAI1 knockdown revealed that SNAI1 induces ZEB1 expression, in turn inducing expression of type I collagen, the major component of retrocorneal membranes, and of cyclin-dependent kinase 2 (CDK2) and cyclin E1, promoting cell proliferation. siRNA-mediated knockdown of SNAI1 or ZEB1, but not of CDK2, inhibited FGF2-dependent expression of fibronectin, vimentin, and type I collagen and of suppression of E-cadherin expression. We conclude that SNAI1 is a key regulator of FGF2-dependent mesenchymal transition in human ex vivo corneal endothelium, with ZEB1 regulating type I collagen expression and CDK2 regulating cell proliferation. These results suggest that SNAI1 promotes fibrosis and cell proliferation in human corneal endothelium through ZEB1 and CDK2.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.RA117.000295
      Issue No: Vol. 293, No. 10 (2018)
  • Vesicular nucleotide transporter mediates ATP release and migration in
           neutrophils [Cell Biology]
    • Authors: Yuika Harada; Yuri Kato, Takaaki Miyaji, Hiroshi Omote, Yoshinori Moriyama, Miki Hiasa
      Pages: 3770 - 3779
      Abstract: Neutrophils migrate to sites infected by pathogenic microorganisms. This migration is regulated by neutrophil-secreted ATP, which stimulates neutrophils in an autocrine manner through purinergic receptors on the plasma membrane. Although previous studies have shown that ATP is released through channels at the plasma membrane of the neutrophil, it remains unknown whether it is also released through alternate secretory systems involving vesicular mechanisms. In this study, we investigated the possible involvement of vesicular nucleotide transporter (VNUT), a key molecule for vesicular storage and nucleotide release, in ATP secretion from neutrophils. RT-PCR and Western blotting analysis indicated that VNUT is expressed in mouse neutrophils. Immunohistochemical analysis indicated that VNUT mainly colocalized with matrix metalloproteinase-9 (MMP-9), a marker of tertiary granules, which are secretory organelles. In mouse neutrophils, ATP release was inhibited by clodronate, which is a potent VNUT inhibitor. Furthermore, neutrophils from VNUT−/− mice did not release ATP and exhibited significantly reduced migration in vitro and in vivo. These findings suggest that tertiary granule-localized VNUT is responsible for vesicular ATP release and subsequent neutrophil migration. Thus, these findings suggest an additional mechanism through which ATP is released by neutrophils.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.M117.810168
      Issue No: Vol. 293, No. 10 (2018)
  • Low-level overexpression of p53 promotes warfarin-induced calcification of
           porcine aortic valve interstitial cells by activating Slug gene
           transcription [Molecular Bases of Disease]
    • Authors: Li Gao; Yue Ji, Yan Lu, Ming Qiu, Yejiao Shen, Yaqing Wang, Xiangqing Kong, Yongfeng Shao, Yanhui Sheng, Wei Sun
      Pages: 3780 - 3792
      Abstract: The most frequently used oral anti-coagulant warfarin has been implicated in inducing calcification of aortic valve interstitial cells (AVICs), whereas the mechanism is not fully understood. The low-level activation of p53 is found to be involved in osteogenic transdifferentiation and calcification of AVICs. Whether p53 participates in warfarin-induced AVIC calcification remains unknown. In this study, we investigated the role of low-level p53 overexpression in warfarin-induced porcine AVIC (pAVIC) calcification. Immunostaining, quantitative PCR, and Western blotting revealed that p53 was expressed in human and pAVICs and that p53 expression was slightly increased in calcific human aortic valves compared with non-calcific valves. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining indicated that apoptosis slightly increased in calcific aortic valves than in non-calcific valves. Warfarin treatment led to a low-level increase of p53 mRNA and protein in both pAVICs and mouse aortic valves. Low-level overexpression of p53 in pAVICs via an adenovirus vector did not affect pAVIC apoptosis but promoted warfarin-induced calcium deposition and expression of osteogenic markers. shRNA-mediated p53 knockdown attenuated the pAVIC calcium deposition and osteogenic marker expression. Moreover, ChIP and luciferase assays showed that p53 was recruited to the slug promoter and activated slug expression in calcific pAVICs. Of note, overexpression of Slug increased osteogenic marker Runx2 expression, but not pAVIC calcium deposition, and Slug knockdown attenuated pAVIC calcification and p53-mediated pAVIC calcium deposition and expression of osteogenic markers. In conclusion, we found that p53 plays an important role in warfarin induced pAVIC calcification, and increased slug transcription by p53 is required for p53-mediated pAVIC calcification.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.M117.791145
