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Analysis of the Biochemical and Cellular Activities of Substrate Binding by the Molecular Chaperone Hsp110/Sse1

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Analysis of the Biochemical and Cellular Activities of Substrate Binding by the Molecular Chaperone Hsp110/Sse1 The Texas Medical Center Library DigitalCommons@TMC The University of Texas MD Anderson Cancer Center UTHealth Graduate School of The University of Texas MD Anderson Cancer Biomedical Sciences Dissertations and Theses Center UTHealth Graduate School of (Open Access) Biomedical Sciences 5-2017 ANALYSIS OF THE BIOCHEMICAL AND CELLULAR ACTIVITIES OF SUBSTRATE BINDING BY THE MOLECULAR CHAPERONE HSP110/SSE1 Veronica M. Garcia Follow this and additional works at: https://digitalcommons.library.tmc.edu/utgsbs_dissertations Part of the Biochemistry Commons, Medicine and Health Sciences Commons, Microbiology Commons, Molecular Biology Commons, and the Molecular Genetics Commons Recommended Citation Garcia, Veronica M., "ANALYSIS OF THE BIOCHEMICAL AND CELLULAR ACTIVITIES OF SUBSTRATE BINDING BY THE MOLECULAR CHAPERONE HSP110/SSE1" (2017). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access). 771. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/771 This Dissertation (PhD) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. ANALYSIS OF THE BIOCHEMICAL AND CELLULAR ACTIVITIES OF SUBSTRATE BINDING BY THE MOLECULAR CHAPERONE HSP110/SSE1 By Veronica Margarita Garcia, B.S. APPROVED: _______________________________ Supervisory Professor Kevin A. Morano, Ph.D. _______________________________ Catherine Denicourt, Ph.D. _______________________________ Theresa M. Koehler, Ph.D. _______________________________ Michael C. Lorenz, Ph.D. _______________________________ Ambro van Hoof, Ph.D. APPROVED: _______________________________ Dean, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences ANALYSIS OF THE BIOCHEMICAL AND CELLULAR ACTIVITIES OF SUBSTRATE BINDING BY THE MOLECULAR CHAPERONE HSP110/SSE1 A Dissertation Presented to the Faculty of The University of Texas M.D. Anderson Cancer Center Graduate School of Biomedical Sciences in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY By Veronica Margarita Garcia, B.S. Houston, TX May, 2017 I dedicate this work to Ozmin Jr., Emily and Victoria Salinas. May you always dream big and recognize that your dreams are achievable. iii Acknowledgments I would like to thank my mentor Dr. Kevin A. Morano for support and guidance throughout my graduate career and for accepting me into your laboratory when I was without a home. I would like to thank Drs. Catherine Denicourt, Theresa M. Koehler, Michael C. Lorenz, and Ambro van Hoof for all the time you invested in me. Thank you for serving on my committee, for all of your input in my research, and for all the recommendation letters you had to write. The successes I’ve had could not have happened without the five of you. To the members of my lab, Yanyu Wang, Jacob Verghese, Jennifer Abrams, Kimberly Cope, Sara Peffer, Amy Ford, Unekwu Yakubu, Julie Heffler, Mike McCarthy, and Justin Nguyen, it has been a pleasure working with and getting to know you. Thank you for all your help and contributions to my research. Each of you has added greatly to my professional and scientific development. I am grateful to the organizations that funded my graduate studies through awards Ruth L. Kirschtein National NRSA Predoctoral Fellowship, Robert D. Watkins Graduate Research Fellowship, Harry S. & Isabel C. Cameron Foundation. Thank you for funding my ideas. Without your support, there would be less scientific exploration. I want to thank the MMG department. The faculty pushed me to do more and think beyond the obvious. The research we conduct as students would be significantly more difficult if we did not have the support of our departmental staff. I also want to thank my fellow students. I have learned so much from interacting with you. Thank you for the fun times, the scientific challenges, and the help you provided when I needed it. I met three amazing people during my time in graduate school. Jennifer Abrams, I love your heart. Thank you for being the friend that I could always count on. Veronica Rowlett, you’re my friend, my workout partner, my collaborator, and my homodimer. Katie McCallum, thank you for keeping me grounded and a little less insane. These women were excellent contemporaries and iv better friends than I deserved. Thank you for always being available for science talks, guidance, venting sessions, and most importantly to share a beer. Dr. Sarah Castro, Dr. Mark Ott, Willy Wong, and Tatyana Modlin your professional as