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Journal of Biological Chemistry
Journal Prestige (SJR): 2.672
Citation Impact (citeScore): 4
Number of Followers: 255  
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ISSN (Print) 0021-9258 - ISSN (Online) 1083-351X
Published by ASBMB Homepage  [3 journals]
  • Reconciling the controversy regarding the functional importance of bullet-
           and football-shaped GroE complexes [Protein Structure and Folding]
    • Authors: Lavi S. Bigman; Amnon Horovitz
      Pages: 13527 - 13529
      Abstract: The chaperonin GroEL and its co-chaperonin GroES form both GroEL–GroES bullet-shaped and GroEL–GroES2 football-shaped complexes. The residence time of protein substrates in the cavities of these complexes is about 10 and 1 s, respectively. There has been much controversy regarding which of these complexes is the main functional form. Here, we show using computational analysis that GroEL protein substrates have a bimodal distribution of folding times, which matches these residence times, thereby suggesting that both bullet-shaped and football-shaped complexes are functional. More generally, co-existing complexes with different stoichiometries are not mutually exclusive with respect to having a functional role and can complement each other.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.AC119.010299
      Issue No: Vol. 294, No. 37 (2019)
  • Transcriptional profiling of single fiber cells in a transgenic paradigm
           of an inherited childhood cataract reveals absence of molecular
           heterogeneity [Gene Regulation]
    • Authors: Suraj P. Bhat; Rajendra K. Gangalum, Dongjae Kim, Serghei Mangul, Raj K. Kashyap, Xinkai Zhou, David Elashoff
      Pages: 13530 - 13544
      Abstract: Our recent single-cell transcriptomic analysis has demonstrated that heterogeneous transcriptional activity attends molecular transition from the nascent to terminally differentiated fiber cells in the developing mouse lens. To understand the role of transcriptional heterogeneity in terminal differentiation and the functional phenotype (transparency) of this tissue, here we present a single-cell analysis of the developing lens, in a transgenic paradigm of an inherited pathology, known as the lamellar cataract. Cataracts hinder transmission of light into the eye. Lamellar cataract is the most prevalent bilateral childhood cataract. In this disease of early infancy, initially, the opacities remain confined to a few fiber cells, thus presenting an opportunity to investigate early molecular events that lead to cataractogenesis. We used a previously established paradigm that faithfully recapitulates this disease in transgenic mice. About 500 single fiber cells, manually isolated from a 2-day-old transgenic lens were interrogated individually for the expression of all known 17 crystallins and 78 other relevant genes using a Biomark HD (Fluidigm). We find that fiber cells from spatially and developmentally discrete regions of the transgenic (cataract) lens show remarkable absence of the heterogeneity of gene expression. Importantly, the molecular variability of cortical fiber cells, the hallmark of the WT lens, is absent in the transgenic cataract, suggesting absence of specific cell-type(s). Interestingly, we find a repetitive pattern of gene activity in progressive states of differentiation in the transgenic lens. This molecular dysfunction portends pathology much before the physical manifestations of the disease.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA119.008853
      Issue No: Vol. 294, No. 37 (2019)
  • The crystal structure of the protein kinase HIPK2 reveals a unique
           architecture of its CMGC-insert region [Signal Transduction]
    • Authors: Christopher Agnew; Lijun Liu, Shu Liu, Wei Xu, Liang You, Wayland Yeung, Natarajan Kannan, David Jablons, Natalia Jura
      Pages: 13545 - 13559
      Abstract: The homeodomain-interacting protein kinase (HIPK) family is comprised of four nuclear protein kinases, HIPK1–4. HIPK proteins phosphorylate a diverse range of transcription factors involved in cell proliferation, differentiation, and apoptosis. HIPK2, thus far the best-characterized member of this largely understudied family of protein kinases, plays a role in the activation of p53 in response to DNA damage. Despite this tumor-suppressor function, HIPK2 is also found overexpressed in several cancers, and its hyperactivation causes chronic fibrosis. There are currently no structures of HIPK2 or of any other HIPK kinase. Here, we report the crystal structure of HIPK2's kinase domain bound to CX-4945, a casein kinase 2α (CK2α) inhibitor currently in clinical trials against several cancers. The structure, determined at 2.2 Å resolution, revealed that CX-4945 engages the HIPK2 active site in a hybrid binding mode between that seen in structures of CK2α and Pim1 kinases. The HIPK2 kinase domain crystallized in the active conformation, which was stabilized by phosphorylation of the activation loop. We noted that the overall kinase domain fold of HIPK2 closely resembles that of evolutionarily related dual-specificity tyrosine-regulated kinases (DYRKs). Most significant structural differences between HIPK2 and DYRKs included an absence of the regulatory N-terminal domain and a unique conformation of the CMGC-insert region and of a newly defined insert segment in the αC–β4 loop. This first crystal structure of HIPK2 paves the way for characterizing the understudied members of the HIPK family and for developing HIPK2-directed therapies for managing cancer and fibrosis.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA119.009725
      Issue No: Vol. 294, No. 37 (2019)
  • The long-awaited structure of HIPK2 [Signal Transduction]
    • Authors: James M. Murphy
      Pages: 13560 - 13561
      Abstract: Homeodomain-interacting protein kinases (HIPKs) are kinases that phosphorylate transcription factors involved in cell proliferation, differentiation, and apoptosis. Their structures have been long sought because of their potential as drug targets in cancers and fibrosis. Agnew and colleagues present the first crystal structure of the HIPK2 kinase domain, complexed with the small-molecule inhibitor CX-4945, revealing important structural differences from related protein kinases of the DYRK family. This structure provides a starting point to exploit HIPK2's distinct structural features to develop selective small-molecule inhibitors of this kinase.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.H119.010675
      Issue No: Vol. 294, No. 37 (2019)
  • Hyaluronan synthase 2 (HAS2) overexpression diminishes the procatabolic
           activity of chondrocytes by a mechanism independent of extracellular
           hyaluronan [Metabolism]
    • Authors: Shinya Ishizuka; Saho Tsuchiya, Yoshifumi Ohashi, Kenya Terabe, Emily B. Askew, Naoko Ishizuka, Cheryl B. Knudson, Warren Knudson
      Pages: 13562 - 13579
