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APOBEC3A and Profilin University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2018-01-31 Investigating the Structural Basis for Human Disease: APOBEC3A and Profilin Tania V. Silvas University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Biochemistry Commons, Enzymes and Coenzymes Commons, Medicinal-Pharmaceutical Chemistry Commons, Nervous System Diseases Commons, and the Structural Biology Commons Repository Citation Silvas TV. (2018). Investigating the Structural Basis for Human Disease: APOBEC3A and Profilin. GSBS Dissertations and Theses. https://doi.org/10.13028/M2KD6T. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/955 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. INVESTIGATING THE STRUCTURAL BASIS FOR HUMAN DISEASE: APOBEC3A AND PROFILIN A Dissertation Presented By TAÑA VANESSA SILVAS Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY JANUARY 31st, 2018 BIOCHEMISTRY AND MOLECULAR PHARMACOLOGY i INVESTIGATING THE STRUCTURAL BASIS FOR HUMAN DISEASE: APOBEC3A AND PROFILIN A Dissertation Presented By TAÑA VANESSA SILVAS This work was undertaken in the Graduate School of Biomedical Sciences BIOCHEMISTRY AND MOLECULAR PHARMACOLOGY Under the mentorship of Celia A. Schiffer, PH.D., Thesis Advisor Jeremy Luban, M.D., Member of Committee Brian A. Kelch, PH.D., Member of Committee Katherine Fitzgerald, PH.D., Member of Committee Catherine L. Drennan, PH.D., External Member of Committee Dan Bolon, PH.D., Chair of Committee ANTHONY CARRUTHERS, PH.D., Dean of the Graduate School of Biomedical Sciences JANUARY 31st, 2018 ii Acknowledgments I would like to thank several people for their support, advice, and friendship throughout my graduate career. First, I thank my family, who instilled in me the importance of education and following my passions in life. I thank my mother, nana, my uncle Ramon, and Ruben for all the sacrifices they endured in order to raise my sister and I. Next, I would like to thank the Biochemistry and Molecular Pharmacology department for providing an amazingly friendly and warm environment to work in. I would like to thank my committee members Dr. Daniel Bolon, Dr. Brian Kelch, Dr. Jeremy Luban, Dr. Mohan Somasundaran, Dr. Katherine Fitzgerald, Dr. Catherine Drennan for their insightful comments and keeping me focused throughout my graduate career. I thank all past and current members of the Schiffer lab. It was an amazing experience working in such a large group of people from many different backgrounds scientifically and culturally. I thank Ellen Nalivaika not only for making sure everything in the lab is running smoothly but also for all you help on the APOBEC project and being the go to person for any questions I had with running experiments in the lab. I thank Dr. Nese Kurt-Yilmaz for an amazing mentor in writing and in patience. You helped turn my convoluted ideas and explanations into simple and easy texts, and taught me how to stay level headed when things got difficult in lab. Thank you to Christine Pruitte, Candice Dufour, iii and Christina Zollo for all of your support and help, I always enjoyed your warm, welcoming, positive energy despite all the crazy paperwork we had to deal with. I thank my rotation mentors Dr. Shivender Shandilya and Dr. Markus Bohn for introducing me to the APOBEC field, I was always amazed by your excitement and enthusiasm for this project even throughout all the challenges. I also thank you for teaching me protein purification, biochemical assays and crystallography. Thank you to Shurong Hou for being the best lab and bay mate ever. For the many hours we spent together trouble shooting experiments to brainstorming ideas about the implications of our results- we made great team. It was always a pleasure working with you and I am grateful for your friendship and optimism. I would like to thank Dr. Bill Royer for being a great mentor especially in crystallography. I thank Dr. Mohan Somasundaran for his mentorship in the APOBEC project and all your support and advice throughout the years. I thank Dr. Brian Kelch for being an incredible mentor since I was a rotation student in his lab. I value all your mentorship in crystallography, biochemistry, structural analysis and entertaining my random and sometimes off- the-wall general scientific enquiries. I also thank his lab members Dr. Brendan Hilbert, Dr. Christl Gaubitz, Janelle Hayes, Nicholas Stone, for your friendship and help with crystallography and electron