DOCSLIB.ORG

The Eif2 Phosphatase: Characterization and Modulation

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Eif2 Phosphatase: Characterization and Modulation The eIF2 phosphatase: characterization and modulation Ana Crespillo Casado Darwin College July 2018 Ana Crespillo Casado The eIF2 phosphatase: characterization and modulation Abstract Cellular needs are fulfilled by the combined activity of functional proteins. Consequently, cells are equipped with a complex proteostatic network that controls protein production in response to cellular requisites. Thereby, protein synthesis is induced or attenuated depending on the particular cellular conditions. One of the mechanisms to control protein synthesis is the phosphorylation of eIF2, which triggers the so-called Integrated Stress Response (ISR). Kinases that sense stresses induce the phosphorylation of eIF2, which, on the one hand, attenuates global rates of protein synthesis and, on the other hand, activates the expression of specific proteins that help to alleviate the stress. One of the proteins preferentially expressed during the ISR is PPP1R15A, a regulatory subunit of Protein Phosphatase 1 (PP1). The PP1/PPP1R15A holophosphatase dephosphorylates eIF2 and terminates the ISR once the stresses are resolved. Hence, eIF2 kinases and phosphatases work together to control levels of phosphorylated eIF2. Maintaining the right balance between the activity of these kinases and phosphatases is important, as is seen by the correlation between their perturbance and the appearance of certain cellular malfunctions or diseases. However, affecting this balance has been also suggested to have beneficial effects. For example, genetic interference with the PPP1R15A regulatory subunit is proposed to confer protection to mice and cells under ER-stress conditions. This observation led to the search for compounds with the ability to modulate the ISR, in particular, by acting on the eIF2 phosphatases. Three compounds (Salubrinal, Guanabenz and Sephin1) have been proposed as eIF2 phosphatase inhibitors with potential use as therapeutic tools in protein misfolding diseases. However, their precise mechanism of action and their direct effect on the enzyme remains an open question. 2 This thesis focuses on the in vitro reconstitution of the eIF2 phosphatase, which served as a platform for characterizing the enzyme (in terms of its structure, activity and assembly) and for studying the proposed inhibitors. This report details key structural features of the PPP1R15/PP1 holophosphatase, the discovery of a cellular cofactor of the enzyme and the conclusions obtained after analysing the effect of its proposed inhibitors. It also includes the development of several in vitro assays, which could potentially be used to screen libraries of compounds in search for modulators of the enzyme. 3 Preface This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration except as declared in the Preface and specified in the relevant parts of the main text or figure legends. It is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. I further state that no substantial part of my dissertation has already been submitted, or, is being concurrently submitted for any such degree, diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text It does not exceed the prescribed word limit for the relevant Degree Committee. Parts of this thesis are included in the following publications: Chen, R., Rato, C., Yan, Y., Crespillo-Casado, A., Clarke, H. J., Heather, P., Marciniak, S.J., Ron, D. (2015). G-actin provides substrate-specificity to eukaryotic initiation factor 2α holophosphatases. eLife Crespillo-Casado, A., Chambers, J. E., Fischer, P. M., Marciniak, S. J., & Ron, D. (2017). PPP1R15A-mediated dephosphorylation of eIF2 a is unaffected by Sephin1 or Guanabenz. eLife Crespillo-Casado, A., Claes, Z., Choy, M. S., Peti, W., Bollen, M., & Ron, D. (2018). A Sephin1-insensitive tripartite holophosphatase dephosphorylates translation initiation factor 2α. Journal of Biological Chemistry 4 Acknowledgements Although the cover of this thesis contains only my name, there is a big list of people that also deserve to be mentioned here, those who have encouraged me over the years and who have made this possible. I want to express my