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The Unavoidable Threat of Aggregation: Implications for Folding and Function of a Β-Rich Protein

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The Unavoidable Threat of Aggregation: Implications for Folding and Function of a Β-Rich Protein University of Massachusetts Amherst ScholarWorks@UMass Amherst Open Access Dissertations 5-2013 The nU avoidable Threat of Aggregation: Implications for Folding and Function of a β-Rich Protein Mylene Hazelle Anne Ferrolino University of Massachusetts Amherst, [email protected] Follow this and additional works at: https://scholarworks.umass.edu/open_access_dissertations Part of the Cell and Developmental Biology Commons, and the Molecular Biology Commons Recommended Citation Ferrolino, Mylene Hazelle Anne, "The nU avoidable Threat of Aggregation: Implications for Folding and Function of a β-Rich Protein" (2013). Open Access Dissertations. 740. https://doi.org/10.7275/1vz6-td16 https://scholarworks.umass.edu/open_access_dissertations/740 This Open Access Dissertation is brought to you for free and open access by ScholarWorks@UMass Amherst. It has been accepted for inclusion in Open Access Dissertations by an authorized administrator of ScholarWorks@UMass Amherst. For more information, please contact [email protected]. THE UNAVOIDABLE THREAT OF AGGREGATION: IMPLICATIONS FOR FOLDING AND FUNCTION OF A β-RICH PROTEIN A Dissertation Presented by MYLENE CASTELL FERROLINO Submitted to the Graduate School of the University of Massachusetts Amherst in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSPHY May 2013 Molecular and Cellular Biology © Copyright by Mylene C. Ferrolino 2013 All Rights Reserved THE UNAVOIDABLE THREAT OF AGGREGATION: IMPLICATIONS FOR FOLDING AND FUNCTION OF A β-RICH PROTEIN A Dissertation Presented by MYLENE CASTELL FERROLINO Approved as to style and content by: _____________________ Lila M. Gierasch, Chair _____________________ Daniel N. Hebert, Member _____________________ Richard W. Vachet, Member _____________________ Peter Chien, Member ____________________________ Barbara A. Osborne, Director Molecular and Cellular Biology Program DEDICATION I’d like to dedicate this thesis to my inspiration – my children, JAMES and JULIANNE. Always remember that your dreams have no bounds. Live your dreams with passion and you will surely accomplish them. Mommy loves you. ACKNOWLEDGEMENTS I am extremely grateful to the people who have been instrumental in accomplishing this dissertation. I’d like to thank my adviser, Dr. Lila Gierasch for mentoring me to become a good scientist while keeping my sanity. Thank you for never doubting my abilities and for generously sharing your secrets to success. Thank you for allowing me to have my own little niche - to explore and learn new things everyday over the past five years. It was indeed a great pleasure to have you as my mentor. I’d like to thank my committee members, Dr. Daniel Hebert, Dr. Peter Chien and Dr. Richard Vachet. Thank you for helping me direct my study to the right path I’d also like to thank my fellow Gieraschians: Dr. Anastasia Zhuravleva who has been there in all my critical NMR experiments. Thank you for generously sharing your expertise and for always having answers to all my questions. None would have been possible without you. Dr. Ivan Budyak and Dr. Beena Krishnan who I have worked with side by side. Thank you for allowing me to compare notes with you and for helping me develop my ideas. It was a pleasure to have worked with wonderful and hardworking scientists like you. Dr. Zoya Ignatova for starting out the aggregation project. Although I never got to meet you, your work was a perfect precedent for my thesis. Dr. Eugenia Clerico for being the first person in the lab who gave me a warm smile while answering my inane questions. You are a jewel in the lab. Kristine Pobre for being my constant companion and friend. It would have been such a bore not to have you in the lab all these years – (Salamat!). Amanda Clouser who has been a wonderful person to work with. I was so lucky to mentor someone so smart who never really needed me! Ilene Magpiong for all the mommy stories we share. Karan Hingorani and Mandy Blackburn, v you just don’t know how it means have those laughs in my most stressful days. Dr. Anne Gershenson, for constantly sharing new ideas on protein aggregation. Other lab members who have been very considerate during my critical last few days in the lab: Dr. Abhay Thakur, Dr. Weiwei Kuo, Dr. Ben Yang. I’d like to especially thank Ellen Kalt for being the person to I run to with so many things! I’d also like to thank my friends who have been my family’s social support since we arrived in the US. My dear friend, Cristina Martin , who has always helped me in so many ways, even way back in the Philippines. Dr. Abigail Guce, Dr. Vanessa Gill, Dr. Annie Marcelino, Dr. Risha Molato, Dr. Princess Wilson, Dr. Bernard