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Pidd Expression in Response to Cellular Stress PIDD EXPRESSION IN RESPONSE TO CELLULAR STRESS Artiom Zatsepin A thesis submitted to the Faculty of Graduate Studies in partial fulfillment of the requirements for the degree of Master of Science Graduate Program in Biology York University Toronto, Ontario Canada December 2009 Library and Archives Bibliothéque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de lédition 395 Wellington Street 395, rue Wellington OttawaONK1A0N4 Ottawa ON K1A0N4 Canada Canada Yourfile Votre reference ISBN: 978-0-494-62466-1 Ourfile Notre reference ISBN: 978-0-494-62466-1 NOTICE: AVIS: The author has granted a non- Uauteur a accordé une licence non exclusive exclusive license allowing Library and permettant å la Bibliothéque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par 1'lnternet, préter, telecommunication or on the Internet, distribuer et vendre des théses partout dans le loan, distribute and seil theses monde, å des fins commerciales ou autres, sur worldwide, for commercial or non- support microforme, papier, électronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriété du droit d'auteur ownership and moral rights in this et des droits moraux qui protege ætte thése. Ni thesis. Neither the thesis nor la thése ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent étre imprimés ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformément å la loi canadienne sur la Privacy Act some supporting forms protection de la vie privée, quelques may have been removed from this formulaires secondaires ont été enlevés de thesis. cette thése. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. 1+1 Canada IV ABSTRACT PIDD (p53-induced protein with death domain [DD]) has been reported to participate in a number of protein complexes involved in the mitigation of various forms of cellular stress through cell survival and apoptosis. PIDD has also been reported to undergo proteolytic processing, resulting in the production of several PIDD-related polypeptides in response to diverse stimuli. Here we demonstrate that TNF-a treatment of MCF7 and Jurkat cells results in production of a 48 kDa PIDD-related polypeptide, with little change in the expression of the full-length PIDD protein. In addition we show that thapsigargin treatment of U20S cells leads to production of a 65 kDa PIDD-related polypeptide. Additionally, we introduce the immunoprecipitation-based PIDD purification technique that could potentially be used to identify the individual PIDD- related polypeptides. Finally, we describe the spatiotemporal as well as biochemical properties of the 48 kDa polypeptide in vivo. v ACKNOWLEDGEMENTS I am deeply grateful to my supervisor, Dr. Samuel Benchimol, for providing me with the opportunity to join his research group and carry out these studies. Completion of this work would not have been possible without his encouragement and exceptional scientific guidance. I would also like to express special thanks to my supervisory committee chair, Dr. Katalin A. Hudak for her unwavering support and guidance, instrumental in completion of my MSc degree. I would also like to thanks my supervisory committee members Dr. Tsushima and Dr. Connor, for their time, support and advice. Furthermore, I would like to thank Yunping Lin, Weili Ma, Wissam Assaily, Elliot Lavi, Arezou Mohajer, Konstantin Savitsky, Daniel Rubinger, Keith Wheaton, Denise Campuzano, and the entire Dr. Hudak's research team for their help and support. Finally, I would also like to thank my family and friends for their patience and untiring support over the years. VI TABLE OF CONTENTS Abstract IV Acknowledgements V Table of contents VI List of figures IX List of abbreviations X CHAPTER 1 - INTRODUCTION 1 1.1 Apoptosis 2 1.1.1 Extrinsic pathway 3 1.1.2 Intrinsic mitochondrial pathway 8 1.1.3 Intrinsic ER stress-induced pathway 12 1.1.4 Caspase-2 dependent pathway 14 1.2 The p53 protein 15 1.2.1 p53-mediated cell survival, cell cycle arrest and senescence 17 1.2.2 p53-mediated apoptosis 20 1.3 p53 induced protein with a death domain (PIDD) 21 1.3.1 Putative in vivo functions of PIDD 22 1.3.2 PIDD: the mouse model 31 1.4 Thesis rationale 31 CHAPTER2-MATERIALS AND METHODS 34 2.1 CellCulture 35 2.2 Gel electrophoresis and immunoblotting 35 2.3 Preparation of Immobilized Antibody Affinity Resin 36 2.4 Antigen Immunoprecipitation 36 2.5 Sample Preparation, SDS-PAGE Analysis, Gel electrophoresis and immunoblotting for immunoprecipitated samples 37 2.6 MALDI MS/MS sample preparation and analysis 38 2.7 Subcellular fractionation 39 2.8 Fractionation of protein samples using solution phase Isoelectric Focusing 39 2.9 One-dimentional gel electrophoresis and immunoblotting for IEF 40 CHAPTER 3 - PIDD EXPRESSION, PURIFICATION AND CHARACTERZATION 42 3.1 PIDD expression patterns in different model systems 43 3.2 