DOCSLIB.ORG

The Role of Endoplasmic Reticulum Stress in Type 1 Diabetes: Identification of Glucose Regulated Protein 78 As the Autoantigen for Bdc-2.5 T Cell Clone

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Role of Endoplasmic Reticulum Stress in Type 1 Diabetes: Identification of Glucose Regulated Protein 78 As the Autoantigen for Bdc-2.5 T Cell Clone THE ROLE OF ENDOPLASMIC RETICULUM STRESS IN TYPE 1 DIABETES: IDENTIFICATION OF GLUCOSE REGULATED PROTEIN 78 AS THE AUTOANTIGEN FOR BDC-2.5 T CELL CLONE. by Sheila Marie Schreiner B.S., Bridgewater State College, 2002 Submitted to the Graduate Faculty of School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2007 UNIVERSITY OF PITTSBURGH THE SCHOOL OF MEDICINE This dissertation was presented by Sheila Marie Schreiner It was defended on November 6th 2007 and approved by Nick Giannoukakis PhD Assistant Professor, Cellular and Molecular Pathology Tim D. Oury MD/PhD Associate Professor, Cellular and Molecular Pathology Massimo Trucco MD Hillman Professor of Pediatric Immunology Division Head of Immunogenetics, Immunology Committee Chair: Wendy M. Mars PhD Associate Professor, Cellular and Molecular Pathology Thesis Director: Jon D. Piganelli PhD Assistant Professor, Cellular and Molecular Pathology ii THE ROLE OF ENDOPLASMIC RETICULUM STRESS IN TYPE 1 DIABETES: IDENTIFICATION OF GLUCOSE REGULATED PROTEIN 78 AS THE AUTOANTIGEN FOR BDC-2.5 T CELL CLONE. Sheila Marie Schreiner, PhD University of Pittsburgh, 2007 Environmental triggers, such as viral infection and environmental toxins, have been proposed to initiate the autoimmune disease of Type 1 Diabetes (T1D), however, the mechanism is unknown. The identification of novel autoantigens may provide insight to the mechanism of environmental triggers and pathogenesis of T1D. I identified the antigen recognized by the diabetogenic BDC- 2.5 T cell clone using a novel in vivo reconstitution system, Restricted Immune System via Adoptive Transfer (RISAT). In RISAT, immunodeficient mice are adoptive transferred with a single T cell clone and an open repertoire of B cells. Reconstituted mice are immunized with an antigenic protein preparation. This system will drive an antibody response to the cognate antigen for the T and B cell through the co-stimulatory pathways involved in linked recognition. For the BDC-2.5 RISAT, non-obese diabetic (NOD).Rag-/- mice were adoptive transferred with the diabetogenic BDC-2.5 T cells and NOD B cells and then immunized with an antigenic beta cell membrane preparation (βmem) to drive an antibody response. The resulting antibodies recognized the endoplasmic reticulum (ER) stress associated protein glucose regulated protein 78 (GRP78) from βmem. To determine if ER stress plays a role in the antigenic response of the BDC-2.5 T cell clone, the non-antigenic NOD insulinoma cell line, NIT-1, were treated with thapsigargin, which induces ER stress. The treatment of NIT-1 with thapsigargin led to increased GRP78 synthesis, correlating with antigenic recognition by the BDC-2.5 T cell clone. iii The antibodies from the BDC-2.5 TCR-Tg recognizes a subset of GRP78 which is modified with phosphoserine. The data presented in this thesis demonstrates a mechanistic link between ER stress and environmental triggers leading to the initiation of TID through the novel autoantigen, GRP78. Also the technique, RISAT, can be used to identify additional potential autoantigens of isolated T cell clones in both T1D and other autoimmune diseases. iv TABLE OF CONTENTS PREFACE............................................................................................................................ XIV 1.0 INTRODUCTION ........................................................................................................... 1 1.1 DIABETES & GLUCOSE METABOLISM........................................................... 1 1.2 NON-AUTOIMMUNE DIABETES........................................................................ 3 1.3 TYPE 1 DIABETES................................................................................................. 4 1.3.1 Insulin Replacement Therapy ...................................................................... 5 1.3.2 Islet Replacement Therapy........................................................................... 6 1.4 GENETICS OF TYPE 1 DIABETES ..................................................................... 7 1.4.1 HLA Susceptibility........................................................................................ 7 1.4.2 Non-MHC Genes........................................................................................... 8 1.5 ENIVRONMENTAL FACTORS............................................................................ 9 1.5.1 Viral Infections ............................................................................................. 9 1.5.2 Noninfectious Environmental Triggers...................................................... 