      Abstract: Osteoarthritis (OA) is a progressive degenerative disease of the joints caused in part by a change in the phenotype of resident chondrocytes within affected joints. This altered phenotype, often termed proinflammatory or procatabolic, features enhanced production of endoproteinases and matrix metallo-proteinases (MMPs) as well as secretion of endogenous inflammatory mediators. Degradation and reduced retention of the proteoglycan aggrecan is an early event in OA. Enhanced turnover of hyaluronan (HA) is closely associated with changes in aggrecan. Here, to determine whether experimentally increased HA production promotes aggrecan retention and generates a positive feedback response, we overexpressed HA synthase-2 (HAS2) in chondrocytes via an inducible adenovirus construct (HA synthase-2 viral overexpression; HAS2-OE). HAS2-OE incrementally increased high-molecular-mass HA>100-fold within the cell-associated and growth medium pools. More importantly, our results indicated that the HAS2-OE expression system inhibits MMP3, MMP13, and other markers of the procatabolic phenotype (such as TNF-stimulated gene 6 protein (TSG6)) and also enhances aggrecan retention. These markers were inhibited in OA-associated chondrocytes and in chondrocytes activated by interleukin-1β (IL1β), but also chondrocytes activated by lipopolysaccharide (LPS), tumor necrosis factor α (TNFα), or HA oligosaccharides. However, the enhanced extracellular HA resulting from HAS2-OE did not reduce the procatabolic phenotype of neighboring nontransduced chondrocytes as we had expected. Rather, HA-mediated inhibition of the phenotype occurred only in transduced cells. In addition, high HA biosynthesis rates, especially in transduced procatabolic chondrocytes, resulted in marked changes in chondrocyte dependence on glycolysis versus oxidative phosphorylation for their metabolic energy needs.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA119.008567
      Issue No: Vol. 294, No. 37 (2019)
  • The murine IgH locus contains a distinct DNA sequence motif for the
           chromatin regulatory factor CTCF [Immunology]
    • Authors: David N. Ciccone; Yuka Namiki, Changfeng Chen, Katrina B. Morshead, Andrew L. Wood, Colette M. Johnston, John W. Morris, Yanqun Wang, Ruslan Sadreyev, Anne E. Corcoran, Adam G. W. Matthews, Marjorie A. Oettinger
      Pages: 13580 - 13592
      Abstract: Antigen receptor assembly in lymphocytes involves stringently-regulated coordination of specific DNA rearrangement events across several large chromosomal domains. Previous studies indicate that transcription factors such as paired box 5 (PAX5), Yin Yang 1 (YY1), and CCCTC-binding factor (CTCF) play a role in regulating the accessibility of the antigen receptor loci to the V(D)J recombinase, which is required for these rearrangements. To gain clues about the role of CTCF binding at the murine immunoglobulin heavy chain (IgH) locus, we utilized a computational approach that identified 144 putative CTCF-binding sites within this locus. We found that these CTCF sites share a consensus motif distinct from other CTCF sites in the mouse genome. Additionally, we could divide these CTCF sites into three categories: intergenic sites remote from any coding element, upstream sites present within 8 kb of the VH-leader exon, and recombination signal sequence (RSS)-associated sites characteristically located at a fixed distance (∼18 bp) downstream of the RSS. We noted that the intergenic and upstream sites are located in the distal portion of the VH locus, whereas the RSS-associated sites are located in the DH-proximal region. Computational analysis indicated that the prevalence of CTCF-binding sites at the IgH locus is evolutionarily conserved. In all species analyzed, these sites exhibit a striking strand-orientation bias, with>98% of the murine sites being present in one orientation with respect to VH gene transcription. Electrophoretic mobility shift and enhancer-blocking assays and ChIP–chip analysis confirmed CTCF binding to these sites both in vitro and in vivo.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA118.007348
      Issue No: Vol. 294, No. 37 (2019)
  • Kinetics of formation and reactivity of the persulfide in the one-cysteine
           peroxiredoxin from Mycobacterium tuberculosis [Signal Transduction]
    • Authors: Ernesto Cuevasanta; Anibal M. Reyes, Ari Zeida, Mauricio Mastrogiovanni, Maria Ines De Armas, Rafael Radi, Beatriz Alvarez, Madia Truȷillo
      Pages: 13593 - 13605
      Abstract: Hydrogen sulfide (H2S) participates in prokaryotic metabolism and is associated with several physiological functions in mammals. H2S reacts with oxidized thiol derivatives (i.e. disulfides and sulfenic acids) and thereby forms persulfides, which are plausible transducers of the H2S-mediated signaling effects. The one-cysteine peroxiredoxin alkyl hydroperoxide reductase E from Mycobacterium tuberculosis (MtAhpE–SH) reacts fast with hydroperoxides, forming a stable sulfenic acid (MtAhpE–SOH), which we chose here as a model to study the interactions between H2S and peroxiredoxins (Prx). MtAhpE–SOH reacted with H2S, forming a persulfide (MtAhpE–SSH) detectable by mass spectrometry. The rate constant for this reaction was (1.4 ± 0.2) × 103 m−1 s−1 (pH 7.4, 25 °C), six times higher than that reported for the reaction with the main low-molecular-weight thiol in M. tuberculosis, mycothiol. H2S was able to complete the catalytic cycle of MtAhpE and, according to kinetic considerations, it could represent an alternative substrate in M. tuberculosis. MtAhpE–SSH reacted 43 times faster than did MtAhpE–SH with the unspecific electrophile 4,4′-dithiodipyridine, a disulfide that exhibits no preferential reactivity with peroxidatic cysteines, but MtAhpE–SSH was less reactive toward specific Prx substrates such as hydrogen peroxide and peroxynitrite. According to molecular dynamics simulations, this loss of specific reactivity could be explained by alterations in the MtAhpE active site. MtAhpE–SSH could transfer its sulfane sulfur to a low-molecular-weight thiol, a process likely facilitated by the low pKa of the leaving thiol MtAhpE–SH, highlighting the possibility that Prx participates in transpersulfidation. The findings of our study contribute to the understanding of persulfide formation and reactivity.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA119.008883
      Issue No: Vol. 294, No. 37 (2019)
  • A conserved region of nonstructural protein 1 from alphacoronaviruses
           inhibits host gene expression and is critical for viral virulence [Protein
           Structure and Folding]
    • Authors: Zhou Shen; Gang Wang, Yiling Yang, Jiale Shi, Liurong Fang, Fang Li, Shaobo Xiao, Zhen F. Fu, Guiqing Peng
      Pages: 13606 - 13618
      Abstract: Coronaviruses are enveloped, single-stranded RNA viruses that are distributed worldwide. They include transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), and the human coronaviruses severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), many of which seriously endanger human health and well-being. Only alphacoronaviruses and betacoronaviruses harbor nonstructural protein 1 (nsp1), which performs multiple functions in inhibiting antiviral host responses. The role of the C terminus of betacoronavirus nsp1 in virulence has been characterized, but the location of the alphacoronavirus nsp1 region that is important for virulence remains unclear. Here, using TGEV nsp1 as a model to explore the function of this protein in alphacoronaviruses, we demonstrate that alphacoronavirus nsp1 inhibits host gene expression. Solving the crystal structure of full-length TGEV at 1.85-Å resolution and conducting several biochemical analyses, we observed that a specific motif (amino acids 91–95) of alphacoronavirus nsp1 is a conserved region that inhibits host protein synthesis. Using a reverse-genetics system based on CRISPR/Cas9 technology to construct a recombinant TGEV in which this specific nsp1 motif was altered, we found that this mutation does not affect virus replication in cell culture but significantly reduces TGEV pathogenicity in pigs. Taken together, our findings suggest that alphacoronavirus nsp1 is an essential virulence determinant, providing a potential paradigm for the development of a new attenuated vaccine based on modified nsp1.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA119.009713