gratitude to them: -My PhD supervisor, David Ron. He gave me the opportunity to join his lab as a Research Assistant, and his contagious passion for science motivated me to pursue this PhD. He has always been willing to teach, explain, listen and discuss. He has taught me how to love (and fight) science, even when confronting hard situations. -The colleagues from the lab. They have created an amazing atmosphere and thanks to them I have learnt what “team work” means (either to share reagents or to organise international dinners). I want extend a special thanks to Ruming and Claudia, who started the work on the phosphatase and taught me everything they knew about it; and to Steffen, for his continuous advice and essential help, especially during the writing of this thesis. Thanks to Yahui Yan for her help during the crystallization attempts and David Winpenny for his help with the TR-FRET assay. I am grateful to all members of the Ron lab, because I have met not only amazing professionals, but also good friends (that’s why they could also be included in the next group). -My friends. They have been extremely supportive and have made me enjoy these years inside and outside of the lab. I want to say a special big thank to Raquel, for restoring my energies when I needed it the most, through the long hours spent on the phone and the brief (but vital) meetings; and Belen, for the good moments spent together and for learning about science just to understand me. -My partner, Santi. He has listened so much about this project that he would be prepared to give a talk about eIF2α dephosphorylation. He has found the way to help me at every single moment (even through special deliveroo services or splitting cells) and his calm and patience have been essential for me. -I want to thank my family, for their faith in me and their unconditional support. Thanks to my sister, for being also a friend and for cheering me up as she has always done. Lastly, I will thank my parents who have always encouraged me to fulfil my 5 wishes, even when it required us to be far from each other. This thesis is dedicated to my grandma (who unfortunately is not here to share this moment) and to my parents, in gratitude for all they have done. (I’ll add this part in Spanish for them) Quiero dar las gracias también a mi familia, por su fe en mí y su apoyo incondicional. Gracias a mi hermana, por ser también una amiga y por animarme como siempre lo ha hecho. Finalmente, quiero agradecer a mis padres, quienes siempre me han empujado a cumplir mis sueños, incluso cuando ha implicado tener que estar en la distancia. Esta tesis se la dedico a mi abuela (quien por desgracia no está aquí para compartir este momento) y a mis padres, por todo lo que han hecho por mí. 6 List of abbreviations 6-FAM 6-carboxyfluorescein ADE1 adenine requiring 1 ADE2 Adenine requiring 2 ADP Adenosine diphosphate AKAP149 A-kinase anchoring protein 149 ATF4 Activating Transcription Factor 4 ATF6 Activated Transcription Factor 6 ATP Adenosine triphosphate AUR Amplex UltraRed BC Binary complex BLI Bio-Layer Interferometry bp base pair BSA Bovine Serum Albumin BxPC3 human pancreatic cancer cell line cDNA complementary DNA CDS CoDing Sequence CHO-K1 Subclone from parental Chinese Hamster Ovary cells CHOP CCAAT-enhancer-binding protein Homologous Protein CIMR Cambridge Institute of Medical Research CREB cAMP Response Element Binding protein CReP Constitutive Reverter of eIF2a Phosphorylation CRISPR Clustered Regularly Interspaced Short Palindromic Repeats CryoEM Electron CryoMicroscopy CUEDC2 CUE (Coupling of ubiquitin conjugation to ER degradation) Domain-Containing 2 DMSO DiMethyl SulfOxide DNA Deoxyribonucleic acid DNAse I Deoxyribonuclease I DTT DiThioThreitol E Enzyme E. coli Escherichia coli EC50 half maximal Effective Concentration EDTA EthyleneDiamineteTraacetic Acid EGTA Ethylene Glycol-bis(β-aminoethyl ether)-N,N,N',N'-Tetraacetic Acid 7 eIF2 eukaryiotic translation Initiation Factor 2 eIF2α α subunit of eIF2 eIF2B eukaryiotic translation Initiation Factor 2B ELISA Enzyme-Linked ImmunoSorbent Assay ER Endoplasmic Reticulum ES Enzyme-Substrate complex F-actin Filamentous actin FALS Familial Amyotropic Lateral Sclerosis G1/S Gap 1 / Synthetic phase G-actin Globular actin GADD34 Growth Arrest and DNA Damage