10 1.6 IMMUNOLOGY OF TYPE 1 DIABETES........................................................... 11 1.6.1 Central Tolerance ....................................................................................... 13 1.6.1.1 Principles of clonal deletion............................................................. 13 1.6.2 Peripheral Tolerance .................................................................................. 15 1.6.2.1 Clonal Anergy and Adaptive Tolerance.......................................... 15 v 1.6.2.2 Regulation. ....................................................................................... 16 1.6.2.3 Ignorance. ........................................................................................ 17 1.6.3 Acquired Tolerance .................................................................................... 17 1.7 B CELL DEVELOPMENT AND ANTIBODY MATURATION........................ 17 1.8 AUTOIMMUNITY: BREAKDOWN OF THE IMMUNE SYSTEM.................. 20 1.9 AUTOANTIBODIES & AUTOANTIGENS ASSOCIATED WITH T1D .......... 20 1.9.1 Glutamic Acid Decarboxylase (GAD65) .................................................... 22 1.9.2 Insulinoma-associated protein 2 (IA-2)...................................................... 22 1.9.3 Insulin.......................................................................................................... 23 1.9.4 Islet Cell Autoantigen of 69 kDA (ICA69) ................................................. 24 1.9.5 Other autoantigens ..................................................................................... 25 1.9.5.1 Heat shock protein 70 (HSP70). ...................................................... 27 1.9.5.2 Islet-specific Glucose-6-phosphatase catalytic subunit-Related Protein (IGRP). .............................................................................................. 27 1.10 TYPE 1 DIABETES MOUSE MODEL .............................................................. 28 1.10.1 Non-Obese Diabetic (NOD) Mouse Model............................................... 28 1.10.2 BDC-2.5 T Cell Clone ............................................................................... 29 1.10.3 BDC-2.5 TCR-Transgenic Mouse Model................................................. 31 1.11 PROTEIN TRAFFICKING ................................................................................ 33 1.12 ENDOPLASMIC RETICULUM STRESS PATHWAY .................................... 34 1.12.1 Unfolded Protein Response (UPR)........................................................... 36 1.12.2 Translational attenuation ......................................................................... 36 1.12.3 ER associated degradation ...................................................................... 37 vi 1.12.4 ER stress-apoptosis ................................................................................... 37 1.13 ER STRESS AND THE BETA CELL ................................................................ 38 1.14 ER STRESS AND DISEASE............................................................................... 39 2.0 HYPOTHESIS AND SPECIFIC AIMS........................................................................ 40 3.0 MATERIALS AND METHOD ..................................................................................... 41 3.1 ANIMALS.............................................................................................................. 41 3.2 T- AND B-CELL ISOLATION, T CELL CLONE CULTURE, AND ANTIGEN- SPECIFIC T CELL ASSAY. ......................................................................................... 41 3.3 RISAT RECONSTITUTION VIA ADOPTIVE TRANSFERS, IMMUNIZATION, AND SERA COLLECTION. ........................................................ 42 3.4 IMMUNOBLOT ANALYSIS................................................................................ 43 3.5 SCREENING SERUM FOR AUTOANTIBODIES ............................................. 44 3.6 IMMUNOPRECIPITATION OF AUTOANTIGENS ......................................... 45 3.7 PROTEOMIC ASSAYS FOR IDENTIFICATION OF CANDIDATE AUTOANTIGEN............................................................................................................ 45 3.8 INDUCTION AND DETECTION OF ER STRESS AND/OR APOPTOSIS IN CELL LINES.................................................................................................................. 46 3.9 GRP78 PEPTIDE LIBRARY................................................................................ 47 3.10 ATP 8-AH SEPHAROSE PURIFICATION OF GRP78 ................................... 48 3.11 REMOVAL OF O-LINKED GLYCOSYLATION ............................................ 48 3.12 CITRULLINATION DETECTION ASSAY ...................................................... 49 4.0 GLUCOSE REGULATED PROTEIN 78 (GRP78): A CANDIDATE ANTIGEN FOR THE DIABETOGENIC T CELL CLONE, BDC-2.5...........................................................
