University of Massachusetts Amherst ScholarWorks@UMass Amherst Open Access Dissertations 5-2012 Regulation of Caspase-9 by Natural and Synthetic Inhibitors Kristen L. Huber University of Massachusetts Amherst, [email protected] Follow this and additional works at: https://scholarworks.umass.edu/open_access_dissertations Part of the Chemistry Commons Recommended Citation Huber, Kristen L., "Regulation of Caspase-9 by Natural and Synthetic Inhibitors" (2012). Open Access Dissertations. 554. https://doi.org/10.7275/jr9n-gz79 https://scholarworks.umass.edu/open_access_dissertations/554 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]. REGULATION OF CASPASE-9 BY NATURAL AND SYNTHETIC INHIBITORS A Dissertation Presented by KRISTEN L. HUBER Submitted to the Graduate School of the University of Massachusetts Amherst in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY MAY 2012 Chemistry © Copyright by Kristen L. Huber 2012 All Rights Reserved REGULATION OF CASPASE-9 BY NATURAL AND SYNTHETIC INHIBITORS A Dissertation Presented by KRISTEN L. HUBER Approved as to style and content by: _________________________________________ Jeanne A. Hardy, Chair _________________________________________ Lila M. Gierasch, Member _________________________________________ Robert M. Weis, Member _________________________________________ Peter Chien, Member ______________________________________ Craig T. Martin, Department Head Department of Chemistry DEDICATION For my mother, Elizabeth Huber, who taught me perseverance and to always follow my dreams, no matter how big or small For my father, Kenneth Huber, who taught me to believe in myself and to remember you can always find a new beginning in tomorrow For my sister, Elyse Huber, for being the person who inspires me every single day and who taught me to be proud of who I am ACKNOWLEDGMENTS To my mentor, Professor Jeanne Hardy, I thank you for guidance and support over this long and somewhat bumpy road. We have both grown in many ways since our first year here at UMass; you as a mentor, me as a budding scientist and both as individuals. Between the memories of our scientific travels and your enthusiastic high fives, you have truly made this an experience I will never forget. To my committee, Dr. Lila Gierasch, Dr. Bob Weis and Dr. Peter Chein, you have pushed me to be a better scientist by teaching me to think outside the box and appreciate the meaning of testing a hypothesis. I thank you. To the Hardy Lab members, my scientific family, both past and present, you will always be held near and dear to my heart. From the Monday morning sports talk to the mid week frustrations followed up by the Friday fireside chats and all of the fun times, you filled my days with laughter and enjoyment. Thank you for being the everyday rock I could always count on. To my all my friends, you have been a shoulder to lean on and a breath of fresh air when I needed it most. To Mike Wilson Jr. and Shannon Coates Flagg, thank you for always listening, understanding, and making me smile. This road would have been difficult without all of your support so let the good times roll! And last but certainly not least, to my family, I would not be where I am today or the person I am today without your unconditional love, support, and understanding. Thank you, not only for being my biggest fans but for supporting me through the tough times and keeping me grounded as a person. Mama, thank you for encouraging me to not only dance to the beat of a different drummer but to polka whenever I had the chance. Pappy, thank you for always finding the right words to make me feel at ease and giving me the encouragement to always stand up for what I believe in. Elyse, my partner in crime, thank you for teaching me all of the important life skills I needed to accomplish my goals. You have taught me how to think on my feet, especially when getting framed for taking cookies out of the kitchen, how to accept constructive criticism by convincing me there is always room for improvement, particularly when it comes to my touchdown dance moves, and patience by forgetting to find me when playing hide-n-go-seek. We are truly two peas in a pod, SBN! I love you all! “You have brains in your head. You have feet in your shoes You can steer yourself any direction you choose. You're on your own. And you know what you know. And YOU are the guy who'll decide where to go.” Oh, the Places You’ll Go! -Dr. Seuss v ABSTRACT REGULATION OF CASPASE-9 BY NATURAL AND SYNTHETIC INHIBITORS MAY 2012 KRISTEN L. HUBER, B.S., QUINNIPIAC UNIVERSITY Ph.D., UNIVERSITY OF MASSACHUSETTS AMHERST Directed by: Professor Jeanne A. Hardy Tight regulation of caspase-9, a key initiator of apoptosis, is required to uphold cellular homeostasis. Although it is controlled on a multifactorial level, misregulation of this process does occur, which is a characteristic of a variety of diseases from ischemic injury to cancer. Therefore it remains important to gain a detailed understanding of the mechanisms behind native caspase-9 regulatory pathways and harness these mechanisms for therapeutic purposes. Based on known mechanisms, such as the unique inhibitory complex of caspase- 9 and XIAP-BIR3, development of synthetic regulators can be envisioned, while other mechanisms such as zinc-mediated inhibition and CARD activation of caspse-9 remain undefined. Intrigued by the multiple ways to control caspase-9’s activity, we sought after designing synthetic caspase-9 inhibitors in addition to defining the mechanistic details metal regulation and CARD domain activation. We report the first stabilized α-helical peptides that harness the native regulatory mechanism of caspase-9 and the BIR3 domain which lead to the understanding of the importance of exosites in inhibitory complexes. Our studies also revealed that there are two distinct zinc binding sites, one at the active site and another at a novel zinc binding site of yet unknown function in caspase-9 however this site may have the potential to vi control caspase-6 based on its regulatory mechanism. Furthermore, an interaction was discovered between CARD and the catalytic core of caspase-9 in the presence of a properly formed substrate binding groove, a potential mechanism utilized by the apoptosome for activation of the enzyme. All in all, the regulation of caspase-9 occurs on a variety of levels that requires almost every surface of the enzyme. Through exploring these underlying molecular details behind the various mechanisms, not only has the field of caspase-9 regulation mechanisms been extended, essential information was gained for further pursuit in an advancement towards the design of caspase-9 activators and inhibitors. vii TABLE OF CONTENTS Page ACKNOWLEDGMENTS ...................................................................................................v ABSTRACT ....................................................................................................................... vi LIST OF TABLES ........................................................................................................... xiii LIST OF FIGURES ......................................................................................................... xiv CHAPTER I. APOPTOSIS, DISEASE AND THE REGULATION OF CASPASE-9 .........................1 1.1. The Roles of Apoptosis in Disease ...................................................................1 1.2. Caspases: Facilatators of Apoptosis..................................................................4 1.3. Caspase Active Site and Catalytic Mechanism .................................................6 1.4. Apoptotic Pathways ..........................................................................................9 1.5. Natural Regulation of Caspase-9 ....................................................................11 1.6. Synthetic Regulation of Caspase-9 .................................................................15 1.7. Caspase-9 and Its Role in Disease ..................................................................19 1.8. Refrences.........................................................................................................21 II. ROBUST PRODUCTION OF A LIBRARY OF CASPASE-9 INHIBITOR PEPTIDES USING METHODOLOGICAL SYNCHRONIZATION .........................30 2.1. Introduction .....................................................................................................31 2.2. Results .............................................................................................................34 2.2.1. Construction of Peptide-Fusion Expression Vector and aPP Variants ....................................................................................34 2.2.2. Expression and Purification of a Recombinant Peptide Library ......36 2.3. Discussion .......................................................................................................41 2.4. Materials and Methods ....................................................................................45 viii 2.4.1. Cloning of Recombinant Peptide-Fusion Variants ..........................45 2.4.2. Expression and Purification of Recombinant Peptide-Fusion Variants ...................................................................46 2.4.3. Expression