Matute, Drs. Ron and Phey Lerum, Dr. Andrea Gomez-Escudero, Kevin and Dolly Dagbay, Nilima Kolli, Bay and Mahalia Serrano, for making us feel that there is home here in Amherst. Most especially, I’d like to thank Drs. Meme and Albert Orquiola and family who have been our foster family in the U.S. I’d like to also thank my family in the Philippines: My dear parents, Mario and Vikki Castell, who have been my pillar all my life. I attribute all my accomplishments to you. My brothers and my sisters-in-law, Gerald, Cindy, Marvin and Angel thanks for believing in me although we are all apart. My in-laws, Drs. Brendan and Joseline Ferrolino , Jong, Joey and Brian - although Mommy Josie is no longer with us, I am sure she is happy to see me also get a doctoral degree. Finally, I’d like to thank my loving husband Dr. Jose Amadeo Ferrolino for being my source of strength throughout my graduate studies. Thank you for sharing with my frustrations and triumphs. All these would not have been possible if not for our Divine Being who has clearly paved the way for me to achieve my dreams. vi ABSTRACT THE UNAVOIDABLE THREAT OF AGGREGATION: IMPLICATIONS ON FOLDING AND FUNCTION OF A β-RICH PROTEIN MAY 2013 MYLENE CASTELL FERROLINO B.S. UNIVERSITY OF THE PHILIPPINES M.S., UNIVERSITY OF THE PHILIPPINES PH.D., UNIVERSITY OF THE MASSACHUSETTS Directed by: Professor Lila M. Gierasch Protein aggregation has been implicated in several catastrophic diseases (neurodegeneration, diabetes, ALS) and its complexity has also become a major obstacle in large-scale production of protein-based therapeutics. Despite the generic behavior of proteins to aggregate, only a few globular proteins have known aggregation mechanisms. At present, there have been no clear connections between protein folding, function and aggregation. We have tackled the challenge of understanding the links between a protein’s natural tendency to fold and function with its propensity to misfold and aggregate. Using a predominantly β-sheet protein whose in vitro folding has been explored in detail: cellular retinoic acid-binding protein I (CRABP 1), as a model, we investigated sequence determinants for folding and aggregation. In addition, we characterized the aggregation-prone intermediate under native conditions. Our studies revealed similar contiguous aggregation cores in in vitro and in vivo aggregates of CRABP 1 validating the importance of sequence information under extremely different conditions. Hydrophobic stretches that comprise the interface in aggregates include vii residues surrounding the ligand binding portal and residues at the C-terminal strands of CRABP 1. Folding studies reveal that docking of the N and C terminals happen in the early stages of barrel closure of CRABP 1 emphasizing the role of folding in preventing exposure of risky aggregation-prone sequences. We further examined the intermediate that initiates aggregation under native conditions. We found that inherent structural fluctuations in the native protein, relevant to ligand binding of CRABP 1, expose aggregation-prone sequences. Binding of the ligand, retinoic acid decreases the aggregation of CRABP 1 illustrating the contribution of functional interactions in avoiding aggregation. Our study implies that because of the evolutionary requirement for proteins to fold and function, aggregation becomes an unavoidable risk. viii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ……………………………………………………..……………….v ABSTRACT ……………………………………………………………………..……………... vii LIST OF TABLES ………………………………………………………………..……………..xii LIST OF FIGURES ……………………………………………………………..…….……….xiii CHAPTER 1. INTRODUCTION .….…………………………………………………………………………1 1.1. Competitive Interactions in Protein Folding and Aggregation ..…………….………1 1.2. Characteristics of Protein Aggregates …..……………………………………………8 1.3. Prediction of Aggregation-Prone Sequences Proteins …..………………………..10 1.4. Aggregation Mechanisms of Globular Proteins …...………………………………..13 1.5. Protein Folding and Aggregation in the Cell…...……………………………………15 1.6. Statement of Dissertation ……..……………………………………………………...17 2. AGGREGATION SEQUENCE DETERMINANTS: IMPLICATIONS FOR FOLDING OF A β-RICH PROTEIN ...………………………………………………….19 2.1. Introduction………………………………………………………………………...……19 2.1.1. Structure, Function and Dynamics of CRABP 1 ……….…..………..…......20 2.1.2. Folding and Aggregation of CRABP 1 ………………...……………………...23 2.1.3. Hydrogen-Deuterium Exchange NMR to Map Aggregation Cores ……………………………………………………………...27 2.2 Results ………………………………………………....…………………….,…………32 2.2.1. Aggregation Propensity of CRABP 1.……...…………………………………32 2.2.2. Sequestered sequences in CRABP 1 in vitro and in vivo aggregates ……………………………………………………………………….39 2.3. Discussion ……………………………………………....…………………………..…..54 ix 2.3.1. Common Sequences Direct Aggregation of CRABP 1 ……………………..54 2.3.2. Common Sequence Motifs Drive Aggregation and Folding
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