Rockefeller University Digital Commons @ RU Student Theses and Dissertations 2009 Regulation of Apoptosis by XIAP Ubiquitin- Ligase Activity Andrew James Schile Follow this and additional works at: http://digitalcommons.rockefeller.edu/ student_theses_and_dissertations Part of the Life Sciences Commons Recommended Citation Schile, Andrew James, "Regulation of Apoptosis by XIAP Ubiquitin- Ligase Activity" (2009). Student Theses and Dissertations. Paper 125. This Thesis is brought to you for free and open access by Digital Commons @ RU. It has been accepted for inclusion in Student Theses and Dissertations by an authorized administrator of Digital Commons @ RU. For more information, please contact [email protected]. REGULATION OF APOPTOSIS BY XIAP UBIQUITIN-LIGASE ACTIVITY A Thesis Presented to the Faculty of The Rockefeller University In Partial Fulfillment of the Requirements for The degree of Doctor of Philosophy by Andrew James Schile June 2009 © Copyright by Andrew James Schile 2009 REGULATION OF APOPTOSIS BY XIAP UBIQUITIN-LIGASE ACTIVITY Andrew James Schile, Ph.D. The Rockefeller University 2009 Virtually all animal cells have the known ability to self-destruct by undergoing apoptosis, a morphologically distinct form of programmed cell death. The proper regulation of apoptosis is critical for both development and tissue homeostasis, and inhibition of apoptosis contributes to the development and progression of cancer. Inhibitor of Apoptosis Proteins (IAPs) can bind to and inhibit caspases, the key executioners of apoptosis. Because IAPs are frequently over-expressed in human tumors, they have become major pharmacological targets for developing new cancer therapeutics. Many IAPs contain RING domains that function as E3 ubiquitin-ligases to regulate the abundance of IAPs themselves and their binding partners by engaging the ubiquitin system. The precise physiological function of individual mammalian IAPs and their role as E3- ubiquitin ligases in situ remain largely obscure. Here, we investigated the function of XIAP ubiquitin-ligase activity by deleting the RING motif via gene targeting in the mouse. Mice expressing XIAP ΔRING were fertile, born in expected proportions, and were not obviously prone to disease in a pathogen-free environment. Removing the RING finger motif stabilized XIAP protein in apoptotic thymocytes, demonstrating that XIAP ubiquitin-ligase activity is a major determinant of XIAP protein stability. However, consistent with earlier reports on XIAP-null mice, we found no detectable abnormalities in apoptosis of mutant thymocytes. On the other hand, ΔRING embryonic stem cells and fibroblasts had elevated caspase-3 enzyme activity and impaired ubiquitination of active caspase-3 during apoptosis. Furthermore, XIAP ΔRING embryonic fibroblasts were strongly sensitized to TNF-α-induced apoptosis. Similar results were obtained with XIAP-null mice. Finally, deletion of the RING also improved the survival of mice in the Eµ-Myc lymphoma model. The improved prognosis corresponded to increased apoptosis and decreased abundance of proliferating B-cells in the bone marrow, and a curtailed incidence of leukemia. This demonstrates a physiological requirement of XIAP E3 ubiquitin-ligase activity for the inhibition of apoptosis and for tumor suppression in vivo. For my parents iii ACKNOWLEDGEMENTS First, I would like to thank my advisor, Hermann Steller, for not discouraging me from taking on a mouse project in a fruit fly lab. I truly benefited from his optimism, insight and guidance. I would also like to thank the members of my thesis committee, Titia de Lange and Shai Shaham, for their perceptive advice and suggestions throughout all stages of my project. I also thank my external reader, Neal Rosen, for his time and comments. I would like to thank María García-Fernández for helping me with some of the Eµ-Myc experiments, and Samara Brown for assisting with mouse genotyping and injections. I also thank Holger Kissel for supervising the early stages of my project, and Joe Rodriguez for supplying many useful plasmids and antibodies. I am indebted to Dónal O’Carroll for teaching me about ES cell culture, and Mohanish Deshmukh for supplying XIAP-null mice. Finally, I thank all of the past and present members of the Steller lab for their camaraderie. iv TABLE OF CONTENTS LIST OF FIGURES ....................................................................................x LIST OF ABBREVIATIONS .....................................................................xi 1 INTRODUCTION ..................................................................................1 1.1 Introductory comment ....................................................................1 1.2 Historical perspective on programmed cell death .........................2 1.3 