Load more

Recommended publications
  • Download (11Mb)
    A Thesis Submitted for the Degree of PhD at the University of Warwick Permanent WRAP URL: http://wrap.warwick.ac.uk/108813/ Copyright and reuse: This thesis is made available online and is protected by original copyright. Please scroll down to view the document itself. Please refer to the repository record for this item for information to help you to cite it. Our policy information is available from the repository home page. For more information, please contact the WRAP Team at: [email protected] warwick.ac.uk/lib-publications STUDIES ON THE TARGETING AND PROCESSING OF PRORICIN. Michael Westby BSc. (Hons) Dunelm A thesis submitted for the degree of Doctor of Philosophy Department of Biological Sciences University of Warwick Coventry. U.K. September, 1991 CONTENTS Page No. Contents i-vii List of Figures viii-xi List of Tables xi Acknowledgements xii Declaration xiii Abbreviations xiv-xvi Dedication xvii Summary xviii î.o ihtbodpctioh 1 1.1 Summary 2 1.2 Plant storage proteins 3 1.2.1 Introduction 3 1.2.2 Classification of plant storage proteins 4 1.2.3 Storage proteins of cereals 4 1.2.4 Storage proteins of dicots. 4 1.2.5 Subcellular site of storage protein deposition- the protein bodies 9 1.2.6 Synthesis as preproproteins 11 1.2.7 Summary 15 1.3 Intracellular trafficking of newly-synthesised proteins 16 1.3.1 Introduction 16 1.3.2 Membrane translocation as a first step in compartmentalisation 16 1.3.3 Translocation across the ER membrane 18 1.3.4 Co-translatlonal modifications 21 1.3.5 Protein folding in the ER 21 1.3.6 Protein export
    [Show full text]
  • Protein's Intracellular Adventure Talking About
    Dr. Mircea Leabu. The ribosome and intracellular adventure of proteins (lecture iconography) Protein’s intracellular adventure Protein biosynthesis Correct protein folding Non-functional protein degradation Intracellular directing of proteins Talking about Ribosome – structure and function Chaperones – definition and role Proteasome – structure and function Import of proteins into organelles 1 Dr. Mircea Leabu. The ribosome and intracellular adventure of proteins (lecture iconography) Protein biosynthesis •Project – mARN • Machinery – ribosome • Raw material – aminoacyl-tARN Genetic code (degenerated or redundant) Project’s structural organization Eukaryotic messenger ARN 2 Dr. Mircea Leabu. The ribosome and intracellular adventure of proteins (lecture iconography) Raw material structure Eukaryotic transfer ARN (clover leaf) Organization of the machinery Ribosome – 3D Structure opened book lateral view top view Functional3 organization Dr. Mircea Leabu. The ribosome and intracellular adventure of proteins (lecture iconography) Organization of the machinery Molecular organization Biosynthetic process development Stages of protein biosynthesis • Initiation: initiation factors • Elongation: elongation factors • End of translation: releasing factors 4 Dr. Mircea Leabu. The ribosome and intracellular adventure of proteins (lecture iconography) Protein biosynthesis initiation Elongation’s steps Repeating stage, n cycles 5 Dr. Mircea Leabu. The ribosome and intracellular adventure of proteins (lecture iconography) Elongation: energetic needs
    [Show full text]
  • Protein Targeting and Degradation 775