Gene 34 GCN2 General Control Non de-repressible 2 GDP Guanosine DiPhosphate GFP Green Fluorescence Protein GSH Glutathione GSK GlaxoSmithKline GST Glutathione S-Transferase GTP Guanosine TriPhosphate HDR Homology Directed Repair HPLC High-Performance Liquid Chromatography HRI Heme-Regulated Inhibitor eIF2α kinase HRP HorseRadish Peroxidase HTS High Throughput Screening iBAQ intensity Based Absolute Quant IC50 half maximal Inhibitory Concentration IKK Inhibitor Of Nuclear Factor Kappa B Kinase IPTG IsoPropyl β-D-1-ThioGalactopyranoside IRE1 Inositol-REquiring protein 1 ISR Integrated Stress Response ISRIB Integrated Stress Response InhiBitor ITC Isothermal Titration Calorimetry Kd dissociation constant KO Knock Out KVxF amino acid sequence: Lysine(K),
Load more

Recommended publications
  • Gene Expression Analysis of Angioimmunoblastic Lymphoma Indicates Derivation from T Follicular Helper Cells and Vascular Endothelial Growth Factor Deregulation
    Research Article Gene Expression Analysis of Angioimmunoblastic Lymphoma Indicates Derivation from T Follicular Helper Cells and Vascular Endothelial Growth Factor Deregulation Pier Paolo Piccaluga,1,2 Claudio Agostinelli,1 Andrea Califano,3 Antonino Carbone,4 Luca Fantoni,6 Sergio Ferrari,5 Anna Gazzola,1 Annunziata Gloghini,6 Simona Righi,1 Maura Rossi,1 Enrico Tagliafico,5 Pier Luigi Zinzani,1 Simonetta Zupo,7 Michele Baccarani,1 and Stefano A. Pileri1 1Institute of Hematology and Medical Oncology ‘‘L. and A. Sera`gnoli,’’ S. Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy; 2Institute for Cancer Genetics and 3Center for Computational Biology and Biochemistry, Columbia University, New York, New York; 4Department of Pathology, Istituto Nazionale Tumori, Milan, Italy; 5Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy; 6Division of Experimental Oncology, Centro di Riferimento Oncologico, Aviano, Italy; and 7S.S.D. Diagnostica Malattie Linfoproliferative, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy Abstract Introduction Angioimmunoblastic lymphoma (AILT) is the second most Peripheral T-cell lymphomas (PTCL) represent 10% to 15% of common subtype of peripheral T-cell lymphoma (PTCL) and all lymphoid neoplasms (1). They are a heterogeneous group of is characterized by dismal prognosis. Thus far, only a fewstu- tumors that in the Revised European-American Lymphoma dies have dealt with its molecular pathogenesis. We performed (REAL)/WHO classification are subdivided into
    [Show full text]
  • The UVB-Induced Gene Expression Profile of Human Epidermis in Vivo Is Different from That of Cultured Keratinocytes
    Oncogene (2006) 25, 2601–2614 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE The UVB-induced gene expression profile of human epidermis in vivo is different from that of cultured keratinocytes CD Enk1, J Jacob-Hirsch2, H Gal3, I Verbovetski4, N Amariglio2, D Mevorach4, A Ingber1, D Givol3, G Rechavi2 and M Hochberg1 1Department of Dermatology, The Hadassah-Hebrew University Medical Center, Jerusalem, Israel; 2Department of Pediatric Hemato-Oncology and Functional Genomics, Safra Children’s Hospital, Sheba Medical Center and Sackler School of Medicine, Tel-Aviv University,Tel Aviv, Israel; 3Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel and 4The Laboratory for Cellular and Molecular Immunology, Department of Medicine, The Hadassah-Hebrew University Medical Center, Jerusalem, Israel In order to obtain a comprehensive picture of the radiation. UVB, with a wavelength range between 290 molecular events regulating cutaneous photodamage of and 320 nm, represents one of the most important intact human epidermis, suction blister roofs obtained environmental hazards affectinghuman skin (Hahn after a single dose of in vivo ultraviolet (UV)B exposure and Weinberg, 2002). To protect itself against the were used for microarray profiling. We found a changed DNA-damaging effects of sunlight, the skin disposes expression of 619 genes. Half of the UVB-regulated genes over highly complicated cellular programs, including had returned to pre-exposure baseline levels at 72 h, cell-cycle arrest, DNA repair and apoptosis (Brash et al., underscoring the transient character of the molecular 1996). Failure in selected elements of these defensive cutaneous UVB response.
