REDOX HOMEOSTASIS AND STRESS IN MOUSE LIVERS LACKING THE NADPH-DEPENDENT DISULFIDE REDUCTASE SYSTEMS by Colin Gregory Miller A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry MONTANA STATE UNIVERSITY Bozeman, Montana November 2019 ©COPYRIGHT by Colin Gregory Miller 2019 All Rights Reserved ii DEDICATION I would like to dedicate this thesis to my dear friends Adrienne Arnold, Jacob Artz, Aoife Casey, Michael Christman, Phil Hartman, Ky Mickelsen, Sarah Partovi, Greg Prussia and Danica Walsh. Your support has been indescribably generous and completely invaluable. My quality of life, especially during graduate school, has been immeasurably improved by you all and I count myself incredibly lucky to call you my friends and colleagues. Most importantly, I would like to dedicate this degree to my mom. Any standard of excellence I have ever held has been inspired by you. Your compassion and generosity to others, your unwavering positivity and resilience are a constant source of inspiration. I would consider myself a complete success if I grow up to be half the person you are. iii ACKNOWLEDGEMENTS First, I would like to acknowledge support for the work presented in this thesis provided by the NIH (AG055022) and Montana State University. I would also like to acknowledge committee members Dr. Mary Cloninger and Dr. Brian Bothner for invaluable scientific support and guidance. I would like to thank Dr. Loretta Dorn, Dr. James Hohman and the Chemistry Department of Fort Hays State University for providing me the training and desire to pursue a life in science. Much of the work presented here has been directly or indirectly improved by the work of our wonderful collaborators: Dr. Gina DeNiclo, Dr. Colin Shearn, Dr. Péter Nagy, Dr. Éva Dóka, Dr. Elias Arnér, and Dr. Arne Holmgren. Members of the Schmidt research group have also been incredibly valuable colleagues and friends. Dr. Justin Prigge is responsible for almost all of my training in molecular biology and has supported and guided me since my first moments in the Schmidt lab. My outstanding trainee, Allison Perez, has made valuable contributions on both the ascorbate and L-(34S)cystine projects. Julia Houston, Eden Manuel, Alex Kubacki, Curtis Bailey, Jean Kundert, Julie Amato, Maria Jerome, Julia Lytchier, Rosana Molina, Logan Johns, Krista Quale, Ian Cavigli, and Michael Bruschwein have all made substantial contributions to the work presented and to my experience in the lab. My scientific journey at MSU began under the tutelage of Dr. Paul Grieco. I am deeply indebted to Paul for setting me on the path that has led to this dissertation. I would also like to acknowledge Dr. Ben Reeves for his patience, guidance and friendship while supervising the work that led to my Master’s degree in 2015. Lastly, I must acknowledge my PI and mentor, Dr. Ed Schmidt. I really don’t have the words to convey how much I’ve enjoyed working for Ed and how incredibly appreciative I am of his unrelenting support. From brainstorming sessions in his office or on the patio of Bridger Brewing to lab retreats with the donkeys, I’ve enjoyed every day of working for Ed. iv TABLE OF CONTENTS 1. INTRODUCTION PART ONE: NAPDH-DEPENDENT AND –INDEPENDENT DISULFIDE REDUCASE SYSTEMS .............................................1 Contribution of Authors and Co-Authors ........................................................................1 Manuscript Information ...................................................................................................2 1. Redox reactions and reducing power ...........................................................................4 2. NADPH: A “universal currency” for anabolic reduction ............................................4 3. Early evolution of biological disulfide reduction ........................................................7 4. Disulfide reductase-driven reactions fuel DNA precursor biosynthesis ................................................................................................................11 5. NADPH-dependent disulfide reductases ...................................................................12 6. NADPH-independent disulfide reductase systems capable of sustaining DNA replication in microbes or plants .....................................................15 7. Sources of disulfide reducing power in eukaryotic organelles ..................................19 8. The identification of a constitutive NADPH-independent cytosolic disulfide reductase system in mammals .....................................................................21 9. Collateral impacts of the mammalian NADPH-independent disulfide reductase system .........................................................................................26 10. Is the Met-driven cytosolic disulfide reductase system truly NADPH-independent? .............................................................................................27 11. Evolution of the Met-dependent disulfide reductase system ..................................28 12. Flux-direction in the transsulfuration pathway ........................................................30 13. Similar phylogenetic distributions of TrxR-“type” and transsulfuration-“direction” .....................................................................................35 14. Roles of the metazoan TrxR1 system in signaling...................................................36 15. Closing perspectives: Balancing antioxidant activities and redox signaling ..................................................................................................38 Chapter 1 References: ....................................................................................................41 2. INTRODUCTION PART TWO: DISULFIDE REDUCTASE SYSTEMS IN THE LIVER ...........................................................................................58 Contribution of Authors and Co-Authors ......................................................................58 Manuscript Information .................................................................................................59 Abstract ..........................................................................................................................61 The redox biology of sulfur and the need for disulfide reductase systems ...........................................................................................................62 The NADPH-dependent disulfide reductase systems ....................................................65 Disulfide/dithiol redox in the cytosol and nucleus ........................................................67 Disulfide/dithiol redox in mitochondria.........................................................................70 Disulfide/dithiol redox in the endoplasmic reticulum ...................................................72 v TABLE OF CONTENTS CONTINUED Genetic disruptions of the cytosolic NADPH-dependent disulfide reductase systems in mice ..............................................................................................74 NADPH-independent disulfide reduction in the liver ...................................................76 Cross-trafficking of disulfide reducing power between cytosolic systems ...................77 Redox perspective on liver physiology ..........................................................................78 Conclusions and perspectives ........................................................................................86 Chapter 2 References: ....................................................................................................87 3. HEPATOCYTE HYPERPROLIFERATION UPON LIVER -SPECIFIC CO-DISRUPTION OF THIOREDOXIN-1, THIOREDOXIN REDUCTASE-1, AND GLUTATHIONE REDUCTASE 95 Contribution of Authors and Co-Authors ......................................................................95 Manuscript Information .................................................................................................98 Summary ......................................................................................................................100 Introduction ..................................................................................................................100 Results ..........................................................................................................................103 Development and Survival of Mice with Trx1-, Trx1/TrxR1-, Trx1/Gsr and Trx1/TrxR1/Gsr-null livers ...........................................................103 Redistribution of Cytosolic Disulfide-Reducing Power ......................................109 Oxidative Stress and Xenobiotic Tolerance in Disulfide Reductase-Deficient Livers ..................................................................................112 Hyperproliferation in Hepatocytes Having Disulfide Reductase System Deficiencies ...........................................................................119 Discussion ....................................................................................................................121 Experimental Procedures .............................................................................................125 Mice, Surgeries, and Harvests .............................................................................125 Immunoblotting and Enzymatic Assays ..............................................................125 Primary Fibroblast Systems .................................................................................126