    Contents CONTENTS CHAPTER • General Considerations • Free and Membrane-bound Ribosomes • Signal Hypothesis • Glycosylation of Proteins at the Level of ER Envelope Carrier Hypothesis 29 • • Proteins with a Carboxyl- terminal KDEL Sequence (=Recycling of Resident Proteins of the ER) Protein • Bacterial Signal Sequences and Protein Targeting • Eukaryotic Protein Transport Targeting and Across Membranes • Protein Import by Receptor- mediated Endocytosis Degradation • Protein Degradation GENERAL CONSIDERATIONS rotein turnover – that is synthesis and degradation – occurs constantly in eukaryotic cells but it is a Phighly selective process with different rates of turnover for various proteins. Turnover of proteins can Nuclear localization signal peptide control the level of certain enzymes, furnish amino acids in times of need and degrade faulty or damaged proteins Protein targeting signal recognition that are generated during synthesis or arise from The structure of the nuclear localization deleterious activities in the cell. Nascent proteins contain signal-binding protein α-karyopherin (or signals that determine their ultimate destination. A newly α-importin) with a nuclear localization signal peptide bound to its major synthesized protein in the prokaryotic Escherichia coli recognition site. cell, for example, can stay in the cytosol or it can be sent to the plasma membrane, the outer membrane, the space between them, or the extracellular medium. The eukaryotic cell is made up of many structures, compartments and organelles, each with specific functions requiring different types of proteins and enzymes. The synthesis of most of these proteins begins on free ribosomes in the cytosol. Therefore, eukaryotic cells must direct proteins to internal sites such as lysosomes, mitochondria, chloroplasts, nucleus etc. How then is sorting accomplished? In eukaryotes, a key choice is made soon after the synthesis of a protein begins.
    [Show full text]
  • 7: Biological Sciences*
    Subject Index to Volume 1'7: Biological Sciences* January-Dece:nber 1980 Introdul ction The terms of the Subject Index for Volu me 77, January-December 1980, of the PROCEEDINGS OF THE NATIONAL ACA] )EMY OF SCIENCES USA (Biological Sciences) were chosen from titles, key terr ns, and abstracts of articles. The index terms are alphabetized by computer; nui abers, conformational prefixes, Greek letters, hyphens, and spaces between words aredisregarded in alphabetization. After each index term is printed the title of the Erticle (or a suitable modification of the title) and the appropriate page number. Tit [es are listed in alphabetical order under the index terms. Corrections to papers in which errors occurred are indexed under the term "Correction"as well as under the index term s selected for the paper itself. Organisms are indexed by their scientific names whe] i scientific names were provided in the papers; suitable cross-references are provi ded. Because the PROCEEDINGSurges autho rs to follow the tentative rules and rec- ommendations of the nomenclature comrmissions (e.g., for biochemistry, those proposed by the International Union of E iochemistry), an effort has been made to construct an index that conforms with t] lis policy. However, correction of errors in nomenclature was not attempted and t he index should not be looked upon as a reference for correct or recommended us. tge. In addition, some exceptions to the recommendations of the commissions were necessary. Index terms themselves are usually not abbreviated even if specific re( 'ommendations have been made by the commissions; and in some instances, wordsI within an index term were rearranged.
    [Show full text]
  • Information to Users
    Probing the plant endomembrane-secretory pathway using heterologous membrane protein markers. Item Type text; Dissertation-Reproduction (electronic) Authors Gong, Fangcheng. Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 30/09/2021 05:30:51 Link to Item http://hdl.handle.net/10150/187119 INFORMATION TO USERS This manuscript ,has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely. event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal SectiOllS with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy.