      Issue No: Vol. 294, No. 37 (2019)
  • Prion disease is accelerated in mice lacking stress-induced heat shock
           protein 70 (HSP70) [Protein Structure and Folding]
    • Authors: Charles E. Mays; Enrique Armijo, Rodrigo Morales, Carlos Kramm, Andrea Flores, Anjana Tiwari, Jifeng Bian, Glenn C. Telling, Tej K. Pandita, Clayton R. Hunt, Claudio Soto
      Pages: 13619 - 13628
      Abstract: Prion diseases are a group of incurable neurodegenerative disorders that affect humans and animals via infection with proteinaceous particles called prions. Prions are composed of PrPSc, a misfolded version of the cellular prion protein (PrPC). During disease progression, PrPSc replicates by interacting with PrPC and inducing its conversion to PrPSc. As PrPSc accumulates, cellular stress mechanisms are activated to maintain cellular proteostasis, including increased protein chaperone levels. However, the exact roles of several of these chaperones remain unclear. Here, using various methodologies to monitor prion replication (i.e. protein misfolding cyclic amplification and cellular and animal infectivity bioassays), we studied the potential role of the molecular chaperone heat shock protein 70 (HSP70) in prion replication in vitro and in vivo. Our results indicated that pharmacological induction of the heat shock response in cells chronically infected with prions significantly decreased PrPSc accumulation. We also found that HSP70 alters prion replication in vitro. More importantly, prion infection of mice lacking the genes encoding stress-induced HSP70 exhibited accelerated prion disease progression compared with WT mice. In parallel with HSP70 being known to respond to endogenous and exogenous stressors such as heat, infection, toxicants, and ischemia, our results indicate that HSP70 may also play an important role in suppressing or delaying prion disease progression, opening opportunities for therapeutic intervention.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA118.006186
      Issue No: Vol. 294, No. 37 (2019)
  • Expansion of base excision repair compensates for a lack of DNA repair by
           oxidative dealkylation in budding yeast [Enzymology]
    • Authors: Suzanne J. Admiraal; Daniel E. Eyler, Michael R. Baldwin, Emily M. Brines, Christopher T. Lohans, Christopher J. Schofield, Patrick J. O'Brien
      Pages: 13629 - 13637
      Abstract: The Mag1 and Tpa1 proteins from budding yeast (Saccharomyces cerevisiae) have both been reported to repair alkylation damage in DNA. Mag1 initiates the base excision repair pathway by removing alkylated bases from DNA, and Tpa1 has been proposed to directly repair alkylated bases as does the prototypical oxidative dealkylase AlkB from Escherichia coli. However, we found that in vivo repair of methyl methanesulfonate (MMS)-induced alkylation damage in DNA involves Mag1 but not Tpa1. We observed that yeast strains without tpa1 are no more sensitive to MMS than WT yeast, whereas mag1-deficient yeast are ∼500-fold more sensitive to MMS. We therefore investigated the substrate specificity of Mag1 and found that it excises alkylated bases that are known AlkB substrates. In contrast, purified recombinant Tpa1 did not repair these alkylated DNA substrates, but it did exhibit the prolyl hydroxylase activity that has also been ascribed to it. A comparison of several of the kinetic parameters of Mag1 and its E. coli homolog AlkA revealed that Mag1 catalyzes base excision from known AlkB substrates with greater efficiency than does AlkA, consistent with an expanded role of yeast Mag1 in repair of alkylation damage. Our results challenge the proposal that Tpa1 directly functions in DNA repair and suggest that Mag1-initiated base excision repair compensates for the absence of oxidative dealkylation of alkylated nucleobases in budding yeast. This expanded role of Mag1, as compared with alkylation repair glycosylases in other organisms, could explain the extreme sensitivity of Mag1-deficient S. cerevisiae toward alkylation damage.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA119.009813
      Issue No: Vol. 294, No. 37 (2019)
  • Magnesium transporter 1 (MAGT1) deficiency causes selective defects in
           N-linked glycosylation and expression of immune-response genes
           [Glycobiology and Extracellular Matrices]
    • Authors: Mami Matsuda-Lennikov; Matthew Biancalana, Juan Zou, Juan C. Ravell, Lixin Zheng, Chrysi Kanellopoulou, Ping Jiang, Giulia Notarangelo, Huie Jing, Evan Masutani, Andrew J. Oler, Lisa Renee Olano, Benjamin L. Schulz, Michael J. Lenardo
      Pages: 13638 - 13656
      Abstract: Magnesium transporter 1 (MAGT1) critically mediates magnesium homeostasis in eukaryotes and is highly-conserved across different evolutionary branches. In humans, loss–of–function mutations in the MAGT1 gene cause X-linked magnesium deficiency with Epstein-Barr virus (EBV) infection and neoplasia (XMEN), a disease that has a broad range of clinical and immunological consequences. We have previously shown that EBV susceptibility in XMEN is associated with defective expression of the antiviral natural-killer group 2 member D (NKG2D) protein and abnormal Mg2+ transport. New evidence suggests that MAGT1 is the human homolog of the yeast OST3/OST6 proteins that form an integral part of the N-linked glycosylation complex, although the exact contributions of these perturbations in the glycosylation pathway to disease pathogenesis are still unknown. Using MS-based glycoproteomics, along with CRISPR/Cas9-KO cell lines, natural killer cell-killing assays, and RNA-Seq experiments, we now demonstrate that humans lacking functional MAGT1 have a selective deficiency in both immune and nonimmune glycoproteins, and we identified several critical glycosylation defects in important immune-response proteins and in the expression of genes involved in immunity, particularly CD28. We show that MAGT1 function is partly interchangeable with that of the paralog protein tumor-suppressor candidate 3 (TUSC3) but that each protein has a different tissue distribution in humans. We observed that MAGT1-dependent glycosylation is sensitive to Mg2+ levels and that reduced Mg2+ impairs immune-cell function via the loss of specific glycoproteins. Our findings reveal that defects in protein glycosylation and gene expression underlie immune defects in an inherited disease due to MAGT1 deficiency.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA119.008903
      Issue No: Vol. 294, No. 37 (2019)
  • Vitamin C-dependent lysine demethylase 6 (KDM6)-mediated demethylation
           promotes a chromatin state that supports the endothelial-to-hematopoietic
           transition [Gene Regulation]
