University of Alberta Hexokinase 1 attenuates type II death receptor-induced apoptosis. by Anja Schindler A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Immunology Medical Microbiology and Immunology ©Anja Schindler Fall 2013 Edmonton, Alberta Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission. To my husband Guy-Armel and my son Menelik, who are a constant source of joy and inspiration ABSTRACT Deregulated TNF signaling with elevated or decreased levels of TNF-induced apoptosis causes numerous inflammatory and cancerous diseases. Thus, there is a clear need to identify cellular proteins that regulate cell fate in the presence of TNF. RNA interference technology provides an excellent tool to address that problem. It allows the rapid generation of transient protein depletion “mutants” in cell culture, whose behaviour in the context of TNF can be examined. I developed a quantitative high-throughput siRNA assay to identify modifiers of TNF-induced cell death in HeLa cells and screened a set of nine hundred eighty six proteins, which includes the entire set of human kinases and phosphatases and several of their binding partners or related proteins. Of all gene products tested, loss of hexokinase 1 (HK1) resulted in the greatest elevation in TNF- induced death. In secondary assays, I demonstrated that the presence of HK1 attenuates TNF-induced apoptosis. Specifically, HK1 attenuates the processing of key caspases and caspase substrates, and decrease of the mitochondrial membrane potential. The predominantly mitochondrial localization of HK1 prompted me to examine whether HK1 impacted TNF-induced apoptosis at the mitochondria. I found that HK1 constitutively stabilized the mitochondrial membrane potential at least in part through the inhibition of the pro-apoptotic Bcl- 2 effector proteins Bax and Bak. In line with these findings, HK1 attenuated Bax translocalization and oligomerization to and at the mitochondria in the absence and presence of an apoptotic stimulus. Finally, I found that attachment of hexokinases to the mitochondria is a prerequisite for mitochondrial integrity and essential for pro-survival functions of hexokinases in TNF-induced apoptosis. These data are the first loss-of-function reports to examine the involvement of HK1 in the transduction of extrinsic apoptotic cues and identify HK1 as a potential target in deregulated TNF signaling. ACKNOWLEDGEMENTS Foremost, I want to thank my supervisor Dr. Edan Foley. Most of my skills as a scientist are the product of his mentorship. I am forever grateful for his tireless effort on that journey, his patience, and the trust that he had in me. I greatly appreciated the freedom he gave me in pursuing my own ideas and his input to realize them, his positive outlook, and infectious passion for science. I also want to express my gratitude for his support during my pregnancy with my son Menelik and the years as a mother of a young child. I am grateful for the mentorship I experienced from my graduate committee members Drs. Michele Barry and Kevin Kane. I valued their insights into my project and their personal support over the years. I also want to thank Dr. Michele Barry for training me in various aspects of human tissue culture and for numerous reagents she generously provided. I want to thank my external examiners Drs. Ing Swie Goping and Robert Screaton for their time and effort in the evaluation of my thesis. I am thankful for the advice and materials I kindly received from Dr. Ing Swie Goping to perform mitochondrial isolations. My thanks also go to Dr. David Evans, who provided the siRNA libraries as the cornerstone of this project. Moreover, I greatly benefited from the excellent working environment in this department that is to a large extent the result of his engagement as the departmental chair. Thank you to the departmental support staff, especially Anne Giles and Tabitha Vasquez. The time as a graduate student would not have been as joyful, were there not the marvellous crowd of colleagues and friends in the department with whom I have shared every aspect of the graduate student experience. I especially want to thank Silvia Guntermann, David Bond, Brendon Parsons, Tanja Lindahl, David Primrose, Derly Benitez, Chelsea Davidson, George Johnson, Brittany Fraser, and Kristina Petkau. I am also grateful for the experience, protocols, and materials that my colleagues in the entire department