well as personal support is greatly appreciated. My years at Johnson Space Center were a pleasure because I was able to work with a great team, and I was lucky to have mentors like you. Sarah, I’m ready for the Fusion Institute! Dr. Serenella Sukno and Dr. Carlos Gonzales thank you for noticing and believing in my potential. Dr. Sukno, your teachings were the foundation of my scientific career something for which I am very grateful. Erin, I want to thank you for your friendship. We met almost 15 years ago, and your support and encouragement has never faltered. In you, I have a friend and another sister. I want to thank my partner. You celebrate my accomplishments and feel my frustrations as if they were your own. Thank you for being there every step of the way and for holding my hand when things get tough. Jay, I love you. Erika, Claudia, Miguel, Ozmin, and Kristin, you’re the best crew. Thank you for pointing out my flaws, as siblings should, and accepting me in spite of them. Thank for supporting me and cheering me on during the difficult times. I hope that I can pay it back someday. I am lucky to have grown up with you, and I look forward to us growing older and weirder together. Mami y Papi, estoy muy agradecida de la educación que me dieron, del apoyo que siempre me brindaron, y del amor que han mostrado. Los quiero con todo mi corazón. Todo lo que eh logrado a sido posible porque tengo los mejores padres del mundo. v ANALYSIS OF THE BIOCHEMICAL AND CELLULAR ACTIVITIES OF SUBSTRATE BINDING BY THE MOLECULAR CHAPERONE HSP110/SSE1 Veronica Margarita Garcia, B.S. Advisory Professor: Kevin A. Morano, Ph.D. Molecular chaperones ensure protein quality during protein synthesis, delivery, damage repair, and degradation. The ubiquitous and highly conserved molecular chaperone 70-kDa heat- shock proteins (Hsp70s) are essential in maintaining protein homeostasis by cycling through high and low affinity binding of unfolded protein clients to facilitate folding. The Hsp110 class of chaperones are divergent relatives of Hsp70 that are extremely effective in preventing protein aggregation but lack the hallmark folding activity seen in Hsp70s. Hsp110s serve as Hsp70 nucleotide exchange factors (NEF) that facilitate the Hsp70 folding cycle by inducing release of protein substrate from Hsp70, thus recycling the chaperone for a sequential round of folding and allowing successfully folded substrates to exit the folding cycle. In the model organism Saccharomyces cerevisiae, Hsp110 is represented by the proteins Sse1 and Sse2, which possess an Hsp70-like substrate binding domain (SBD), making them unique among other functionally similar, but structurally distinct, NEFs. Studies of Hsp110 and Sse1 have demonstrated that this chaperone/NEF family can bind polypeptides and prevent proteins from aggregating in vitro and that this ability is conferred by the SBD. However, attempts to study Hsp110 protein binding in vivo have not been successful. To date, the impact of peptide binding by Hsp110 is unknown. This study elucidates and defines substrate binding by the yeast Hsp110 and addresses the contributions of this activity toward protein and cellular homeostasis as well as begins inquiries into substrate binding by the Drosophila melanogaster Hsp110, Hsc70cb. As a major partner of Hsp70, determining cellular Hsp110 activities is a prerequisite to a full understanding of chaperone- mediated protein homeostasis. By studying chaperone functions and activities in yeast and animal vi models, we can understand human cellular protein quality control systems which can then be pharmacologically targeted to combat protein conformational disorders, including Alzheimer’s, Huntington’s, and Parkinson’s diseases. vii Table of Contents Approval Sheet i Title Page ii Dedication iii Acknowledgements iv Abstract vi Table of Contents viii List of Figures x List of Tables xii Chapter I: Introduction 1 Stress and protein quality control 2 The Hsp70 machine 4 Hsp70•Hsp40•NEF complexes throughout the yeast cell 9 Sse1/Hsp110 as a nucleotide exchange factor and holdase 12 Biomedical Significance 13 Chapter II: Materials and Methods 18 Chapter III: Semi-automated microplate monitoring of protein polymerization and aggregation 30 Introduction 31 Results 32 Discussion 39 Chapter IV: Substrate binding by the yeast Hsp110 nucleotide exchange factor and molecular chaperone, Sse1, is not obligate for its biological activities 40 Introduction 41 viii Results 43 Discussion 61 Chapter V: A carboxy-terminus regulated fluorescence affinity tag affects Sse1 functions 64 Introduction 65 Results 67 Discussion 85 Chapter VI: Characterization
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