    [Show full text]
  • PPP1R15A-Mediated Dephosphorylation of Eif2a
    RESEARCH ARTICLE PPP1R15A-mediated dephosphorylation of eIF2a is unaffected by Sephin1 or Guanabenz Ana Crespillo-Casado1*, Joseph E Chambers1, Peter M Fischer2,3, Stefan J Marciniak1, David Ron1* 1Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom; 2Division of Biomolecular Science and Medicinal Chemistry, School of Pharmacy, University of Nottingham, Nottingham, United Kingdom; 3Centre for Biomolecular Sciences, University of Nottingham, Nottingham, United Kingdom Abstract Dephosphorylation of translation initiation factor 2 (eIF2a) terminates signalling in the mammalian integrated stress response (ISR) and has emerged as a promising target for modifying the course of protein misfolding diseases. The [(o-chlorobenzylidene)amino]guanidines (Guanabenz and Sephin1) have been proposed to exert protective effects against misfolding by interfering with eIF2a-P dephosphorylation through selective disruption of a PP1-PPP1R15A holophosphatase complex. Surprisingly, they proved inert in vitro affecting neither stability of the PP1-PPP1R15A complex nor substrate-specific dephosphorylation. Furthermore, eIF2a-P dephosphorylation, assessed by a kinase shut-off experiment, progressed normally in Sephin1-treated cells. Consistent with its role in defending proteostasis, Sephin1 attenuated the IRE1 branch of the endoplasmic reticulum unfolded protein response. However, repression was noted in both wildtype and Ppp1r15a deleted cells and in cells rendered ISR-deficient by CRISPR editing of the Eif2s1 locus to encode a non-phosphorylatable eIF2a (eIF2aS51A). These findings challenge the view that [(o- chlorobenzylidene)amino]guanidines restore proteostasis by interfering with eIF2a-P *For correspondence: ac880@ dephosphorylation. cam.ac.uk (AC-C); dr360@ DOI: 10.7554/eLife.26109.001 medschl.cam.ac.uk (DR) Competing interest: See page 26 Introduction Funding: See page 26 Protein folding homeostasis (proteostasis) is achieved by balancing the rate of production, folding Received: 16 February 2017 and protein degradation.
