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UC San Francisco Electronic Theses and Dissertations UCSF UC San Francisco Electronic Theses and Dissertations Title Uncovering virulence pathways facilitated by proteolysis in HIV and a HIV associated fungal pathogen, Cryptococcus neoformans Permalink https://escholarship.org/uc/item/7vv2p2fh Author Clarke, Starlynn Cascade Publication Date 2015 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California ii Acknowledgments I would first like to thank my thesis advisor, Dr. Charles Craik. Throughout my PhD Charly has been unfailingly optimistic and enthusiastic about my projects, but most importantly, he has always had confidence in my abilities. During the course of graduate school I have doubted myself and my capabilities almost daily, but Charly has always believed that I would be successful in my scientific endeavors and I cannot thank him enough for that. Charly is the most enthusiastic scientist that I have ever met and he has the capacity to find a silver lining to almost any event. He also understands the importance of presentation and has the resourcefulness to transform almost any situation into an opportunity. These are all traits that do not come naturally to me, but having Charly as a mentor has helped me to learn some of these important skills. I would also like to thank my thesis committee members, Dr. Raul Andino and Dr. John Gross who have both been incredibly supportive over the years. Despite the fact that my research ended up veering away from the original focus that was more in line with their expertise, they have continued to provide me with encouragement and thoughtful feedback during my thesis committee meetings as well as at other times that I sought their advice. Dr. Anthony O’Donoghue was my friend and mentor in the Craik lab for five years. His guidance and knowledge were instrumental in my research success. Besides that, Anthony is one of the most good-natured people that I have ever met and in spite of the long list of demands on his time, he always answered my questions and even went out of his way to check up on me and offer to discuss my project. When I was struggling with a project that was not working he helped me to find a new direction, something that I will always be grateful to him for. I would like to thank Dr. Hiten Madhani, Dr. Phillip Dumesic and Christina Homer, my collaborators on my research project studying peptidases secreted by Cryptococcus neoformans. I first proposed the project to Phillip and despite his extremely busy schedule with iii his numerous and successful projects, he made time to plan experiments with me and help me with reagents, experiments, and edits to our manuscript. Likewise, Christina has been a wealth of advice on the project as well as generously contributing her time to helping me with mouse experiments and editing our manuscript. Hiten has been supportive and helpful on the project as well. I am very fortunate to have had an expert in the field to help me construct the story and determine how best to present it for publication. My favorite aspect of the Craik lab is the wonderful people that have worked in the lab over the years. In addition to Anthony, Dr. Dan Hostetter, Dr. Jungmin Kim and Dr. Michael Winter were my mentors and friends in the Craik lab. All extremely knowledgeable, they have always been more than willing to answer any questions I have had, about science as well as career choices or life in general. However, there has always been a strong sense of camaraderie and support amongst all of the Craik lab members. At some point, I have sought help from almost everyone in the lab and been surprised and touched by how generously everyone has shared their time and expertise. In particular, I would like to thank Dr. Cheryl Tajon for helping me make and test nanosensors for HIV protease and the graduate students of my same year Dr. Jonathan Gable and Dr. Melody Lee for their support as we each achieved the mile stones of graduate school. I have met so many wonderful people at UCSF, first and foremost, my class mate, room mate and friend, Kelly Nissen. Kelly is the most supportive person that I have ever known and also a wonderful scientist. She has given me pep talks countless times throughout graduate school as well as thoughtful advice on my scientific writings and presentations. Dr. Manon Eckhardt is also a wonderful friend that I have made at UCSF. She is an excellent scientist and a fun and interesting person and has cheered me up many times. Nicole Olsen, a graduate student in the Craik lab has been a great friend to me and made lab a more enjoyable place. Finally, I would like to thank my mother Felice Clarke and my non-scientist friends for their support and love during this long process. They have always been there for me