      Issue No: Vol. 293, No. 10 (2018)
  • The CD300e molecule in mice is an immune-activating receptor [Immunology]
    • Authors: Masamichi Isobe; Kumi Izawa, Masahiro Sugiuchi, Tamami Sakanishi, Ayako Kaitani, Ayako Takamori, Akie Maehara, Toshihiro Matsukawa, Mariko Takahashi, Yoshinori Yamanishi, Toshihiko Oki, Shino Uchida, Koichiro Uchida, Tomoaki Ando, Keiko Maeda, Nobuhiro Nakano, Hideo Yagita, Toshiyuki Takai, Hideoki Ogawa, Ko Okumura, Toshio Kitamura, Jiro Kitaura
      Pages: 3793 - 3805
      Abstract: CD300 molecules (CD300s) belong to paired activating and inhibitory receptor families, which mediate immune responses. Human CD300e (hCD300e) is expressed in monocytes and myeloid dendritic cells and transmits an immune-activating signal by interacting with DNAX-activating protein 12 (DAP12). However, the CD300e ortholog in mice (mCD300e) is poorly characterized. Here, we found that mCD300e is also an immune-activating receptor. We found that mCD300e engagement triggers cytokine production in mCD300e-transduced bone marrow–derived mast cells (BMMCs). Loss of DAP12 and another signaling protein, FcRγ, did not affect surface expression of transduced mCD300e, but abrogated mCD300e-mediated cytokine production in the BMMCs. Co-immunoprecipitation experiments revealed that mCD300e physically interacts with both FcRγ and DAP12, suggesting that mCD300e delivers an activating signal via these two proteins. Binding and reporter assays with the mCD300e extracellular domain identified sphingomyelin as a ligand of both mCD300e and hCD300e. Notably, the binding of sphingomyelin to mCD300e stimulated cytokine production in the transduced BMMCs in an FcRγ- and DAP12-dependent manner. Flow cytometric analysis with an mCD300e-specific Ab disclosed that mCD300e expression is highly restricted to CD115+Ly-6Clow/int peripheral blood monocytes, corresponding to CD14dim/+CD16+ human nonclassical and intermediate monocytes. Loss of FcRγ or DAP12 lowered the surface expression of endogenous mCD300e in the CD115+Ly-6Clow/int monocytes. Stimulation with sphingomyelin failed to activate the CD115+Ly-6Clow/int mouse monocytes, but induced hCD300e-mediated cytokine production in the CD14dimCD16+ human monocytes. Taken together, these observations indicate that mCD300e recognizes sphingomyelin and thereby regulates nonclassical and intermediate monocyte functions through FcRγ and DAP12.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.RA117.000696
      Issue No: Vol. 293, No. 10 (2018)
  • Oxysterol-binding protein-related protein 5 (ORP5) promotes cell
           proliferation by activation of mTORC1 signaling [Cell Biology]
    • Authors: Ximing Du; Armella Zadoorian, Ivan E. Lukmantara, Yanfei Qi, Andrew J. Brown, Hongyuan Yang
      Pages: 3806 - 3818
      Abstract: Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a large family of proteins that mainly function in lipid transport and sensing. ORP5 is an endoplasmic reticulum (ER)-anchored protein implicated in lipid transfer at the contact sites between the ER and other membranes. Recent studies indicate that ORP5 is also involved in cancer cell invasion and tumor progression. However, the molecular mechanism underlying ORP5's involvement in cancer is unclear. Here, we report that ORP5 promotes cell proliferation and motility of HeLa cells, an effect that depends on its functional OSBP-related domain (ORD). We also found that ORP5 depletion or substitutions of key residues located within ORP5–ORD and responsible for interactions with lipids interfered with cell proliferation, migration, and invasion. ORP5 interacted with the protein mechanistic target of rapamycin (mTOR), and this interaction also required ORP5–ORD. Of note, whereas ORP5 overexpression induced mTOR complex 1 (mTORC1) activity, ORP5 down-regulation had the opposite effect. Finally, ORP5-depleted cells exhibited impaired mTOR localization to lysosomes, which may have accounted for the blunted mTORC1 activation. Together, our results suggest that ORP5 expression is positively correlated with mTORC1 signaling and that ORP5 stimulates cell proliferation, at least in part, by activating mTORC1.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.RA117.001558
      Issue No: Vol. 293, No. 10 (2018)
  • In vitro reconstitution, functional dissection, and mutational analysis of
           metal ion transport by mitoferrin-1 [Molecular Biophysics]
    • Authors: Eric T. Christenson; Austin S. Gallegos, Anirban Banerjee
      Pages: 3819 - 3828
