Molecular Basis of Angiogenesis and Neuroprotection by Angiogenin By Trish T. Hoang A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biochemistry) at the UNIVERSITY OF WISCONSIN–MADISON 2016 Date of final oral examination: May 09, 2016 The dissertation is approved by the following members of the Final Oral Committee: Ronald T. Raines, Henry Lardy Professor of Biochemistry, Biochemistry and Chemistry Jeffrey A. Johnson, Professor, Pharmaceutical Sciences David J. Pagliarini, Associate Professor, Biochemistry Samuel E. Butcher, Professor, Biochemistry Nader Sheibani, Professor, Ophthalmology and Visual Sciences ProQuest Number:10189602 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. ProQuest 10189602 Published by ProQuest LLC ( 2018). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, MI 48106 - 1346 i Molecular Basis of Angiogenesis and Neuroprotection by Angiogenin Trish Truc Hoang Under the Supervision of Professor Ronald T. Raines at the University of Wisconsin–Madison Cancer and neurodegeneration are disorders with profound impact on human health. Cancer results from uncontrolled cell growth, whereas neurodegeneration is caused by premature neuronal cell death. Although these disease mechanisms seem to be at opposite ends of a spectrum, an increasing number of cellular and molecular studies have linked the two disorders. Activation and deregulation of the cell cycle appear to be core features of both diseases; thus, many genes associated with cell cycle control, DNA repair, and kinase signaling have been studied extensively. Despite continuing efforts to understand the underlying pathophysiology of both diseases, key regulatory factors that modulate balance between cell growth and cell death remain unknown. In addition to cell cycle regulation, another cellular process implicated in both cancer and neurodegeneration is angiogenesis—the process of establishing new blood vessels from pre- existing vasculature. Angiogenesis is a vital physiological event that supplies cells with oxygen and nutrients. Up-regulation of angiogenesis in cancer promotes progression of the disease while insufficient angiogenic signals contribute to neurodegeneration. There are two angiogenic factors–vascular endothelial growth factor (VEGF) and angiogenin (ANG)–that play roles in ii both diseases. While the roles of VEGF have been well-documented, ANG functions and signaling pathway are understood less completely. ANG is a prevalent protein that acts directly on the proliferation of endothelial cells to promote neovascularization. ANG is up-regulated in cancer cells, but also functions to protect neurons against oxidative stress. Hence, drugs that target this pro-tumorigenic protein could also promote neurological damage, as loss-of-function mutations of the ANG gene are associated with amyotrophic lateral sclerosis. My efforts have been focused on understanding the molecular mechanisms of ANG underlying these diseases. The findings could offer novel therapeutic strategies that target one disorder without advancing the other. ANG belongs to the pancreatic-type ribonuclease superfamily. ANG relies on its ribonucleolytic activity to affect protein translation during cell growth and cell death. To meet the high demand for protein synthesis during cell proliferation, cells increase ribosome biogenesis. ANG promotes rRNA production to satisfy this demand. In contrast, cells that suffer toxic insults will undergo cell death without activation of anti-apoptotic signals. Those cells urge energy conservation to repress global protein translation and focus on production of cytoprotective factors. ANG stalls protein translation by cleaving a subset of tRNAs to generate RNA products that specifically inhibit translation initiation. Indeed, ANG acts as a double-edged sword; overproduction of this protein is correlated with cancer progression, but deprivation is associated with neurological disorders. Although the molecular roles of ANG are not understood completely in either diseases, a central theme emerging from ANG actions is its re-programming of protein synthesis. The purposes of this thesis are to explore the effect of ANG on protein synthesis in the context of cancer and neurodegeneration, to determine how ANG promotes cell proliferation, to pinpoint the role of iii ANG neuroprotection, and ultimately, to lay a foundation for the development of new ANG- based therapeutics for treating these diseases. In CHAPTER 1, I summarize the large body of evidence indicating the important role of ANG in cancer and neurodegenerative diseases. I discuss the role of ribosome biogenesis in cancer development. I also highlight the importance of stress granule formation in repressing protein translation and its pathological connection to neurodegeneration. Depending on the state of the cell, ANG navigates itself into two distant and distinctive cellular foci: nucleoli during proliferation or stress granules during oxidative stress. My studies report molecular details of ANG actions in these foci. In CHAPTER 2, I report on the unprecedented mechanism of signal transduction by ANG that promotes cell proliferation. This mechanism allows the protein to execute its angiogenic activity. In CHAPTER 3, I discuss another pathway that ANG manifests in astrocytes–brain cells that regulate neuronal function–to employ its neuroprotection. By elucidating the elegant network of ANG actions, features of ANG required for a particular pathway might be used to design ANG-based therapeutics. CHAPTER 4 outlines several future directions for developing ANG therapeutics that can selectively target one disease without exacerbating the other. Taken together, the results of this thesis offer a deeper understanding of the molecular basis of the divergence of cancer and neurodegeneration and inspire innovative treatment approaches. iv Acknowledgments I would like to express my deepest gratitude towards all the people who have generously provided their support and assistance during my graduate career. First and foremost, I would like to thank my advisor, Professor Ron Raines. I am grateful for the opportunity he has given me to pursue my Ph.D. thesis work in his research group. His enthusiasm and optimism for science, breadth of knowledge, as well as his extraordinary ability with words have been truly inspirational. Ron has cultivated a collaborative environment, where we are encouraged to pursue interesting questions from a number of different angles. As a result, I have had 7 in total of both intra- and inter-collaboration projects. He has taught me much, especially regarding science communication and writing. I am also grateful to my thesis committee: Professor Jeffrey Johnson, Professor Dave Pagliarini, Professor Nadia Sheibani, Professor Alan Attie and Professor Sam Butcher. Their guidance, patience, constructive criticism, and excellent advice, have all contributed to my development as a scientist. I would like to give special thanks to Professor Jeffrey Johnson and Professor Delinda Johnson for “adopting” me to their lab and have treated me like their graduate student. Now, I must acknowledge those individuals that initially inspired me to pursue science as a career. Their enthusiasm for their respective fields and passion for teaching were the reason I chose to study biochemistry. First is my undergraduate supervisor, Dr. Mariangela Segre, whom I worked for 3 years as a lab technician. She consistently told me that “Trish! You need to go to graduate school. Research is the right career path for you.” And surely, she was right. Second, I thank my undergraduate mentor, Professor Marie Spies. She has provided great opportunities for me to work independently as an undergrad and often encouraged me to attend graduate school. v Third, I was fortunate to work in Professor Lauren Trepanier’s lab at UW-Madison in summer 2008 as a REU student. She introduced me to clinically relevant type of research. It was definitely an eye opening experience for me. Certainly, I felt in love to Madison and decided to come back for graduate school. It can often be quite easy to overlook all of the individuals in core facilities and biochemistry media lab that make our research possible. I have special thanks to Laura Vanderploeg for her patience and willingness to teach me how to use Illustrator and Photoshop. I thank Dr. Darrel McCaslin for his assistance and insightful discussion in the Biophysics Instrumentation Facility. I also want to thank Dr. Elle Grevstad for her continued service and passion for science. I could have not made it thus far without the help from many talented individuals in the Raines lab. Thanks to all members of the Raines Laboratory, past and present, who have been invaluable source of advice, support, and friendship to me during those years. Moreover, I also had the privilege to work alongside Greg Jakubczak, Amit Choudhary, Ben Caes, Greg Ellis, Mike Palte, John Lukesh, Nadia Sundlass, Raso Biswas, Ho-Hsuan Chou, Christine