microscopy. I would like to thank Dr. Daryl Bosco and Dr. Siva Boopathy for a wonderful collaboration in the Profilin project. I would also like to thank Dr. iv Hiroshi Matsuo and Dr. Wazo Myint for their collaboration in the APOBEC project. Thank you to Dr. Kristina Prachanranarong, Dr. Djade Soumana, and Dr. Kuan Hung Lin for being amazing lab mates and all your support and friendship over the years. From consolation ramen dinners to dragging couches up my 3rd story apartment window, you guys were always there for me. I would also like to thank Dr. Furkan Ayaz, Dr. Mary Munson, Ashley Mathew, Florian Leidner Dr. Madhvi Koli, Dr. Sagar Kathuria, Dr. Caroline Duffy, Dr. Laura Deveau, for their advice, friendship and encouragement. I would like to thank Dr. Aneth Canale and Pamela Cote for being such great friends throughout grad school. We met as classmates our first year have stuck together since. I will miss our lunch dates and our girl’s night out. I’ll be forever grateful for our friendship. Finally, I would like to thank my thesis advisor, Celia Schiffer for being an amazing mentor. I am so grateful for your support not only in my scientific endeavors but also in my personal growth throughout the past six years. Your encouragement and compassion even through the difficult times is nothing like I have ever experienced from a mentor. I grew up in an environment where aggression and intimidation is the main mode to gain power and respect in a situation. You have opened my eyes to the strength that is in kindness and I strive to put what I learned from you in practice in my career and also in my personal relationships. Thank you for believing in me, even when I didn’t. v Abstract Analyzing protein tertiary structure is an effective method to understanding protein function. In my thesis study, I aimed to understand how surface features of protein can affect the stability and specificity of enzymes. I focus on 2 proteins that are involved in human disease, Profilin (PFN1) and APOBEC3A (A3A). When these proteins are functioning correctly, PFN1 modulates actin dynamics and A3A inhibits retroviral replication. However, mutations in PFN1 are associated with amyotrophic lateral sclerosis (ALS) while the over expression of A3A are associated with the development of cancer. Currently, the pathological mechanism of PFN1 in this fatal disease is unknown and although it is known that the sequence context for mutating DNA vary among A3s, the mechanism for substrate sequence specificity is not well understood. To understand how the mutations in Profilin could lead to ALS, I solved the structure of WT and 2 ALS-related mutants of PFN1. Our collaborators demonstrated that ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported loss-of-function phenotypes in cell-based assays. The source of this destabilization was illuminated by my X-ray crystal structures of several PFN1 proteins. I found an expanded cavity near the protein core of the destabilized M114T variant. In contrast, the E117G mutation only modestly perturbs the structure and stability of PFN1, an observation that reconciles the occurrence of this mutation in the control population. These findings suggest that a destabilized form of PFN1 underlies PFN1-mediated ALS pathogenesis. To characterize A3A’s substrate specificity, we solved the structure of apo and bound A3A. I then used a systematic approach to quantify affinity for substrate as a function of sequence context, pH and substrate secondary structure. I found that A3A preferred ssDNA binding motif is T/CTCA/G, and that A3A can bind RNA in a sequence specific manner. The affinity for substrate increased with a decrease in pH. Furthermore, A3A binds tighter to its substrate binding motif when in the loop region of folded nucleic acid compared to a linear sequence. This result suggests that the structure of DNA, and not just its chemical identity, modulates A3 affinity and specificity for substrate. vi Table of Contents Title Page...............................................................................................................i Acknowledgment................................................................................................iii Abstract...............................................................................................................vi Table of Contents...............................................................................................vii
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