when things iv were not going well and when good things happen they are ready to celebrate with me. I have had the opportunity to work on several great projects at UCSF and publish with some amazing scientists. I am sixth author on Dr. Stefanie Jager’s paper “Global landscape of HIV-human protein complexes” Pubmed ID: PMC3310911 where I contributed studies of the interactions between HIV protease and the eukaryotic translation initiation complex 3 (eIF3). I am fourth author on Christina Homer’s paper “Convergent evolution of a peptide-based cell-cell signaling system required for the virulence of a eukaryotic pathogen” (submitted to Cell). For this project I helped to determine that the peptidase secretion of C. neoformans is affected by a quorum sensing pathway discovered by Christina. I am second author on Dr. Steven Jones’s paper “Evolutionary selection on barrier activity: The aspartyl protease Bar1 is an enzyme with novel substrate specificity” (submitted to mBio) where I contributed to identifying the unique specificity of a peptidase secreted by Candida albicans called Bar1. Finally, my findings on the secreted peptidases of C. neoformans will be published in collaboration with Dr. Phillip Dumesic, Dr. Christina Homer, Dr. Anthony O’Donoghue, Lenka, Pallova, Dr. Pavel Maher, Dr. Hiten Madhani and Dr. Charles Craik, “Integrated activity and genetic profiling of secreted peptidases in Cryptococcus neoformans reveals an asparty peptidase required for low pH survival and virulence” (submitted to PLOS Pathogens). v Uncovering virulence pathways facilitated by proteolysis in HIV and a HIV associated fungal pathogen, Cryptococcus neoformans By Starlynn Clarke Abstract Peptidases are enzymes that cleave proteins or peptides, irreversibly modifying their substrates. Proteolytic activity is essential for many biological processes, however these enzymes are also associated with many pathological states. In particular, peptidases are frequently used by pathogenic organisms for purposes such as host invasion, immune system evasion and nutrient acquisition. Both HIV and the HIV/AIDS associated fungal pathogen, Cryptococcus neoformans, are known to employ peptidases to promote virulence. HIV expresses a protease that has a well characterized role in maturation of infectious viral particles by cleaving viral polyproteins into their individual constituents. However, in this work we present evidence that this enzyme has a dual purpose. Our results indicate that in addition to processing viral proteins, HIV protease cleaves numerous host-proteins to optimize the host environment for viral replication. We conducted a study of HIV protease host-protein interactions using an affinity purification-mass spectrometry based approach with tagged, inactive HIV protease. Over 50 candidate substrate proteins were identified through this technique. We then individually studied over 40 of these proteins, revealing that eight are cleaved by HIV protease in vitro. Through experiments assaying viral replication in combination with siRNA knock-down of host proteins, we discovered one vi of these host-proteins, the eukaryotic translation initiation factor 3d (eIF3d) restricts viral reverse transcription. Therefore, we hypothesize that cleavage of this protein by HIV protease may promote reverse transcription during viral infection. In this work we also investigate the secreted peptidases of C. neoformans, an opportunistic fungal pathogen responsible for over 600,000 deaths every year. This organism is known to secrete peptidases but the functions, regulation and characteristics of these enzymes have remained mysterious. We used an unbiased activity based approach to detail the specificity and regulation of extracellular peptidase activity produced by this pathogen grown under two different culture conditions. We pinpoint a principle enzyme responsible for secreted peptidase activity and find that this enzyme is important for tolerance to low pH environments as well as virulence in a mouse model of cryptococcus infection. Using knowledge of the substrate specificity of this enzyme, we screen a focused library of small molecule inhibitors, identifying four potent compounds. Our study of the functions and characteristics of peptidases from viral and fungal pathogens reveals important information regarding the host-pathogen biology of these organisms. Our results may be informative for the development of alternative therapeutic strategies for combating these intractable pathogens. Furthermore, the approaches and techniques we describe are useful for the investigation of proteolytic activity secreted by other disease-causing organisms. vii TABLE OF CONTENTS CHAPTER 1: INTRODUCTION 1 1.1 Peptidases: an overview 1 1.2 Investigating substrate specificity
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