kindly shared in support of my work. The work in this thesis would have been impossible without the human cell lines derived from individuals such as Henrietta Lacks. Immeasurable thanks goes to my parents Rita and Hans-Albrecht, who fostered my curiosity and encouraged me to persue a career path that aligns with my passion. Finally, the strongest rocks along the road of my PhD were my husband Guy- Armel and my son Menelik. Without your loving support, I would not have been able to conduct the work presented in this thesis. TABLE OF CONTENTS CHAPTER 1. INTRODUCTION…………………………… 1 1.1. Death receptor-induced apoptosis…………………………………… 2 1.1.1. Apoptosis……………………………………………………………. 2 1.1.2. Apoptotic caspases………………………………………………... 3 1.1.3. Death receptors……………………………………………………. 6 1.1.4. TNF receptor 1…………………………………………………….. 8 1.1.4.1. The physiological role of TNF…………………………… 8 1.1.4.2. TNF…………………………………………………………. 9 1.1.4.3. The TNF receptor complex………………………………. 9 1.1.4.4. TNF-induced NF-κB signaling…………………………… 12 1.1.4.5. TNF-induced MAPK signaling…………………………… 13 1.1.4.6. TNF-induced apoptosis…………………………………... 15 1.1.5. Type I and type II death receptor-induced apoptosis…………... 15 1.1.6. Type II mitochondrial apoptosis………………………………….. 20 1.1.6.1. The mitochondrial membranes………………………….. 20 1.1.6.2. Bcl-2 proteins and the permeabilization of the mitochondrial outer membrane………………………….. 22 1.1.6.3. Permeabilization of the mitochondrial inner membrane. 24 1.2. Hexokinases………………………………………………………………. 25 1.2.1. Hexokinase isozymes……………………………………………. 25 1.2.2. Hexokinase function……………………………………………... 29 1.2.2.1. Hexokinases and metabolism…………………………… 29 1.2.2.2. Hexokinases and apoptosis……………………………… 31 1.2.2.3. Hexokinases and cancer…………………………………. 35 1.3. RNA interference………………………………………………………… 36 1.4. Project objectives……………………………………………………….. 39 CHAPTER 2. MATERIALS AND METHODS…………….. 41 2.1. Buffers and media……………………………………………………….. 42 2.2. DNA cloning………………………………………………………………. 46 2.2.1. Expression constructs……………………………………………... 46 2.2.2. Preparation of bacterial culture plates with selective antibiotics…………………………………………………………… 49 2.2.3. Preparation of fluid media for bacterial cultures………………... 49 2.2.4. Generation of chemically competent bacteria…………………... 50 2.2.5. Set up of bacterial cultures on culture plates from a glycerol stock………………………………………………………………… 50 2.2.6. Set up of bacterial mini cultures from bacterial colonies………. 50 2.2.7. Set up of bacterial midi cultures from mini cultures……………. 50 2.2.8. Plasmid midi preparation………………………………………….. 51 2.2.9. Measurement of DNA concentration and purity………………… 52 2.2.10. Polymerase chain reaction (PCR) with Pfx polymerase……… 52 2.2.11. Agarose gel electrophoresis…………………………………….. 52 2.2.12. Polymerase chain reaction (PCR) purification………………… 53 2.2.13. Preparative restriction digest……………………………………. 53 2.2.14. Preparative agarose gel electrophoresis………………………. 54 2.2.15. DNA isolation from excised gel fragments…………………….. 54 2.2.16. Ligation…………………………………………………………….. 55 2.2.17. Transformation of bacteria………………………………………. 55 2.2.18. Analytical polymerase chain reaction (PCR) from bacterial colonies……………………………………………………………. 56 2.2.19. Plasmid mini preparations……………………………………….. 56 2.2.20. Analytical restriction digest……………………………………… 57 2.2.21. Preparation of DNA samples for sequencing………………….. 57 2.2.22. Site directed mutagenesis of plasmid constructs……………... 58 2.2.23. Preparation of glycerol stocks…………………………………... 59 2.3. Cell lines and cell culture………………………………………………. 59 2.3.1. Cell lines…………………………………………………………….. 59 2.3.2. Passaging…………………………………………………………… 60 2.3.3. Determination of the concentration of viable cells in a cell suspension………………………………………………………….. 60 2.4. Cell treatments…………………………………………………………… 61 2.5. siRNA and siRNA screening…………………………………………… 61 2.5.1. siRNA transfections………………………………………………... 61 2.5.2. GAPDH assay for the optimization of siRNA transfection efficiency……………………………………………………………. 64 2.5.3. High-throughput siRNA screen for modifiers of TNF-induced death………………………………………………………………… 65 2.5.4. Screen analysis……………………………………………………. 66 2.5.4.1. Plate normalization and data scoring…………………… 66 2.5.4.2. Visualization of 96-well plates…………………………… 67 2.5.4.3. Kernel density estimates for behaviour of positive controls…………………………………………………….. 67 2.5.4.4. Heat maps of viability values and death indices in the screen……………………………………………………… 67