    [Show full text]
  • SHOC2–MRAS–PP1 Complex Positively Regulates RAF Activity and Contributes to Noonan Syndrome Pathogenesis
    SHOC2–MRAS–PP1 complex positively regulates RAF activity and contributes to Noonan syndrome pathogenesis Lucy C. Younga,1, Nicole Hartiga,2, Isabel Boned del Ríoa, Sibel Saria, Benjamin Ringham-Terrya, Joshua R. Wainwrighta, Greg G. Jonesa, Frank McCormickb,3, and Pablo Rodriguez-Vicianaa,3 aUniversity College London Cancer Institute, University College London, London WC1E 6DD, United Kingdom; and bHelen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA 94158 Contributed by Frank McCormick, September 18, 2018 (sent for review November 22, 2017; reviewed by Deborah K. Morrison and Marc Therrien) Dephosphorylation of the inhibitory “S259” site on RAF kinases CRAF/RAF1 mutations are also frequently found in NS and (S259 on CRAF, S365 on BRAF) plays a key role in RAF activation. cluster around the S259 14-3-3 binding site, enhancing CRAF ac- The MRAS GTPase, a close relative of RAS oncoproteins, interacts tivity through disruption of 14-3-3 binding (8) and highlighting the with SHOC2 and protein phosphatase 1 (PP1) to form a heterotri- key role of this regulatory step in RAF–ERK pathway activation. meric holoenzyme that dephosphorylates this S259 RAF site. MRAS is a very close relative of the classical RAS oncoproteins MRAS and SHOC2 function as PP1 regulatory subunits providing (H-, N-, and KRAS, hereafter referred to collectively as “RAS”) the complex with striking specificity against RAF. MRAS also func- and shares most regulatory and effector interactions as well as tions as a targeting subunit as membrane localization is required transforming ability (9–11). However, MRAS also has specific for efficient RAF dephosphorylation and ERK pathway regulation functions of its own, and uniquely among RAS family GTPases, it in cells.
    [Show full text]
  • Intrinsic Disorder of the BAF Complex: Roles in Chromatin Remodeling and Disease Development
    International Journal of Molecular Sciences Article Intrinsic Disorder of the BAF Complex: Roles in Chromatin Remodeling and Disease Development Nashwa El Hadidy 1 and Vladimir N. Uversky 1,2,* 1 Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, 12901 Bruce B. Downs Blvd. MDC07, Tampa, FL 33612, USA; [email protected] 2 Laboratory of New Methods in Biology, Institute for Biological Instrumentation of the Russian Academy of Sciences, Federal Research Center “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”, Pushchino, 142290 Moscow Region, Russia * Correspondence: [email protected]; Tel.: +1-813-974-5816; Fax: +1-813-974-7357 Received: 20 September 2019; Accepted: 21 October 2019; Published: 23 October 2019 Abstract: The two-meter-long DNA is compressed into chromatin in the nucleus of every cell, which serves as a significant barrier to transcription. Therefore, for processes such as replication and transcription to occur, the highly compacted chromatin must be relaxed, and the processes required for chromatin reorganization for the aim of replication or transcription are controlled by ATP-dependent nucleosome remodelers. One of the most highly studied remodelers of this kind is the BRG1- or BRM-associated factor complex (BAF complex, also known as SWItch/sucrose non-fermentable (SWI/SNF) complex), which is crucial for the regulation of gene expression and differentiation in eukaryotes. Chromatin remodeling complex BAF is characterized by a highly polymorphic structure, containing from four to 17 subunits encoded by 29 genes. The aim of this paper is to provide an overview of the role of BAF complex in chromatin remodeling and also to use literature mining and a set of computational and bioinformatics tools to analyze structural properties, intrinsic disorder predisposition, and functionalities of its subunits, along with the description of the relations of different BAF complex subunits to the pathogenesis of various human diseases.
