PROBING LYMPHOCYTE RESPONSES TO MODULATION OF THE F0F1-ATPASE BY BZ-423 by Li Wang A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemistry) in the University of Michigan 2009 Doctoral Committee: Professor Gary D. Glick, Chair Professor Dennis Keith Bishop Professor Robert T. Kennedy Associate Professor Philip D. King Associate Professor Anna K. Mapp Li Wang © 2009 All Rights Reserved This dissertation is dedicated to my loving husband Dr. Jun Cheng ii ACKNOWLEDGEMENTS This thesis could not have been finished without the help and support from everyone I have worked with over the past six years. First, I am really thankful to my advisor Prof. Gary Glick for supporting me in all aspects of my graduate career. His guidance, intellect and expertise on my thesis projects as well as providing a world-class scientific training environment are priceless. I would also like to thank Dr. Tony Opipari for his countless in-depth discussions regarding my research data, which have greatly enhanced my intellectual development. In addition, I would like to thank all members of my dissertation committee: Dennis K Bishop, Robert T Kennedy, Philip D King, and Anna K Mapp. I appreciate their time taken out of busy schedules to serve on my committee as well as provide constructive feedback through the dissertation process. I would like to thank all current and former Glick lab members for their friendships and contributions: Lara Swenson, Tom Sundberg, Joanne Cleary, Neal Blatt, Tony Boitano, Katie Groendyke, Gina Ney, Shawn Stevens, Richard Frazee, Xueni Chen, Tasha Francis, Dan Wahl, Melissa Bobeck, Ben Farrar, Jenny Rush and Costas Lyssiotis. I am also grateful for the Jim Mobley, Lara Swanson, and Rod Morgan to isolate splenic lymphocytes from mice for me. I also would like to thank Chitra for the protocol of nucleus isolation. iii Special thanks go to Gary, Lara, and Amberlyn Wands for their time and effort to read and critique my thesis. Finally, I would thank my friends and family. I must also thank my parents Xianqin and Jinsheng, parents-in-law Fulan and Xianlin, my brothers Changqing and Changhong and my sister Xiufang for their love, encouragements and understandings. But most of all, it would have been impossible to complete this thesis without the tremendous love and support from my husband Jun throughout the whole graduate school. iv TABLE OF CONTENTS DEDICATION............................................................................................................ii ACKNOWLEDGMENTS ........................................................................................iii LIST OF TABLES ....................................................................................................xi LIST OF FIGURES .................................................................................................xii LIST OF ABBREVIATIONS…………………………...……………………....xviii ABSTRACT..………………………………………………………………...…xxviii CHAPTER 1 INTRODUCTION...........................................................................................1 Autoimmune disease results from loss of peripheral tolerance to self- antigens .....................................................................................1 Immunological tolerance is critical for maintaining normal immune function ...........................................................2 Pathogenic lymphocytes derived from defective peripheral tolerance are critical for SLE pathogenesis ..................4 Mechanisms of deletion, anergy and receptor editing in maintaining peripheral tolerance ...................................................................6 Apoptosis ..................................................................................6 Anergy.......................................................................................9 Receptor editing ......................................................................11 The role of B cells in autoimmunity ...................................................11 During development, B cells are regulated to maintain tolerance for self-antigens ...........................................13 BCR signaling in peripheral tolerance ....................................16 Other factors that contribute to abnormal activation of self-reactive B cells .....................................................17 SLE treatment .....................................................................................19 The challenge of SLE treatment and potential novel lupus treatments ....................................................................19 v B cell targeted SLE treatment .................................................20 Bz-423, a pro-apoptotic benzodiazepine, improves lupus disease .....22 Bz-423 is a novel chemical for lupus treatment with efficacy and specificity .............................................................22 Bz-423 induces superoxide as a result of inhibiting mitochondrial F0F1-ATPase ........................................23 Statement of Goals ..............................................................................26 Bibliography… ..............................................................................................28 CHAPTER 2 MECHANISM OF ANTI-IGM INDUCED SYNERGY WITH BZ-423 ..45 Introduction ....................................................................................................45 Apoptotic signaling pathway ...............................................................45 Mechanisms for mitochondrial pro-apoptotic factors release ..............48 Involvement of MPT pore in cytochrome c release .................48 Invovlement of mitochondrial outer membrane permeablization (MOMP) in mitochondrial pro-apoptotic factors release ..........................................................................52 Bcl-2 family proteins regulate MOMP through Bax and Bak ...............................................................................55 B cell receptor mediated signaling pathway ........................................63 BCR-induced survival and proliferation ..................................65 Co-receptor of BCR signaling pathway ...................................67 B cell receptor mediated apoptosis ..........................................70 Defective B cell receptor signaling and apoptosis in SLE ...................74 Defective BCR signaling in SLE .............................................75 Defective BCR-mediated apoptosis in SLE .............................77 Bz-423 mediated apoptotic signaling pathway in Ramos B cells ........80 Bz-423 and anti-IgM co-treatment results in synergistic cell death ....82 Statement of problem ...........................................................................83 Results .............................................................................................................84 Bz-423 selectively kills activated B cells with either a GC phenotype or immature B characteristics .................................84 The roles of Bz-423 and anti-IgM in cell death induced by co-treatment…………………………………………………………..89 Bz-423 is not required for early proximal activation signals to establish synergy ..........................................................89 Anti-IgM pre-stimulation sensitizes Ramos B cells to vi Bz-423 ..........................................................................91 Basic characterizations of cell death induced by anti-IgM and Bz-423 co-treatment .............................................................................93 12 h anti-IgM and Bz-423 co-treatment is required to establish full cell death by co-treatment .....................................93 Cell death induced by co-treatment is kinetically different from cell death induced by Bz-423 or anti-IgM alone .........95 The involvement of superoxide in cell death induced by anti-IgM and Bz-423 co-treatment .................................................................97 ROS is an early signal to cell death induced by co-treatment .................................................................97 Sustained superoxide increase is required to establish cell death induced by co-treatment .................................................99 General ROS-inducing reagents do not synergize with anti-IgM ......................................................................99 Antioxidant and an extra-cellular Ca2+ chelator together block cell death induced by anti-IgM and Bz-423 co-treatment ............101 Ca2+ involvement in the cell death by co-treatment ...........................102 Early Ca2+ change is dispensable for cell death induced by anti-IgM and Bz-423 co-treatment.............................103 Ca2+ is sufficient to sensitize Ramos cells to Bz-423 ............106 Anti-IgM induced Ca2+ increase is required for cell death induced by anti-IgM and Bz-423 co-treatment ........106 Detailed cellular signaling pathway of cell death induced by Bz-423 and anti-IgM co-treatment .....................................................110 New protein is synthesized within 6 h of co-treatment to establish cell death .....................................................110 Cell death induced by co-treatment adopts Mitochondria-dependent pathway .............................111 Mitochondrial pro-apoptotic factors are released by co-treatment ...............................................................114 Bax and Bak are activatd by anti-IgM and Bz-423 co-treatment ....................................................................................117