    • Authors: Tian Zhang; Ke Huang, Yanling Zhu, Tianyu Wang, Yongli Shan, Bing Long, Yuhang Li, Qianyu Chen, Pengtao Wang, Shaoyang Zhao, Dongwei Li, Chuman Wu, Baoqiang Kang, Jiaming Gu, Yuchan Mai, Qing Wang, Jinbing Li, Yanqi Zhang, Zechuan Liang, Lin Guo, Fang Wu, Shuquan Su, Junwei Wang, Minghui Gao, Xiaofen Zhong, Baojian Liao, Jiekai Chen, Xiao Zhang, Xiaodong Shu, Duanqing Pei, Jinfu Nie, Guangjin Pan
      Pages: 13657 - 13670
      Abstract: Hematopoietic stem cells (HSCs)/progenitor cells (HPCs) are generated from hemogenic endothelial cells (HECs) during the endothelial-to-hematopoietic transition (EHT); however, the underlying mechanism remains poorly understood. Here, using an array of approaches, including CRSPR/Cas9 gene knockouts, RNA-Seq, ChIP-Seq, ATAC-Seq etc., we report that vitamin C (Vc) is essential in HPC generation during human pluripotent stem cell (hPSC) differentiation in defined culture conditions. Mechanistically, we found that the endothelial cells generated in the absence of Vc fail to undergo the EHT because of an apparent failure in opening up genomic loci essential for hematopoiesis. Under Vc deficiency, these loci exhibited abnormal accumulation of histone H3 trimethylation at Lys-27 (H3K27me3), a repressive histone modification that arose because of lower activities of demethylases that target H3K27me3. Consistently, deletion of the two H3K27me3 demethylases, Jumonji domain-containing 3 (JMJD3 or KDM6B) and histone demethylase UTX (UTX or KDM6A), impaired HPC generation even in the presence of Vc. Furthermore, we noted that Vc and jmjd3 are also important for HSC generation during zebrafish development. Together, our findings reveal an essential role for Vc in the EHT for hematopoiesis, and identify KDM6-mediated chromatin demethylation as an important regulatory mechanism in hematopoietic cell differentiation.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA119.009757
      Issue No: Vol. 294, No. 37 (2019)
  • The cell adhesion molecule IGPR-1 is activated by and regulates responses
           of endothelial cells to shear stress [Cell Biology]
    • Authors: Rachel Xi-Yeen Ho; Rawan Tahboub, Razie Amraei, Rosana D. Meyer, Nitinun Varongchayakul, Mark Grinstaff, Nader Rahimi
      Pages: 13671 - 13680
      Abstract: Vascular endothelial cells respond to blood flow-induced shear stress. However, the mechanisms through which endothelial cells transduce mechanical signals to cellular responses remain poorly understood. In this report, using tensile-force assays, immunofluorescence and atomic force microscopy, we demonstrate that immunoglobulin and proline-rich receptor-1 (IGPR-1) responds to mechanical stimulation and increases the stiffness of endothelial cells. We observed that IGPR-1 is activated by shear stress and tensile force and that flow shear stress–mediated IGPR-1 activation modulates remodeling of endothelial cells. We found that under static conditions, IGPR-1 is present at the cell–cell contacts; however, under shear stress, it redistributes along the cell borders into the flow direction. IGPR-1 activation stimulated actin stress fiber assembly and cross-linking with vinculin. Moreover, we noted that IGPR-1 stabilizes cell–cell junctions of endothelial cells as determined by staining of cells with ZO1. Mechanistically, shear stress stimulated activation of AKT Ser/Thr kinase 1 (AKT1), leading to phosphorylation of IGPR-1 at Ser-220. Inhibition of this phosphorylation prevented shear stress–induced actin fiber assembly and endothelial cell remodeling. Our findings indicate that IGPR-1 is an important player in endothelial cell mechanosensing, insights that have important implications for the pathogenesis of common maladies, including ischemic heart diseases and inflammation.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA119.008548
      Issue No: Vol. 294, No. 37 (2019)
  • Triple-negative breast cancer-derived microvesicles transfer microRNA221
           to the recipient cells and thereby promote epithelial-to-mesenchymal
           transition [Signal Transduction]
    • Authors: Kaushik Das; Subhojit Paul, Arpana Singh, Arnab Ghosh, Abhishek Roy, Shabbir Ahmed Ansari, Ramesh Prasad, Ashis Mukherjee, Prosenjit Sen
      Pages: 13681 - 13696
      Abstract: The triple-negative phenotype is the most prevalent form of human breast cancer worldwide and is characterized by poor survival, high aggressiveness, and recurrence. Microvesicles (MV) are shredded plasma membrane components and critically mediate cell–cell communication, but can also induce cancer proliferation and metastasis. Previous studies have revealed that protease-activated receptor 2 (PAR2) contributes significantly to human triple-negative breast cancer (TNBC) progression by releasing nano-size MV and promoting cell proliferation, migration, and invasion. MV isolated from highly aggressive human TNBC cells impart metastatic potential to nonmetastatic cells. Over-expression of microRNA221 (miR221) has also been reported to enhance the metastatic potential of human TNBC, but miR221's relationship to PAR2-induced MV is unclear. Here, using isolated MV, immunoblotting, quantitative RT-PCR, FACS analysis, and enzymatic assays, we show that miR221 is translocated via human TNBC-derived MV, which upon fusion with recipient cells, enhance their proliferation, survival, and metastasis both in vitro and in vivo by inducing the epithelial-to-mesenchymal transition (EMT). Administration of anti-miR221 significantly impaired MV-induced expression of the mesenchymal markers Snail, Slug, N-cadherin, and vimentin in the recipient cells, whereas restoring expression of the epithelial marker E-cadherin. We also demonstrate that MV-associated miR221 targets phosphatase and tensin homolog (PTEN) in the recipient cells, followed by AKT Ser/Thr kinase (AKT)/NF-κB activation, which promotes EMT. Moreover, elevated miR221 levels in MV derived from human TNBC patients' blood could induce cell proliferation and metastasis in recipient cells. In summary, miR221 transfer from TNBC cells via PAR2-derived MV induces EMT and enhances the malignant potential of recipient cells.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA119.008619
      Issue No: Vol. 294, No. 37 (2019)
  • MtpB, a member of the MttB superfamily from the human intestinal acetogen
           Eubacterium limosum, catalyzes proline betaine demethylation
    • Authors: Jonathan W. Picking; Edward J. Behrman, Liwen Zhang, Joseph A. Krzycki
      Pages: 13697 - 13707
      Abstract: The trimethylamine methyltransferase MttB is the founding member of a widely distributed superfamily of microbial proteins. Genes encoding most members of the MttB superfamily lack the codon for pyrrolysine that distinguishes previously characterized trimethylamine methyltransferases, leaving the function(s) of most of the enzymes in this superfamily unknown. Here, investigating the MttB family member MtpB from the human intestinal isolate Eubacterium limosum ATCC 8486, an acetogen that excretes N-methyl proline during growth on proline betaine, we demonstrate that MtpB catalyzes anoxic demethylation of proline betaine. MtpB along with MtqC (a corrinoid protein) and MtqA (a methylcorrinoid:tetrahydrofolate methyltransferase) was much more abundant in E. limosum cells grown on proline betaine than on lactate. We observed that recombinant MtpB methylates Co(I)-MtqC in the presence of proline betaine and that other quaternary amines are much less preferred substrates. MtpB, MtqC, and MtqA catalyze tetrahydrofolate methylation with proline betaine, thereby forming a key intermediate in the Wood–Ljungdahl acetogenesis pathway. To our knowledge, MtpB methylation of Co(I)-MtqC for the subsequent methylation of tetrahydrofolate represents the first described anoxic mechanism of proline betaine demethylation. The activities of MtpB and associated proteins in acetogens or other anaerobes provide a possible mechanism for the production of N-methyl proline by the gut microbiome. MtpB's activity characterized here strengthens the hypothesis that much of the MttB superfamily comprises quaternary amine-dependent methyltransferases.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA119.009886