    [Show full text]
  • Sephin1 Protects Neurons Against Excitotoxicity Independently of the Integrated Stress Response
    International Journal of Molecular Sciences Article Sephin1 Protects Neurons against Excitotoxicity Independently of the Integrated Stress Response Asier Ruiz *, Jone Zuazo , Carolina Ortiz-Sanz, Celia Luchena, Carlos Matute and Elena Alberdi Departamento de Neurociencias, Universidad del País Vasco (UPV/EHU), Achucarro Basque Center for Neuroscience and Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED), 48940 Leioa, Spain; [email protected] (J.Z.); [email protected] (C.O.-S.); [email protected] (C.L.); [email protected] (C.M.); [email protected] (E.A.) * Correspondence: [email protected] Received: 30 July 2020; Accepted: 20 August 2020; Published: 24 August 2020 Abstract: Sephin1 is a derivative of guanabenz that inhibits the dephosphorylation of the eukaryotic initiation factor 2 alpha (eIF2α) and therefore may enhance the integrated stress response (ISR), an adaptive mechanism against different cellular stresses, such as accumulation of misfolded proteins. Unlike guanabenz, Sephin1 provides neuroprotection without adverse effects on the α2-adrenergic system and therefore it is considered a promising pharmacological therapeutic tool. Here, we have studied the effects of Sephin1 on N-methyl-D-aspartic acid (NMDA) receptor signaling which may modulate the ISR and contribute to excitotoxic neuronal loss in several neurodegenerative conditions. Time-course analysis of peIF2α levels after NMDA receptor overactivation showed a delayed dephosphorylation that occurred in the absence of activating transcription factor 4 (ATF4) and therefore independently of the ISR, in contrast to that observed during endoplasmic reticulum (ER) stress induced by tunicamycin and thapsigargin. Similar to guanabenz, Sephin1 completely blocked NMDA-induced neuronal death and was ineffective against AMPA-induced excitotoxicity, whereas it did not protect from experimental ER stress.
    [Show full text]
  • Protein Phosphatases in Pancreatic Islets
    H ORTSA¨ TER and others Islet protein phosphatases 221:3 R121–R144 Review Protein phosphatases in pancreatic islets Henrik Ortsa¨ter1,2, Nina Grankvist3, Richard E Honkanen4 and Åke Sjo¨holm1,4,5 1Biovation Park Telge, So¨ derta¨ lje, Sweden 2Research Unit, So¨ derta¨ lje Hospital, SE-152 86 So¨ derta¨ lje, Sweden Correspondence 3Degenerative Disease Program, Sanford-Burnham Medical Research Institute, Del E. Webb Neuroscience, should be addressed Aging and Stem Cell Research Center, 10901 North Torrey Pines Road, La Jolla, California 92037, USA to A˚ Sjo¨ holm 4Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, Email Mobile, Alabama 36688, USA ake.sjoholm@ 5Department of Internal Medicine, So¨ derta¨ lje Hospital, So¨ derta¨ lje, Sweden sodertaljesjukhus.se Abstract The prevalence of diabetes is increasing rapidly worldwide. A cardinal feature of most forms Key Words of diabetes is the lack of insulin-producing capability, due to the loss of insulin-producing " islet cells b-cells, impaired glucose-sensitive insulin secretion from the b-cell, or a combination thereof, " insulin secretion the reasons for which largely remain elusive. Reversible phosphorylation is an important and " apoptosis versatile mechanism for regulating the biological activity of many intracellular proteins, " diabetes which, in turn, controls a variety of cellular functions. For instance, significant changes in " phosphatase protein kinase activities and in protein phosphorylation patterns occur subsequent to the stimulation of insulin release by glucose. Therefore, the molecular mechanisms regulating the phosphorylation of proteins involved in the insulin secretory process by the b-cell have Journal of Endocrinology been extensively investigated.
    [Show full text]
  • Higher Order Phosphatase-Substrate Contacts Terminate the Integrated Stress Response
    bioRxiv preprint doi: https://doi.org/10.1101/2021.06.18.449003; this version posted June 18, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. The eIF2P pre-dephosphorylation complex (V0.5.1F; BioRxiv) Higher order phosphatase-substrate contacts terminate the Integrated Stress Response Yahui Yan, Heather P. Harding and David Ron* Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom *Correspondence should be addressed to DR [email protected] KEYWORDS Actins/*metabolism Eukaryotic Initiation Factor-2/*metabolism Phosphorylation Protein Phosphatase 1/*metabolism Substrate Specificity 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.18.449003; this version posted June 18, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. The eIF2P pre-dephosphorylation complex (V0.5.1F; BioRxiv) ABSTRACT Many regulatory PPP1R subunits join few catalytic PP1c subunits to mediate phosphoserine and phosphothreonine dephosphorylation in metazoans. Regulatory subunits are known to engage PP1c’s surface, locally affecting flexible phosphopeptides access to the active site. However, catalytic efficiency of holophosphatases towards their natively-folded phosphoprotein substrates is largely unexplained. Here we present a Cryo-EM structure of the tripartite PP1c/PPP1R15A/G-actin holophosphatase that terminates signalling in the Integrated Stress Response (ISR) in pre-dephosphorylation complex with its substrate, translation initiation factor 2 (eIF2).