Caspases...........................................................................................5 1.4 Activation of caspases......................................................................9 1.4.1 Activation through mitochondrial factors.....................................9 1.4.2 Activation through death receptors ............................................13 1.5 Regulation of cell death by anti-apoptotic proteins.....................15 1.6 IAPs: negative regulators of caspases...........................................19 1.6.1 Inhibition of caspases by direct binding .....................................21 1.6.2 Inhibition of caspases through ubiquitination.............................23 1.7 IAP antagonists..............................................................................27 1.8 Mouse models of IAP function......................................................32 1.9 Roles of IAPs outside of apoptosis ................................................38 1.10 IAPs in cancer and other diseases...............................................39 1.11 Concluding remarks ....................................................................41 1.12 Prologue to the XIAP ΔRING project .........................................43 2 ENGINEERING THE ΔRING ALLELE BY GENE TARGETING ...45 2.1 SUMMARY ...................................................................................45 2.2 INTRODUCTION.........................................................................45 2.3 EXPERIMENTAL PROCEDURES.............................................47 2.3.1 Design and construction of the XIAP ΔRING targeting vector ..47 2.3.2 Gene targeting, generation of chimeric mice, and establishment of a ΔRING colony .................................................................................49 2.3.3 Aging and irradiation experiments .............................................51 2.3.4 TNF-α and Fas-dependent liver apoptosis..................................52 2.3.5 Statistical treatments..................................................................53 2.4 RESULTS ......................................................................................53 v 2.4.1 Generation of ΔRING mice by gene targeting and blastocyst injection..............................................................................................53 2.4.2 Analysis of an aging cohort of mice...........................................57 2.4.3 Whole-body irradiation..............................................................58 2.4.4 In vivo hepatocyte apoptosis......................................................58 2.5 DISCUSSION ................................................................................62 3 APOPTOSIS IN ΔRING THYMOCYTES AND OTHER PRIMARY HEMATOPOETIC CELLS.......................................................................63 3.1 SUMMARY ...................................................................................63 3.2 INTRODUCTION.........................................................................63 3.2.1 General comment on apoptosis in immunity ..............................63 3.2.2 Apoptosis in developing lymphocytes........................................64 3.2.3 Apoptosis in peripheral T- and B-cells.......................................67 3.2.4 Roles of IAPs in thymocyte apoptosis........................................68 3.3 EXPERIMENTAL PROCEDURES.............................................70 3.3.1 Isolation and culture of primary lymphocytes ............................70 3.3.2 Apoptosis assays........................................................................72 3.3.3 FLICA labeling of the caspase-3 active site ...............................72 3.3.4 Annexin-V/propidium iodide and TUNEL apoptosis assays ......73 3.3.5 Caspase-3-like (DEVDase) enzyme activity assay.....................74 3.3.6 Immunoblotting .........................................................................74 3.3.7 Analyzing the cellular compositions of the thymus, spleen and bone marrow.......................................................................................75 3.4 RESULTS ......................................................................................76 3.4.1 RING ubiquitin-ligase activity was the primary determinant of XIAP stability during thymocyte apoptosis.........................................76 3.4.2 Genetic deletion of the XIAP RING did not influence thymocyte apoptosis.............................................................................................79 3.4.3 Lymphoid compartments were distributed normally in ΔRING mice ..................................................................................................84 3.5 DISCUSSION ................................................................................88