UNIVERSITY OF CINCINNATI _____________ , 20 _____ I,______________________________________________, hereby submit this as part of the requirements for the degree of: ________________________________________________ in: ________________________________________________ It is entitled: ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ Approved by: ________________________ ________________________ ________________________ ________________________ ________________________ Role of Glutamate-Cysteine Ligase in Maintaining Glutathione Homeostasis and Protecting against Oxidative Stress A dissertation submitted to the Division of Research and Advanced Studies University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTORATE OF PHILOSOPHY (Ph.D.) in the Department of Environmental Health of the College of Medicine 2003 by Yi Yang M.D., Sun Yat-sen University of Medical Sciences, 1995 M.S., Sun Yat-sen University of Medical Sciences, 1997 Committee Chair: Daniel W. Nebert, M.D. Professor Department of Environmental Health University of Cincinnati Abstract Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme catalyzing the first step of glutathione (GSH) biosynthesis. In higher eukaryotes, this enzyme is a heterodimer comprising a catalytic subunit (GCLC) and a modifier subunit (GCLM); the latter changes the catalytic characteristics of the holoenzyme. In the first part of this dissertation, the heterodimer formation between GCLC and GCLM was investigated, using the yeast two-hybrid system and affinity chromatography. A strong and specific interaction between GCLC and GCLM was observed in both systems. Deletion analysis showed that most regions, except part of the C-terminal region in GCLC and part of the N-terminal region in GCLM, are required for the interaction to occur. Point mutations on selected amino acids were also tested for the binding activity. GCLC C248A- C249A and P158L mutants had the same strength of binding to GCLM as the full-length GCLC, yet the catalytic activity was dramatically decreased in these mutations. The functions of GCLC and GCLM were further studied in knockout mouse lines with the targeted disruption of the gene encoding either subunit. Deletion of the Gclc gene specifically in hepatocytes led to a ~98% loss of GSH in liver and plasma. The affected livers showed significant oxidative damage, mitochondrial degeneration, and hepatocyte death. These mice died of hepatic failure around post-partum day 30 (d30). Supplementing N-acetylcysteine in the drinking water, starting at d21, was able to prevent early death; however, these mice developed pathology characteristic of liver cirrhosis. Gclm(-/-) mice, on the other hand, showed no overt phenotype. However, GSH levels decreased to 9-16% of normal in tissues and plasma. Compared to the GCL holoenzyme partially purified from livers of Gclm(+/+) mice, hepatic GCLC in Gclm(-/-) mice showed a ~2-fold increase in Km for glutamate and a dramatically enhanced sensitivity to GSH inhibition. The major decrease in GSH, combined with diminished GCL activity, rendered Gclm(-/-) fetal fibroblasts more strikingly sensitive to chemical oxidants such as hydrogen peroxide. These data demonstrate that both GCLC and GCLM are critical in maintaining GSH homeostasis. Disruption of either component is strikingly detrimental to the cell’s or animal’s protection system against oxidative stress. Acknowledgments I would like to thank members of my dissertation committee, Drs. Daniel W. Nebert, Timothy P. Dalton, Howard G. Shertzer, and Anil Menon for all the suggestions and criticisms. I especially want to express my sincere gratitude to my advisor, Dr. Dan Nebert, and my lab mentor, Dr. Timothy Dalton, for the wonderful opportunity to work on this project, for the advice, support, and encouragement over the past 6 years. You have made my graduate education a fruitful experience. My gratitude also goes to Dr. Alvaro Puga and Dr. Mario Medvedovic for the advice and inspiration during my graduate study. I would also liker to thank Dr. Marian Miller for her technical assistance. Special thanks to the former graduates from our lab, Dr. Matthew Z. Dieter and Dr. Willy A. Solis, for sharing the good and bad times in our graduate student lives, for valuable discussions and suggestions, and for the friendship and encouragement. I would also like to thank for the rest of the members in my lab for various help. You have made the lab an enjoyable place to work. My deepest gratitude goes to my father, Zhilin Yang, my mother, Jinzhong Yang, and my brother, Genghua Yang. Your unconditional love, your endless support, and your tremendous sacrifice have always been the greatest resources of my motivation. I also want to thank my husband, Feng Wang, for your love, patience, understanding, and encouragement. Nothing would have been possible without a strong support from all of you, my dear family. 