      Issue No: Vol. 294, No. 37 (2019)
  • Nonnative structure in a peptide model of the unfolded state of superoxide
           dismutase 1 (SOD1): Implications for ALS-linked aggregation [Protein
           Structure and Folding]
    • Authors: Noah R. Cohen; Jill A. Zitzewitz, Osman Bilsel, C. Robert Matthews
      Pages: 13708 - 13717
      Abstract: Dozens of mutations throughout the sequence of the gene encoding superoxide dismutase 1 (SOD1) have been linked to toxic protein aggregation in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). A parsimonious explanation for numerous genotypes resulting in a common phenotype would be mutation-induced perturbation of the folding free-energy surface that increases the populations of high-energy states prone to aggregation. The absence of intermediates in the folding of monomeric SOD1 suggests that the unfolded ensemble is a potential source of aggregation. To test this hypothesis, here we dissected SOD1 into a set of peptides end-labeled with FRET probes to model the local behavior of the corresponding sequences in the unfolded ensemble. Using time-resolved FRET, we observed that the peptide corresponding to the Loop VII-β8 sequence at the SOD1 C terminus was uniquely sensitive to denaturant. Utilizing a two-dimensional form of maximum entropy modeling, we demonstrate that the sensitivity to denaturant is the surprising result of a two-state–like transition from a compact to an expanded state. Variations of the peptide sequence revealed that the compact state involves a nonnative interaction between the disordered N terminus and the hydrophobic C terminus of the peptide. This nonnative intramolecular structure could serve as a precursor for intermolecular association and result in aggregation associated with ALS. We propose that this precursor would provide a common molecular target for therapeutic intervention in the dozens of ALS-linked SOD1 mutations.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA119.008765
      Issue No: Vol. 294, No. 37 (2019)
  • Methyltransferase-like 21c methylates and stabilizes the heat shock
           protein Hspa8 in type I myofibers in mice [Cell Biology]
    • Authors: Chao Wang; Justine Arrington, Anna C. Ratliff, Jingjuan Chen, Hannah E. Horton, Yaohui Nie, Feng Yue, Christine A. Hrycyna, W. Andy Tao, Shihuan Kuang
      Pages: 13718 - 13728
      Abstract: Protein methyltransferases mediate posttranslational modifications of both histone and nonhistone proteins. Whereas histone methylation is well-known to regulate gene expression, the biological significance of nonhistone methylation is poorly understood. Methyltransferase-like 21c (Mettl21c) is a newly classified nonhistone lysine methyltransferase whose in vivo function has remained elusive. Using a Mettl21cLacZ knockin mouse model, we show here that Mettl21c expression is absent during myogenesis and restricted to mature type I (slow) myofibers in the muscle. Using co-immunoprecipitation, MS, and methylation assays, we demonstrate that Mettl21c trimethylates heat shock protein 8 (Hspa8) at Lys-561 to enhance its stability. As such, Mettl21c knockout reduced Hspa8 trimethylation and protein levels in slow muscles, and Mettl21c overexpression in myoblasts increased Hspa8 trimethylation and protein levels. We further show that Mettl21c-mediated stabilization of Hspa8 enhances its function in chaperone-mediated autophagy, leading to degradation of client proteins such as the transcription factors myocyte enhancer factor 2A (Mef2A) and Mef2D. In contrast, Mettl21c knockout increased Mef2 protein levels in slow muscles. These results identify Hspa8 as a Mettl21c substrate and reveal that nonhistone methylation has a physiological function in protein stabilization.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA119.008430
      Issue No: Vol. 294, No. 37 (2019)
  • Retinal guanylyl cyclase activation by calcium sensor proteins mediates
           photoreceptor degeneration in an rd3 mouse model of congenital human
           blindness [Signal Transduction]
    • Authors: Alexander M. Dizhoor; Elena V. Olshevskaya, Igor V. Peshenko
      Pages: 13729 - 13739
      Abstract: Deficiency of RD3 (retinal degeneration 3) protein causes recessive blindness and photoreceptor degeneration in humans and in the rd3 mouse strain, but the disease mechanism is unclear. Here, we present evidence that RD3 protects photoreceptors from degeneration by competing with guanylyl cyclase-activating proteins (GCAPs), which are calcium sensor proteins for retinal membrane guanylyl cyclase (RetGC). RetGC activity in rd3/rd3 retinas was drastically reduced but stimulated by the endogenous GCAPs at low Ca2+ concentrations. RetGC activity completely failed to accelerate in rd3/rd3GCAPs−/− hybrid photoreceptors, whose photoresponses remained drastically suppressed compared with the WT. However, ∼70% of the hybrid rd3/rd3GCAPs−/− photoreceptors survived past 6 months, in stark contrast to
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA119.009948
      Issue No: Vol. 294, No. 37 (2019)
  • The anti-parasitic drug suramin potently inhibits formation of seminal
           amyloid fibrils and their interaction with HIV-1 [Microbiology]
    • Authors: Suiyi Tan; Jin-Qing Li, Hongyan Cheng, Zhaofeng Li, Yan Lan, Ting-Ting Zhang, Zi-Chao Yang, Wenjuan Li, Tao Qi, Yu-Rong Qiu, Zhipeng Chen, Lin Li, Shu-wen Liu
      Pages: 13740 - 13754
      Abstract: Seminal amyloid fibrils are made up of naturally occurring peptide fragments and are key targets for the development of combination microbicides or antiviral drugs. Previously, we reported that the polysulfonic compound ADS-J1 is a potential candidate microbicide that not only inhibits HIV-1 entry, but also seminal fibrils. However, the carcinogenic azo moieties in ADS-J1 preclude its clinical application. Here, we screened several ADS-J1–like analogs and found that the antiparasitic drug suramin most potently inhibited seminal amyloid fibrils. Using various biochemical methods, including Congo red staining, CD analysis, transmission EM, viral infection assays, surface plasmon resonance imaging, and molecular dynamics simulations, we investigated suramin's inhibitory effects and its putative mechanism of action. We found that by forming a multivalent interaction, suramin binds to proteolytic peptides and mature fibrils, thereby inhibiting seminal fibril formation and blocking fibril-mediated enhancement of viral infection. Of note, suramin exhibited potent anti-HIV activities, and combining suramin with several antiretroviral drugs produced synergistic effects against HIV-1 in semen. Suramin also displayed a good safety profile for vaginal application. Moreover, suramin inhibited the semen-derived enhancer of viral infection (SEVI)/semen-mediated enhancement of HIV-1 transcytosis through genital epithelial cells and the subsequent infection of target cells. Collectively, suramin has great potential for further development as a combination microbicide to reduce the spread of the AIDS pandemic by targeting both viral and host factors involved in HIV-1 sexual transmission.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA118.006797