      Abstract: Iron is universally important to cellular metabolism, and mitoferrin-1 and -2 have been proposed to be the iron importers of mitochondria, the cell's assembly plant of heme and iron–sulfur clusters. These iron-containing prosthetic groups are critical for a host of physiological processes ranging from oxygen transport and energy consumption to maintaining protein structural integrity. Mitoferrin-1 (Mfrn1) belongs to the mitochondrial carrier (MC) family and is atypical given its putative metallic cargo; most MCs transport nucleotides, amino acids, or other small- to medium-size metabolites. Despite the clear importance of Mfrn1 in iron utilization, its transport activity has not been demonstrated unambiguously. To bridge this knowledge gap, we have purified recombinant Mfrn1 under non-denaturing conditions and probed its metal ion–binding and transport functions. Isothermal titration calorimetry indicates that Mfrn1 has micromolar affinity for Fe(II), Mn(II), Co(II), and Ni(II). Mfrn1 was incorporated into defined liposomes, and iron transport was reconstituted in vitro, demonstrating that Mfrn1 can transport iron. Mfrn1 can also transport manganese, cobalt, copper, and zinc but discriminates against nickel. Experiments with candidate ligands for cellular labile iron reveal that Mfrn1 transports free iron and not a chelated iron complex and selects against alkali divalent ions. Extensive mutagenesis identified multiple residues that are crucial for metal binding, transport activity, or both. There is a clear abundance of residues with side chains that can coordinate first-row transition metal ions, suggesting that these could form primary or auxiliary metal-binding sites during the transport process.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.M117.817478
      Issue No: Vol. 293, No. 10 (2018)
  • Histone variant H3.3-mediated chromatin remodeling is essential for
           paternal genome activation in mouse preimplantation embryos [Cell Biology]
    • Authors: Qingran Kong; Laura A. Banaszynski, Fuqiang Geng, Xiaolei Zhang, Jiaming Zhang, Heng Zhang, Claire L. O'Neill, Peidong Yan, Zhonghua Liu, Koji Shido, Gianpiero D. Palermo, C. David Allis, Shahin Rafii, Zev Rosenwaks, Duancheng Wen
      Pages: 3829 - 3838
      Abstract: Derepression of chromatin-mediated transcriptional repression of paternal and maternal genomes is considered the first major step that initiates zygotic gene expression after fertilization. The histone variant H3.3 is present in both male and female gametes and is thought to be important for remodeling the paternal and maternal genomes for activation during both fertilization and embryogenesis. However, the underlying mechanisms remain poorly understood. Using our H3.3B-HA–tagged mouse model, engineered to report H3.3 expression in live animals and to distinguish different sources of H3.3 protein in embryos, we show here that sperm-derived H3.3 (sH3.3) protein is removed from the sperm genome shortly after fertilization and extruded from the zygotes via the second polar bodies (PBII) during embryogenesis. We also found that the maternal H3.3 (mH3.3) protein is incorporated into the paternal genome as early as 2 h postfertilization and is detectable in the paternal genome until the morula stage. Knockdown of maternal H3.3 resulted in compromised embryonic development both of fertilized embryos and of androgenetic haploid embryos. Furthermore, we report that mH3.3 depletion in oocytes impairs both activation of the Oct4 pluripotency marker gene and global de novo transcription from the paternal genome important for early embryonic development. Our results suggest that H3.3-mediated paternal chromatin remodeling is essential for the development of preimplantation embryos and the activation of the paternal genome during embryogenesis.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.RA117.001150
      Issue No: Vol. 293, No. 10 (2018)
  • An essential thioredoxin is involved in the control of the cell cycle in
           the bacterium Caulobacter crescentus [Protein Synthesis and Degradation]
    • Authors: Camille V. Goemans; Francois Beaufay, Khadiȷa Wahni, Inge Van Molle, Joris Messens, Jean–Francois Collet
      Pages: 3839 - 3848
      Abstract: Thioredoxins (Trxs) are antioxidant proteins that are conserved among all species. These proteins have been extensively studied and perform reducing reactions on a broad range of substrates. Here, we identified Caulobacter crescentus Trx1 (CCNA_03653; CcTrx1) as an oxidoreductase that is involved in the cell cycle progression of this model bacterium and is required to sustain life. Intriguingly, the abundance of CcTrx1 varies throughout the C. crescentus cell cycle: although the expression of CcTrx1 is induced in stalked cells, right before DNA replication initiation, CcTrx1 is actively degraded by the ClpXP protease in predivisional cells. Importantly, we demonstrated that regulation of the abundance of CcTrx1 is crucial for cell growth and survival as modulating CcTrx1 levels leads to cell death. Finally, we also report a comprehensive biochemical and structural characterization of this unique and essential Trx. The requirement to precisely control the abundance of CcTrx1 for cell survival underlines the importance of redox control for optimal cell cycle progression in C. crescentus.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.RA117.001042