    [Show full text]
  • Glycosylation Phosphorylation
    Protein structure and function The Function of Proteins Enzymes biological catalysts. Immuno- antibodies of immune system. globulins Transport move materials around hemoglobin for O2. Regulatory hormones, control metabolism. Structural coverings and support skin, tendons, hair, nails, bone. Movement muscles, cilia, flagella. 1 DNA Protein The life cycle of a protein 2 Functional protein Families Science (2001), 15 % intermediary and nucleic acids metabolism 15-20 % structure, protein metabolism (cytoskeleton, chaperones, mediator of degradation) 20-25 % signal transduction or DAN binding protein 40 % gene encodes protein product are unknown function General flow scheme for proteomic analysis Experiment Sample Proteome Clinical Proteins mixture separation digestion protein Peptide mixture digestion peptide Mass Spectrometer identification Data analysis 3 Amino acid 2 amino acids peptide polypeptide 1o 2 o 3o 4o 一級primary 二級secondary 三級tertiary 四級quaternary Nelson & Cox (2000) Lehninger Principles of Biochemistry H pK α-carboxylic acid α-amino group pK2 1 H2N C COOH R The general structural formula of amino acids - pK1 values of the α-carboxylic acid groups lie in a small range around 2.2, above 3.5 COOH COO 2+ pK2 values of the α-amino group lie in a small range around 9.4, below 8.0 NH2 NH H pK pK2 1 +- + H N C COO Amino group = -NH3 3 Carboxyl group = -COO- R • Under normal cellular conditions amino acids are zwitterions (dipolar ions): How well it happens is based on pH and the type of amino acid. Called a zwitterion 4 Acidic environment Neutral environment Alkaline environment pK2 ~ 9 + + NH2 H NH2 H NH2 R-C-H R-C-H R-C-H COOH COO- COO- pK ~ 2 1 5.5 +1 0 -1 Isoelectric point Juang RH (2004) BCbasics 5 Amino Acids Have Buffering Effect pH 12 pK 9 ★ 2 + NH2 H 6 H-C-R Isoelectric point = pI COO- pK1 + pK2 3 ★ 2 pK1 0 [OH] → Juang RH (2004) BCbasics Four aliphatic (脂肪族) amino acid structures (non-polar) CH CH3 Isoleucine (I) CH2 (Ile) CH3 6 Aromatic (芳香族) amino acid structures 7 Methionine and cysteine First a.a.
    [Show full text]
  • ABSTRACT a Close Look at the Electrostatic Properties of Cu, Zn
    ABSTRACT A Close Look at the Electrostatic Properties of Cu, Zn-Superoxide Dismutase Yunhua Shi, Ph.D. Mentor: Bryan F. Shaw, Ph.D. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Mutations in the gene encoding Cu, Zn-superoxide dismutase (SOD1) are responsible for 1-2% of ALS cases. Numerous studies have proved that SOD1 forms neurotoxic aggregates, which add toxicity to motor neurons through a widely accepted “gain of function” mechanism. The electrostatic potential is an overlooked important biophysical property that can affect the aggregation propensity of SOD1. Protein net charge, a good representative of the electrostatic surface potential, is tightly regulated by a network of solvent accessible ionizable amino acid residues and coordinated small molecules such as water and metal ions. Few tools exist to detailed study the electrostatic potential of proteins, however, in this dissertation, we introduced capillary electrophoresis in conjunction with protein charge ladders to i) experimentally measure the net charge of WT and ALS-variant mutant SOD1 under various conditions, and ii) investigate the effect of electrostatic potential on the interaction between SOD1 and sodium dodecyl sulfate (SDS) molecules. Also, we predict and detect the deamidation of asparagine residues of Cu, Zn-SOD1 (human erythrocytes). With our novel tools, we successfully detect this sub-Dalton post-translational modification (< 1 Da) in aged Cu, Zn-SOD1 purified from human erythrocytes. The deamidation of SOD1 was proved to produce an ALS mutant analog which shares similar biochemical and biophysical properties to ALS mutant N139D. This solvent catalyzed spontaneous deamidation of SOD1 is incentive for understanding the mechanism of sporadic ALS.
    [Show full text]