      Issue No: Vol. 294, No. 37 (2019)
  • Structure-based optimization of a PDZ-binding motif within a viral peptide
           stimulates neurite outgrowth [Neurobiology]
    • Authors: Zakir Khan; Elouan Terrien, Florent Delhommel, Cynthia Lefebvre-Omar, Delphine Bohl, Sandrine Vitry, Clara Bernard, Juan Ramirez, Alain Chaffotte, Kevin Ricquier, Renaud Vincentelli, Henri Buc, Christophe Prehaud, Nicolas Wolff, Monique Lafon
      Pages: 13755 - 13768
      Abstract: Protection of neuronal homeostasis is a major goal in the management of neurodegenerative diseases. Microtubule-associated Ser/Thr kinase 2 (MAST2) inhibits neurite outgrowth, and its inhibition therefore represents a potential therapeutic strategy. We previously reported that a viral protein (G-protein from rabies virus) capable of interfering with protein–protein interactions between the PDZ domain of MAST2 and the C-terminal moieties of its cellular partners counteracts MAST2-mediated suppression of neurite outgrowth. Here, we designed peptides derived from the native viral protein to increase the affinity of these peptides for the MAST2–PDZ domain. Our strategy involved modifying the length and flexibility of the noninteracting sequence linking the two subsites anchoring the peptide to the PDZ domain. Three peptides, Neurovita1 (NV1), NV2, and NV3, were selected, and we found that they all had increased affinities for the MAST2–PDZ domain, with Kd values decreasing from 1300 to 60 nm, while target selectivity was maintained. A parallel biological assay evaluating neurite extension and branching in cell cultures revealed that the NV peptides gradually improved neural activity, with the efficacies of these peptides for stimulating neurite outgrowth mirroring their affinities for MAST2–PDZ. We also show that NVs can be delivered into the cytoplasm of neurons as a gene or peptide. In summary, our findings indicate that virus-derived peptides targeted to MAST2–PDZ stimulate neurite outgrowth in several neuron types, opening up promising avenues for potentially using NVs in the management of neurodegenerative diseases.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA119.008238
      Issue No: Vol. 294, No. 37 (2019)
  • C-terminal proteolysis of the collagen VI {alpha}3 chain by BMP-1 and
           proprotein convertase(s) releases endotrophin in fragments of different
           sizes [Glycobiology and Extracellular Matrices]
    • Authors: Stefanie Elisabeth Heumuller; Maya Talantikite, Manon Napoli, Jean Armengaud, Matthias Morgelin, Ursula Hartmann, Gerhard Sengle, Mats Paulsson, Catherine Moali, Raimund Wagener
      Pages: 13769 - 13780
      Abstract: The assembly of collagen VI microfibrils is a multistep process in which proteolytic processing within the C-terminal globular region of the collagen VI α3 chain plays a major role. However, the mechanisms involved remain elusive. Moreover, C5, the short and most C-terminal domain of the α3 chain, recently has been proposed to be released as an adipokine that enhances tumor progression, fibrosis, inflammation, and insulin resistance and has been named “endotrophin.” Serum endotrophin could be a useful biomarker to monitor the progression of such disorders as chronic obstructive pulmonary disease, systemic sclerosis, and kidney diseases. Here, using biochemical and isotopic MS-based analyses, we found that the extracellular metalloproteinase bone morphogenetic protein 1 (BMP-1) is involved in endotrophin release and determined the exact BMP-1 cleavage site. Moreover, we provide evidence that several endotrophin-containing fragments are present in various tissues and body fluids. Among these, a large C2–C5 fragment, which contained endotrophin, was released by furin-like proprotein convertase cleavage. By using immunofluorescence microscopy and EM, we also demonstrate that these proteolytic maturations occur after secretion of collagen VI tetramers and during microfibril assembly. Differential localization of N- and C-terminal regions of the collagen VI α3 chain revealed that cleavage products are deposited in tissue and cell cultures. The detailed information on the processing of the collagen VI α3 chain reported here provides a basis for unraveling the function of endotrophin (C5) and larger endotrophin-containing fragments and for refining their use as biomarkers of disease progression.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA119.008641
      Issue No: Vol. 294, No. 37 (2019)
  • Mitochondrial cysteinyl-tRNA synthetase is expressed via alternative
           transcriptional initiation regulated by energy metabolism in yeast cells
           [Gene Regulation]
    • Authors: Akira Nishimura; Ryo Nasuno, Yuki Yoshikawa, Minkyung Jung, Tomoaki Ida, Tetsuro Matsunaga, Masanobu Morita, Hiroshi Takagi, Hozumi Motohashi, Takaaki Akaike
      Pages: 13781 - 13788
      Abstract: Eukaryotes typically utilize two distinct aminoacyl-tRNA synthetase isoforms, one for cytosolic and one for mitochondrial protein synthesis. However, the genome of budding yeast (Saccharomyces cerevisiae) contains only one cysteinyl-tRNA synthetase gene (YNL247W, also known as CRS1). In this study, we report that CRS1 encodes both cytosolic and mitochondrial isoforms. The 5′ complementary DNA end method and GFP reporter gene analyses indicated that yeast CRS1 expression yields two classes of mRNAs through alternative transcription starts: a long mRNA containing a mitochondrial targeting sequence and a short mRNA lacking this targeting sequence. We found that the mitochondrial Crs1 is the product of translation from the first initiation AUG codon on the long mRNA, whereas the cytosolic Crs1 is produced from the second in-frame AUG codon on the short mRNA. Genetic analysis and a ChIP assay revealed that the transcription factor heme activator protein (Hap) complex, which is involved in mitochondrial biogenesis, determines the transcription start sites of the CRS1 gene. We also noted that Hap complex–dependent initiation is regulated according to the needs of mitochondrial energy production. The results of our study indicate energy-dependent initiation of alternative transcription of CRS1 that results in production of two Crs1 isoforms, a finding that suggests Crs1's potential involvement in mitochondrial energy metabolism in yeast.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA119.009203
      Issue No: Vol. 294, No. 37 (2019)
  • Regulation of flagellar motor switching by c-di-GMP phosphodiesterases in
           Pseudomonas aeruginosa [Microbiology]
    • Authors: Lingyi Xin; Yukai Zeng, Shuo Sheng, Rachel Andrea Chea, Qiong Liu, Hoi Yeung Li, Liang Yang, Linghui Xu, Keng-Hwee Chiam, Zhao-Xun Liang
      Pages: 13789 - 13799