    [Show full text]
  • A Single-Cell Transcriptome Atlas of the Mouse Glomerulus
    RAPID COMMUNICATION www.jasn.org A Single-Cell Transcriptome Atlas of the Mouse Glomerulus Nikos Karaiskos,1 Mahdieh Rahmatollahi,2 Anastasiya Boltengagen,1 Haiyue Liu,1 Martin Hoehne ,2 Markus Rinschen,2,3 Bernhard Schermer,2,4,5 Thomas Benzing,2,4,5 Nikolaus Rajewsky,1 Christine Kocks ,1 Martin Kann,2 and Roman-Ulrich Müller 2,4,5 Due to the number of contributing authors, the affiliations are listed at the end of this article. ABSTRACT Background Three different cell types constitute the glomerular filter: mesangial depending on cell location relative to the cells, endothelial cells, and podocytes. However, to what extent cellular heteroge- glomerular vascular pole.3 Because BP ad- neity exists within healthy glomerular cell populations remains unknown. aptation and mechanoadaptation of glo- merular cells are key determinants of kidney Methods We used nanodroplet-based highly parallel transcriptional profiling to function and dysregulated in kidney disease, characterize the cellular content of purified wild-type mouse glomeruli. we tested whether glomerular cell type sub- Results Unsupervised clustering of nearly 13,000 single-cell transcriptomes identi- sets can be identified by single-cell RNA fied the three known glomerular cell types. We provide a comprehensive online sequencing in wild-type glomeruli. This atlas of gene expression in glomerular cells that can be queried and visualized using technique allows for high-throughput tran- an interactive and freely available database. Novel marker genes for all glomerular scriptome profiling of individual cells and is cell types were identified and supported by immunohistochemistry images particularly suitable for identifying novel obtained from the Human Protein Atlas.
    [Show full text]
  • GADD34-Deficient Mice Develop Obesity, Nonalcoholic Fatty Liver
    www.nature.com/scientificreports OPEN GADD34-deficient mice develop obesity, nonalcoholic fatty liver disease, hepatic carcinoma and Received: 24 February 2015 Accepted: 15 July 2015 insulin resistance Published: 28 August 2015 Naomi Nishio1 & Ken-ichi Isobe1,2 The prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing in parallel with the prevalence of obesity. DNA damage-inducible protein 34 (GADD34/Ppp1r15a), originally isolated from UV-inducible transcripts in Chinese hamster ovary (CHO) cells, dephosphorylates several kinases that function in important signaling cascades, including dephosphorylation of eIF2α. We examined the effects of GADD34 on natural life span by using GADD34-deficient mice. Here we observed for the first time that with age GADD34-deficient mice become obese, developing fatty liver followed by liver cirrhosis, hepatocellular carcinoma, and insulin resistance. We found that myofibroblasts and immune cells infiltrated the portal veins of aged GADD34-deficient mouse livers. A high-fat diet (HFD) induced a higher level of steatosis in young GADD34-deficient mice compared with WT mice. Differentiation into fat is dependent on insulin signaling. Insulin signaling in young GADD34- deficient mice was higher than that in WT mice, which explained the higher fat differentiation of mouse embryonic fibroblasts (MEFs) observed in GADD34-deficient mice. Through aging or a HFD, insulin signaling in GADD34-deficient liver converted to be down regulated compared with WT mice. We found that a HFD or palmitate treatment converted insulin signaling by up-regulating TNF-α and JNK. The prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing in parallel with the prevalence of obesity1. NAFLD progresses to nonalcoholic steatohepatitis (NASH) featured by exacerbated intrahe- patic inflammation, more intense steatosis, and hepatocellular injury.