1 Table of Contents List of tables ………………………………………………………………………………...…. 3 List of figures …………………………………………………………………………………...4 Abbreviations ………………..……………………………………………………………..….. 5 Introduction ……………….…………………………………………………………….…...… 6 Chapter I Characterization of the Interaction between Glutamate-Cysteine Ligase Subunits Abstract ……….……………………………………………………………………………… 21 Introduction ……………….………………………………………………………………..… 22 Materials and Methods ………………………………………………………………..……… 23 Results ………………..………………………………………………………………….…… 26 Discussion ………………………………………………………………………………..…... 29 References ……………………………………………………………………………….…… 32 Chapter II Hepatocyte-Specific Knockout of Glutamate-Cysteine Ligase Catalytic Subunit Gclc(-/-) in Mouse: Early Death with Progressive Liver Degeneration and Rescue by N-acetylcysteine Abstract …………………………………………………………………………….………. 47 Introduction ………………………………………………………………………………….. 49 Materials and Methods ………………...……………………………………………….……. 51 Results ………………………………………………………….…………………….……… 54 Discussion ………………………………………………………………..…………….……. 59 2 References ……………………………………………………………………….………….. 63 Chapter III Initial Characterization of the Glutamate-Cysteine Ligase Modifier Subunit Gclm(-/-) Knockout Mouse: Novel Model System for a Severely Compromised Oxidative Stress Response ….... 84 Discussion …………………………………..…………………………………….…………. 85 References for introduction and discussion sections …….…………………….……………. 93 3 List of Tables Chapter I Table 1 - Plasmids constructs used in this study ……………………………………………. 39 Table 2 - Enzymatic activity of wild-type and mutant GCLC ……………………………… 40 Chapter II Table 1 - GSH levels in tissues of Gclc(f/f) mice and Gclc(h/h) mice ………………..…..... 74 Table 2 - Mitochondria GSH levels in livers from Gclc(f/f) mice and Gclc(h/h) mice …….. 75 Table 3 - Cysteine levels in tissues of Gclc(f/f) mice and Gclc(h/h) mice ……………..…... 76 Table 4 - Liver biochemical functions in Gclc(f/f) mice and Gclc(h/h) mice …..……..…… 77 4 List of Figures Chapter I Figure 1 - GCLC and GCLM interaction in yeast …………………………………………… 41 Figure 2 - Interaction of GCLC and GCLM on Ni-NTA resin ……………………………… 42 Figure 3 - GCLC deletion studies in yeast two-hybrid system ……………………….....….... 43 Figure 4 - GCLM deletion studies in yeast two-hybrid system ……………………………… 44 Figure 5 - Interaction of GCLC mutations with GCLM ……………………………….…….. 45 Chapter II Figure 1 - Generation of Gclc floxed mice ……………………………………………..…….. 78 Figure 2 - Hepatocyte-specific conversion of Gclc(f) allele to Gclc(h) allele ……………...… 79 Figure 3 - Liver histology of Gclc(f/f) mice and Gclc(h/h) mice …………………………….. 80 Figure 4 - Liver morphology and histology of Gclc(f/f) mice and Gclc(h/h) mice after NAC supplement ………………………………………………………………………………….... 81 Figure 5 - Liver mRNA levels in Gclc(f/f) and Gclc(h/h) mice ……………………………… 82 Figure 6 - Lipid peroxidation in livers of Gclc(f/f) mice and Gclc(h/h) mice …...……..…….. 83 5 Abbreviations ALT - alanine aminotransferase AST- aspartate aminotransferase BSO - buthionine sulfoximine γ-GC - γ-glutamylcysteine GCL - glutamate-cysteine ligase GCLC – glutamate-cysteine ligase catalytic subunit (Gclc = mouse gene) GCLM – glutamate-cysteine ligase modifier subunit (Gclm = mouse gene) GD - gestational day GGT - γ-glutamyltranspeptidase GSH - reduced glutathione GSH-EE – glutathione monoethyl ester GSSG - glutathione disulfide GPX - glutathione peroxidase H2O2 - hydrogen peroxide MFF – mouse fetal fibroblasts NAC – N-acetylcysteine . - O2 - superoxide anion .OH – hydroxyl radical ROS - reactive oxygen species SOD - superoxide dismutase TBARS - thiobarbituric acid-reactive substances 6 Introduction Oxygen is essential for most forms of life. Its reduction to water provides the energy that allows virtually all complex functions to proceed in aerobic organisms. However, oxygen can be potentially dangerous if not completely reduced. The reactive oxygen species (ROS), such as . - . superoxide anion ( O2 ), hydrogen peroxide (H2O2), and hydroxyl radical ( OH), can arise from normal metabolic reactions as well as environmental insult. The accumulation of ROS may perturb the cell’s natural antioxidant defense systems, resulting in damage to a variety of biochemical and physiological processes. Oxidative stress, by definition, refers to a disturbance in the prooxidant-antioxidant balance in favor of the former [1]. Oxidative stress has been implicated in a variety of diseases and degenerative conditions such as aging, inflammation, carcinogenesis, diabetes,