      Abstract: The second messenger cyclic diguanylate (c-di-GMP) plays a prominent role in regulating flagellum-dependent motility in the single-flagellated pathogenic bacterium Pseudomonas aeruginosa. The c-di-GMP–mediated signaling pathways and mechanisms that control flagellar output remain to be fully unveiled. Studying surface-tethered and free-swimming P. aeruginosa PAO1 cells, we found that the overexpression of an exogenous diguanylate cyclase (DGC) raises the global cellular c-di-GMP concentration and thereby inhibits flagellar motor switching and decreases motor speed, reducing swimming speed and reversal frequency, respectively. We noted that the inhibiting effect of c-di-GMP on flagellar motor switching, but not motor speed, is exerted through the c-di-GMP–binding adaptor protein MapZ and associated chemotactic pathways. Among the 22 putative c-di-GMP phosphodiesterases, we found that three of them (DipA, NbdA, and RbdA) can significantly inhibit flagellar motor switching and swimming directional reversal in a MapZ-dependent manner. These results disclose a network of c-di-GMP–signaling proteins that regulate chemotactic responses and flagellar motor switching in P. aeruginosa and establish MapZ as a key signaling hub that integrates inputs from different c-di-GMP–signaling pathways to control flagellar output and bacterial motility. We rationalized these experimental findings by invoking a model that postulates the regulation of flagellar motor switching by subcellular c-di-GMP pools.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA119.009009
      Issue No: Vol. 294, No. 37 (2019)
  • Conserved residue His-257 of Vibrio cholerae flavin transferase ApbE plays
           a critical role in substrate binding and catalysis [Bioenergetics]
    • Authors: Xuan Fang; Jerzy Osipiuk, Srinivas Chakravarthy, Ming Yuan, William M. Menzer, Devin Nissen, Pingdong Liang, Daniel A. Raba, Karina Tuz, Andrew J. Howard, Andrzej Joachimiak, David D. L. Minh, Oscar Juarez
      Pages: 13800 - 13810
      Abstract: The flavin transferase ApbE plays essential roles in bacterial physiology, covalently incorporating FMN cofactors into numerous respiratory enzymes that use the integrated cofactors as electron carriers. In this work we performed a detailed kinetic and structural characterization of Vibrio cholerae WT ApbE and mutants of the conserved residue His-257, to understand its role in substrate binding and in the catalytic mechanism of this family. Bi-substrate kinetic experiments revealed that ApbE follows a random Bi Bi sequential kinetic mechanism, in which a ternary complex is formed, indicating that both substrates must be bound to the enzyme for the reaction to proceed. Steady-state kinetic analyses show that the turnover rates of His-257 mutants are significantly smaller than those of WT ApbE, and have increased Km values for both substrates, indicating that the His-257 residue plays important roles in catalysis and in enzyme-substrate complex formation. Analyses of the pH dependence of ApbE activity indicate that the pKa of the catalytic residue (pKES1) increases by 2 pH units in the His-257 mutants, suggesting that this residue plays a role in substrate deprotonation. The crystal structures of WT ApbE and an H257G mutant were determined at 1.61 and 1.92 Å resolutions, revealing that His-257 is located in the catalytic site and that the substitution does not produce major conformational changes. We propose a reaction mechanism in which His-257 acts as a general base that deprotonates the acceptor residue, which subsequently performs a nucleophilic attack on FAD for flavin transfer.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA119.008261
      Issue No: Vol. 294, No. 37 (2019)
  • Hypoxic ER stress suppresses {beta}-catenin expression and promotes
           cooperation between the transcription factors XBP1 and HIF1{alpha} for
           cell survival [Signal Transduction]
    • Authors: Zhixiong Xia; Shiyong Wu, Xin Wei, Yifei Liao, Ping Yi, Yong Liu, Jianmiao Liu, Jianfeng Liu
      Pages: 13811 - 13821
      Abstract: Hypoxia occurs in many human solid tumors and activates multiple cellular adaptive-response pathways, including the unfolded protein response (UPR) in the endoplasmic reticulum (ER). Wnt/β-catenin signaling plays a critical role in tumorigenesis, and β-catenin has been shown to enhance hypoxia-inducible factor 1α (HIF1α)-activated gene expression, thereby supporting cell survival during hypoxia. However, the molecular interplay between hypoxic ER stress, Wnt/β-catenin signaling, and HIF1α-mediated gene regulation during hypoxia remains incompletely understood. Here, we report that hypoxic ER stress reduces β-catenin stability, which, in turn, enhances the activity of spliced X-box–binding protein 1 (XBP1s), a transcription factor and signal transducer of the UPR, in HIF1α-mediated hypoxic responses. We observed that in the RKO colon cancer cell line, which possesses a Wnt-stimulated β-catenin signaling cascade, increased ER stress during hypoxia is accompanied by a reduction in low-density lipoprotein receptor-related protein 6 (LRP6), and this reduction in LRP6 decreased β-catenin accumulation and impaired Wnt/β-catenin signaling. Of note, β-catenin interacted with both XBP1s and HIF1α, suppressing XBP1s-mediated augmentation of HIF1α target gene expression. Furthermore, Wnt stimulation or β-catenin overexpression blunted XBP1s-mediated cell survival under hypoxia. Together, these results reveal an unanticipated role for the Wnt/β-catenin pathway in hindering hypoxic UPR-mediated responses that increase cell survival. Our findings suggest that the molecular cross-talks between hypoxic ER stress, LRP6/β-catenin signaling, and the HIF1α pathway may represent an unappreciated mechanism that enables some tumor subtypes to survive and grow in hypoxic conditions.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA119.008353
      Issue No: Vol. 294, No. 37 (2019)
  • Solution structure and flexibility of the condensin HEAT-repeat subunit
           Ycg1 [DNA and Chromosomes]
    • Authors: Karen Manalastas-Cantos; Marc Kschonsak, Christian H. Haering, Dmitri I. Svergun
      Pages: 13822 - 13829
      Abstract: High-resolution structural analysis of flexible proteins is frequently challenging and requires the synergistic application of different experimental techniques. For these proteins, small-angle X-ray scattering (SAXS) allows for a quantitative assessment and modeling of potentially flexible and heterogeneous structural states. Here, we report SAXS characterization of the condensin HEAT-repeat subunit Ycg1Cnd3 in solution, complementing currently available high-resolution crystallographic models. We show that the free Ycg1 subunit is flexible in solution but becomes considerably more rigid when bound to its kleisin-binding partner protein Brn1Cnd2. The analysis of SAXS and dynamic and static multiangle light scattering data furthermore reveals that Ycg1 tends to oligomerize with increasing concentrations in the absence of Brn1. Based on these data, we present a model of the free Ycg1 protein constructed by normal mode analysis, as well as tentative models of Ycg1 dimers and tetramers. These models enable visualization of the conformational transitions that Ycg1 has to undergo to adopt a closed rigid shape and thereby create a DNA-binding surface in the condensin complex.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA119.008661
      Issue No: Vol. 294, No. 37 (2019)
  • Withdrawal: Thromboxane A2 receptor activates a Rho-associated
           kinase/LKB1/PTEN pathway to attenuate endothelium insulin signaling.