      Issue No: Vol. 293, No. 10 (2018)
  • Systems analysis of the glycoside hydrolase family 18 enzymes from
           Cellvibrio japonicus characterizes essential chitin degradation functions
    • Authors: Estela C. Monge; Tina R. Tuveng, Gustav Vaaje-Kolstad, Vincent G. H. Eijsink, Jeffrey G. Gardner
      Pages: 3849 - 3859
      Abstract: Understanding the strategies used by bacteria to degrade polysaccharides constitutes an invaluable tool for biotechnological applications. Bacteria are major mediators of polysaccharide degradation in nature; however, the complex mechanisms used to detect, degrade, and consume these substrates are not well-understood, especially for recalcitrant polysaccharides such as chitin. It has been previously shown that the model bacterial saprophyte Cellvibrio japonicus is able to catabolize chitin, but little is known about the enzymatic machinery underlying this capability. Previous analyses of the C. japonicus genome and proteome indicated the presence of four glycoside hydrolase family 18 (GH18) enzymes, and studies of the proteome indicated that all are involved in chitin utilization. Using a combination of in vitro and in vivo approaches, we have studied the roles of these four chitinases in chitin bioconversion. Genetic analyses showed that only the chi18D gene product is essential for the degradation of chitin substrates. Biochemical characterization of the four enzymes showed functional differences and synergistic effects during chitin degradation, indicating non-redundant roles in the cell. Transcriptomic studies revealed complex regulation of the chitin degradation machinery of C. japonicus and confirmed the importance of CjChi18D and CjLPMO10A, a previously characterized chitin-active enzyme. With this systems biology approach, we deciphered the physiological relevance of the glycoside hydrolase family 18 enzymes for chitin degradation in C. japonicus, and the combination of in vitro and in vivo approaches provided a comprehensive understanding of the initial stages of chitin degradation by this bacterium.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.RA117.000849
      Issue No: Vol. 293, No. 10 (2018)
  • Modular domain swapping among the bacterial cytotoxic necrotizing factor
           (CNF) family for efficient cargo delivery into mammalian cells
    • Authors: Elizabeth E. Haywood; Mengfei Ho, Brenda A. Wilson
      Pages: 3860 - 3870
      Abstract: Modular AB-type bacterial protein toxins target mammalian host cells with high specificity and deliver their toxic cargo into the cytosol. Hence, these toxins are being explored as agents for targeted cytosolic delivery in biomedical and research applications. The cytotoxic necrotizing factor (CNF) family is unique among these toxins in that their homologous sequences are found in a wide array of bacteria, and their activity domains are packaged in various delivery systems. Here, to study how CNF cargo and delivery modules can be assembled for efficient cytosolic delivery, we generated chimeric toxins by swapping functional domains among CNF1, CNF2, CNF3, and CNFy. Chimeras with a CNFy delivery vehicle were more stably expressed, but were less efficient at cargo delivery into HEK293-T cells. We also found that CNFy cargo is the most universally compatible and that CNF3 delivery vehicle is the most flexible and efficient at delivering cargo. These findings suggest that domains within proteins can be swapped and accommodate each other for efficient function and that an individual domain could be engineered for compatibility with multiple partner domains. We anticipate that our insights could help inform chemical biology approaches to develop toxin-based cargo-delivery platforms for cytosolic cargo delivery of therapeutics or molecular probes into mammalian cells.
      PubDate: 2018-03-09T00:05:58-08:00
      DOI: 10.1074/jbc.RA117.001381
      Issue No: Vol. 293, No. 10 (2018)
School of Mathematical and Computer Sciences
Heriot-Watt University
Edinburgh, EH14 4AS, UK
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Fax: +00 44 (0)131 4513327
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