    [Show full text]
  • Inhibiting the Integrated Stress Response Pathway Prevents
    RESEARCH ARTICLE Inhibiting the integrated stress response pathway prevents aberrant chondrocyte differentiation thereby alleviating chondrodysplasia Cheng Wang1†, Zhijia Tan1†, Ben Niu1, Kwok Yeung Tsang1, Andrew Tai1, Wilson C W Chan1, Rebecca L K Lo1, Keith K H Leung1, Nelson W F Dung1, Nobuyuki Itoh2, Michael Q Zhang3,4, Danny Chan1, Kathryn Song Eng Cheah1* 1School of Biomedical Sciences, University of Hong Kong, Hong Kong, China; 2Graduate School of Pharmaceutical Sciences, University of Kyoto, Kyoto, Japan; 3Department of Biological Sciences, Center for Systems Biology, The University of Texas at Dallas, Richardson, United States; 4MOE Key Laboratory of Bioinformatics, Center for Synthetic and Systems Biology, Tsinghua University, Beijing, China Abstract The integrated stress response (ISR) is activated by diverse forms of cellular stress, including endoplasmic reticulum (ER) stress, and is associated with diseases. However, the molecular mechanism(s) whereby the ISR impacts on differentiation is incompletely understood. Here, we exploited a mouse model of Metaphyseal Chondrodysplasia type Schmid (MCDS) to provide insight into the impact of the ISR on cell fate. We show the protein kinase RNA-like ER kinase (PERK) pathway that mediates preferential synthesis of ATF4 and CHOP, dominates in causing dysplasia by reverting chondrocyte differentiation via ATF4-directed transactivation of Sox9. Chondrocyte survival is enabled, cell autonomously, by CHOP and dual CHOP-ATF4 *For correspondence: transactivation of Fgf21. Treatment of mutant mice with a chemical inhibitor of PERK signaling [email protected] prevents the differentiation defects and ameliorates chondrodysplasia. By preventing aberrant †These authors contributed differentiation, titrated inhibition of the ISR emerges as a rationale therapeutic strategy for stress- equally to this work induced skeletal disorders.
    [Show full text]
  • Inactivation of Ppp1r15a Minimises Weight Gain and Insulin Resistance
    bioRxiv preprint doi: https://doi.org/10.1101/313940; this version posted May 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Inactivation of Ppp1r15a minimises weight gain and insulin resistance during caloric excess in mice Vruti Patel1,2, Guillaume Bidault3, Joseph E. Chambers1, Stefania Carobbio3, Angharad J. T. Everden1, Concepción Garcés1, Lucy E. Dalton1, Fiona M. Gribble3, Antonio Vidal-Puig3,4 and Stefan J. Marciniak1,2 1. Cambridge Institute for Medical Research (CIMR), University of Cambridge Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY, UK. 2. Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Hills Rd, Cambridge CB2 0SP, UK 3. Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge, CB2 0QQ, UK 4. Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, CB10 1SA, UK Correspondence should be addressed to: Professor Stefan J. Marciniak, Cambridge Institute for Medical Research (CIMR), University of Cambridge, CB2 0XY, UK Email: [email protected] Telephone: +44 (0) 1223 762660 Abstract 210 words Main text: 3911 1 bioRxiv preprint doi: https://doi.org/10.1101/313940; this version posted May 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Phosphorylation of the translation initiation factor eIF2α within the mediobasal hypothalamus is known to suppress food intake, but the role of the eIF2α phosphatases in regulating body weight is poorly understood.
    [Show full text]