    • Authors: Ping Song; Miao Zhang, Shaungxi Wang, Jian Xu, Hyoung Chul Choi, Ming-Hui Zou
      Pages: 13830 - 13830
      Abstract: VOLUME 284 (2009) PAGES 17120–17128This article has been withdrawn by the authors. The Journal raised questions that the Akt immunoblot in Fig. 2A was reused in Fig. 5B; lanes 1, 2, and 4 of the actin immunoblot in Fig. 4A are the same; the LKB1 immunoblot in Fig. 7B was reused in Fig. 7D; and lane 2 of the tubulin immunoblot in Fig. 9A was reused in lane 1 for Fig. 9C. The authors were able to locate some of the original data and were able to locate some repeated experiments performed at the time of the original work, which the authors state support the conclusions of the paper. The authors offered to publish amended figures based upon that data and, alternatively, offered to repeat the experiments. However, the Journal declined both offers, a decision with which the authors disagree. Further, the authors state that the results of this paper are confirmed by the results of complementary experiments presented in the article and that some of the principal observations of this article were further confirmed in publications from other laboratories (Soliman, H. et al. (2015) Am. J. Physiol. Heart Circ. Physiol. 309, H70–H81; Lee, S-H. et al. (2014) Am. J. Physiol. Endocrinol. Metab. 306, E332–E343). The authors stand by the conclusions of the paper.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.W119.010660
      Issue No: Vol. 294, No. 37 (2019)
  • Withdrawal: Protein kinase C{zeta}-dependent LKB1 serine 428
           phosphorylation increases LKB1 nucleus export and apoptosis in endothelial
           cells. [Withdrawals/Retractions]
    • Authors: Ping Song; Zhonglin Xie, Yong Wu, Yunzhou Dong, Ming-Hui Zou
      Pages: 13831 - 13831
      Abstract: VOLUME 283 (2008) PAGES 12446–12455This article has been withdrawn by the authors. The Journal raised questions that the Akt immunoblot in Fig. 2B was reused in Fig. 6E, lanes 2 and 5 of the Akt immunoblot in Fig. 2C were duplicated, lanes 4 and 5 of the Akt immunoblot in Fig. 2C were reused in lanes 5 and 6 of the Akt immunoblot in Fig. 5C, lanes 2 and 5 of the Akt immunoblot were duplicated in Fig. 5C, the first lane of the PKCζ immunoblot in Fig. 4B was reused in Fig. 7A as Akt, and lanes 1 and 6 of the Akt immunoblot in Fig. 4C were duplicated. The authors were able to locate some of the original data and repeated 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 article, and some of the principal observations of this paper were further confirmed in publications from other laboratories (Elshaer, S. L. et al. (2018) Antioxidants 7, 47; Heo, K. S. et al. (2011) J. Cell Biol. 193, 867–884; Tommasini, I. et al. (2008) J. Immunol. 181, 5637–5645). The authors stand by the conclusions of the paper.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.W119.010661
      Issue No: Vol. 294, No. 37 (2019)
  • Withdrawal: A novel germ line-specific gene of the phosducin-like protein
           (PhLP) family: A meiotic function conserved from yeast to mice.
    • Authors: Pascal Lopez; Ruken Yaman, Luis A. Lopez–Fernandez, Frederique Vidal, Daniel Puel, Philippe Clertant, Francois Cuzin, Minoo Rassoulzadegan
      Pages: 13832 - 13832
      Abstract: VOLUME 278 (2003) PAGES 1751–1757This article has been withdrawn by Pascal Lopez, Luis A. Lopez-Fernandez, François Cuzin, and Minoo Rassoulzadegan. Ruken Yaman, Frédérique Vidal, Daniel Puel, and Philippe Clertant could not be reached. The Hss26 Northern blots from Fig. 3 (A and D) were previously published in Lopez et al. (2002) Mol. Cell. Biol. 22, 3488–3496, with some data representing different experimental conditions. The withdrawing authors state that the same nitrocellulose filter was reused for two different probes and so the control is the same for both publications. The image in Fig. 6B was inappropriately manipulated. The withdrawing authors state that after 20 years, they cannot provide the original data.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.W119.010673
      Issue No: Vol. 294, No. 37 (2019)
  • Ega3 from the fungal pathogen Aspergillus fumigatus is an
           endo-{alpha}-1,4-galactosaminidase that disrupts microbial biofilms
    • Authors: Natalie C. Bamford; Francois Le Mauff, Adithya S. Subramanian, Patrick Yip, Claudia Millan, Yongzhen Zhang, Caitlin Zacharias, Adam Forman, Mark Nitz, Jeroen D. C. Codee, Isabel Uson, Donald C. Sheppard, P. Lynne Howell
      Pages: 13833 - 13849
      Abstract: Aspergillus fumigatus is an opportunistic fungal pathogen that causes both chronic and acute invasive infections. Galactosaminogalactan (GAG) is an integral component of the A. fumigatus biofilm matrix and a key virulence factor. GAG is a heterogeneous linear α-1,4–linked exopolysaccharide of galactose and GalNAc that is partially deacetylated after secretion. A cluster of five co-expressed genes has been linked to GAG biosynthesis and modification. One gene in this cluster, ega3, is annotated as encoding a putative α-1,4-galactosaminidase belonging to glycoside hydrolase family 114 (GH114). Herein, we show that recombinant Ega3 is an active glycoside hydrolase that disrupts GAG-dependent A. fumigatus and Pel polysaccharide-dependent Pseudomonas aeruginosa biofilms at nanomolar concentrations. Using MS and functional assays, we demonstrate that Ega3 is an endo-acting α-1,4-galactosaminidase whose activity depends on the conserved acidic residues, Asp-189 and Glu-247. X-ray crystallographic structural analysis of the apo Ega3 and an Ega3-galactosamine complex, at 1.76 and 2.09 Å resolutions, revealed a modified (β/α)8-fold with a deep electronegative cleft, which upon ligand binding is capped to form a tunnel. Our structural analysis coupled with in silico docking studies also uncovered the molecular determinants for galactosamine specificity and substrate binding at the −2 to +1 binding subsites. The findings in this study increase the structural and mechanistic understanding of the GH114 family, which has>600 members encoded by plant and opportunistic human pathogens, as well as in industrially used bacteria and fungi.
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.RA119.009910
      Issue No: Vol. 294, No. 37 (2019)
  • Celebrating and cultivating excellent peer review at JBC [Editorials]
    • Authors: Lila M. Gierasch
      Pages: 13850 - 13851
      Abstract: We are living through a transformational time in scientific publishing, with lively and healthy discussions of mechanisms for improved transparency in review, increased emphasis on pre- and post-publication consultation about papers, and high pressure for rigor and rapidity in publishing. We are surrounded by a myriad of devices, apps, and tools to facilitate these exchanges. And yet, at least for now, scientists still primarily communicate their results through scholarly publications that have been rigorously evaluated by their colleagues in a tried and true peer review system that we revere and defend. Indeed, peer review is the cornerstone of the trust we place in each other, enabling us to be inspired by and build on the literature to achieve ever greater advances and insights. Impressively, peer review relies on selfless dedication of time and effort by the scientific community, primarily based on the shared belief that peer review is an essential service to the community to maintain quality in the published literature. There are also additional benefits: Peer review provides a mechanism for feedback and mentorship and an opportunity to use one's critical mind to digest and assess new science. But the bottom line is that scientists generously give of their time to enable our scientific structure to stand on a firm foundation. We take this opportunity to heartily thank all of our reviewers for the time they have donated to JBC to allow us to proceed with confidence that our published papers will engage the community and stand the test...
      PubDate: 2019-09-13T00:05:56-07:00
      DOI: 10.1074/jbc.E119.010822
      Issue No: Vol. 294, No. 37 (2019)
School of Mathematical and Computer Sciences
Heriot-Watt University
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