Purification of 48 kda pidd-related polypeptide 47 3.3 Characterization of the 48 kda PIDD-related polypeptide 52 CHAPTER 4 - DISCUSSION AND FUTURE DIRECTIONS 58 4.1 DISCUSSION 59 4.2 FUTURE DIRECTIONS 64 4.2.1 Characterization of the 48 kDa polypeptide 64 4.2.2 Investigationg the physiological relevance of the 48 kDa polypeptide in TNF-a-induced apoptosis 65 4.2.3 Evaluating the role of p53 in PIDD-mediated cell death 66 VIII 4.2.4 Investigating the role of PIDD complexes in p53-mediated apoptosis 67 4.2.5 Investigating the involvement of PIDD in TNF-a-induced celldeath 69 APPENDIX: Sample MS report 70 REFERENCES 75 LIST OF FIGURES 1. Schematic representation of the extrinsic apoptotic signalling network 7 2. Schematic representation of the intrinsic (mitochondrial) apoptotic signalling network 11 3. PIDD in the global context of cellular apoptosis, survival and cell cycle arrest signalling networks 28 4. Schematic representation of the putative PIDD fragments and cleavage sites 30 5. Immunogen comparison of 19-2 and AL233 antibodies 33 6. Expression of endogenous PIDD in response to various stress stimuli 45,46 7. Western blot of the immunoprecipitation of PIDD and related polypeptides 51 8. Coomassie total protein staining of the region of the SDS-PAGE gel to be analysed for presence of PIDD-related polypeptides 51 9. Subcellular fractionation of TNF-alpha treated MCF7 cells 55 10. Fractionation of protein samples using solution phase Isoelectric Focusing 56 x LIST OF ABBREVIATIONS AIF Apoptosis Inducing Factor ALDH4 Aldehyde dehydrogenase 4 APAF-1 Apoptotic Protease Activating Factor-1 ATF6 Activating transcription factor 6 ATM Ataxia telangiectasia mutated ATR ATM and Rad3 related BH Bcl-2 homology Bid Bcl2-Interacting Protein c-FLIP FLICE-(FADD-like IL-ip-converting enzyme) inhibitory protein CAD Caspase-Activated DNase CAF CREBBP-associated factor CAK Cyclin/Cyclin-dependent kinase -activating kinase CARD Caspase recruitment domain CDK Cyclin/Cyclin-dependent kinase cIAPl/2 Cellular Inhibitor of Apoptosis-1/2 CytoC Cytochrome-C DD Death domain DED Death Effector Domain DISC Death Inducing Signaling Complex DL Death Ligand DNA-PK DNA-dependent protein kinase DR Death Receptor eIF2a Eukaryotic initiation factor 2a EndoG Endonuclease-G ER Endoplasmic reticulum FÅDD Fas-Associated via Death Domain FasL Fas Ligand GAPDH Glyceraldehyde-3 -phosphate dehydrogenase GML Glycosylphosphatidylinositol-anchoredmolecule-like protein gene GPX Glutathione peroxidase HS Heat shock HTRA2 High Temperature Requirement Protein-A2 IAP Inhibitor of Apoptosis Protein ICAD Inhibitor of Caspase-Activated DNase IEF Isoelectic focusing IKK I-KB-Kinase IR Ionizing radiation IRE1 Inositol-requiring enzyme 1 JNK c-Jun n-terminal kinase LRDD Leucine repeat death domain containing protein LRR Leucine rich repeats MALDI Matrix-assisted laser desorption/ionization MEF Mouse embryonic fibroblast MGMT 06-methylguanine-DNAmethyltransferase MnSOD Manganese superoxide dismutase MOMP Mitochondrial outer membrane permiabilization MS Mass spectrometry NF-KB Nuclear Factor-KB NHEJ Non-homologous end-joining p53AIPl p53-regulated apoptosis-inducing protein-1 PBS Phosphate-buffered saline PCNA Proliferating Cell Nuclear Antigen PERK PKR-like ER kinase Isoelectic point PI3K PI-3 kinase PIDD p53-induced protein with death domain PIDD-C C-terminal fragment of PIDD containing the death domain PIDD-FL Full-length PIDD PIDD-N N-terminal fragment of PIDD containing 7 leucine-rich repeats PIG3 p53-inducible gene 3 PVDF Polyvinylidene Fluoride RAIDD Receptor-interacting protein (RlP)-associated ICHl/CED- 3-homologous protein with a death domain Rb Retinoblastoma RIP1 Receptor-interacting Protein-1 ROS Reactive oxygen species SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis shRNa Short Hairpin RNA siRNA Short Interfering RNA SMAC/Diablo Second Mitochondria-Derived Activator of Caspase TACE Tumour Necrosis Factor alpha converting enzyme TIGAR TP53-Induced Glycolysis and Apoptosis Regulator TG Thapsigargin TNF-a Tumour Necrosis Factor a TNFR1/2 Tumour Necrosis Factor Receptor 1/2 TRÅDD TNF-Receptor Associated protein with Death Domain TRAF2 TNF-Receptor Associated Factor 2 TRAIL TNF-Receptor Apoptosis Inducing Ligand UPR Unfolded Protein Response XPC Xeroderma pigmentosum group C 1 CHAPTER 1 INTRODUCTION 2 1.1 Apoptosis Apoptosis is a naturally occurring form of programmed cell death by which a cell receives a "directive" to dismantle in a controlled and orderly manner. Cells that are no longer useful or detrimental to the organism are disposed of in an organized manner that prevents the development of inflammatory responses, typically associated with necrotic cell death (Edinger et al., 2004; Fink and Cookson 2005). Apoptosis plays a central and indispensable role in embryonic development and maintenance of adult tissue homeostasis.
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