University of Massachusetts Amherst ScholarWorks@UMass Amherst Doctoral Dissertations Dissertations and Theses December 2020 Engineered proteins as tools to understand ubiquitin signaling Lin Hui Chang University of Massachusetts Amherst Follow this and additional works at: https://scholarworks.umass.edu/dissertations_2 Part of the Chemistry Commons, and the Life Sciences Commons Recommended Citation Chang, Lin Hui, "Engineered proteins as tools to understand ubiquitin signaling" (2020). Doctoral Dissertations. 2001. https://doi.org/10.7275/18414115 https://scholarworks.umass.edu/dissertations_2/2001 This Open Access Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks@UMass Amherst. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of ScholarWorks@UMass Amherst. For more information, please contact [email protected]. Engineered Proteins As Tools To Understand Ubiquitin Signaling A Dissertation Presented by LIN HUI CHANG Submitted to the Graduate School of the University of Massachusetts Amherst in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY September 2020 Chemistry © Copyright by Lin Hui Chang 2020 All Rights Reserved Engineered Proteins As Tools To Understand Ubiquitin Signaling A Dissertation Presented by LIN HUI CHANG Approved as to style and content by: ______________________________ Eric R. Strieter, Chair ______________________________ Lynmarie K. Thompson, Member ______________________________ Min Chen, Member ______________________________ Scott C. Garman, Outside Member ______________________________ Ricardo B. Metz, Department Head Chemistry Department ACKNOWLEDGEMENT I would like to thank my advisor, Eric Strieter, for his guidance and encouragement. I still remember vividly that after my transfer to University of Massachusetts-Amherst, that was a time when I was struggling with my research; he has asked me to focus on yeast display and has accompanied me to seek advice from other faculty member in the field. Eric is very passion in science and will always come into the lab with different ideas, sometimes they could be weird, but most of the times, his ideas are thought-provoking. His attitude in science will continue motivate me to strive harder in my future career. I would also like to thank my committee members, Prof. Lynmarie Thompson, Prof. Min Chen and Prof. Scott Garman for their advices in my research. I really appreciate working with the core facilities manager, especially for Dr. Amy Burnside, Dr. Lizz Bartlett, Dr. Ravi Ranjan, Dr. James Chambers, and Dr. Steve Eyles for being always available every time I have questions with the instruments or experiments. I would like to especially thank Prof. Sarah Moore from Smith College and her student, Allison Sirois for helping me with yeast display. I am fortunate to work with my group members. They have taught me how to perform experiments and helped me with my writing and presentation for my exam. Robert, Ambar, Sean, Kirandeep and Jiale, whom I know from Madison. Because of them, I have the courage to move to Amherst to challenge the new environment. I would like to thank the group members in Amherst, Sandor, iv Yanfeng, Heather, Fangying, Rehab and Rakesh. Thank you all for the numerous supports, and sharing the ideas and materials with me. Also, I would like to thank the Chemistry Department. DV, jms, and Chandra who help me with the administrative documents. Vicki and Kris for the helps with the issues regarding purchasing. Marv, Bob, and Ryan, for fixing our instruments and help us every time something wrong in the lab. John, Gary, and Nikki who come into rescue when my computer decided to “have an attitude”. Dennis who is so friendly, and a trip to the stockroom is always a good short break from the experiments. Last but not least, I am very grateful for families and friends. Dad and mum, who always encourage me every time I am doubtful of myself and my sisters, Lin Zhi, Lin June and Lin-li who support me to pursuit my graduate study. My relatives in New York, who is like a second family to me and make sure I adapt to the life in USA. Also, I would like to thank to my friends, especially Ying Ji, Kong, and 7th floor members for all the morale support throughout my graduate career. v ABSTRACT ENGINEERED PROTEINS AS TOOLS TO UNDERSTAND UBIQUITIN SIGNALING SEPTEMBER 2020 LIN HUI CHANG, B. S., UNIVERSITY OF TOKYO M.S., UNIVERSITY OF WISCONSIN-MADISON PH. D., UNIVERSITY OF MASSACHUSETTS AMHERST Directed by Professor Eric R. Strieter Ubiquitin is a 76 amino acids protein that is evolutionary conserved in eukaryotes. It is an important signaling molecule in a plethora biological events, such as protein degradation, DNA damage response, and transcription. This thesis aims to develop engineered protein as a tool to study ubiquitin signaling. Through targeted mutagenesis and directed evolution, a deubiquitinase is reprogrammed into a transamidase, which lead to the generation of ubiquitin- protein conjugates with discrete ubiquitin linkages through auto-ubiquitination. These ubiquitin-protein conjugates could be used as a model substrate to profile their interaction of different ubiquitin interacting proteins. In addition, using directed evolution and deep sequencing, a nanobody-based binder targeting a deubiquitinase is engineered. This nanobody could be utilized as an intracellular probe to study the regulation of the deubiquitinase with great spatial and temporal control. vi TABLE OF CONTENTS Page ACKNOWLEDGEMENT .......................................................................................iv ABSTRACT ..........................................................................................................vi LIST OF TABLES ................................................................................................. x LIST OF FIGURES ...............................................................................................xi CHAPTER 1: INTRODUCTION .............................................................................................. 1 Ubiquitination machinery ................................................................................... 1 How are different ubiquitin conjugates recognized by their interacting proteins? ........................................................................................................... 3 How the specificities of ubiquitin interacting protein are regulated? .................. 5 Regulation of the proteasome associated ubiquitin receptors and deubiquitinases .............................................................................................. 5 Protein engineering in ubiquitin system ........................................................... 12 Rational design and directed evolution ........................................................ 12 Engineered protein as tools to study ubiquitin system ................................. 16 Forward to thesis ............................................................................................. 18 References ...................................................................................................... 20 2: REPROGRAMMING A DEUBIQUITINASE INTO A TRANSAMIDASE .......... 26 Abstract ........................................................................................................... 26 vii Page Introduction ..................................................................................................... 27 Result .............................................................................................................. 32 Targeted mutagenesis ................................................................................. 32 Autoubiquitination of Yuh1m ........................................................................ 38 Directed evolution of Yuh1qm ...................................................................... 43 Profiling DUBs activities using Ubiquitin-Yuh1 conjugates ........................... 49 Discussion and Future Directions.................................................................... 55 Materials and Methods .................................................................................... 58 References ...................................................................................................... 68 3: ENGINEER A NANOBODY SPECIFIC TOWARDS PROTEASOME- ASSOCIATED UCH37 ........................................................................................ 72 Abstract ........................................................................................................... 72 Introduction ..................................................................................................... 73 Result .............................................................................................................. 77 Generation of the UCH37 nanobodies ......................................................... 77 Affinity maturation of the proteasome-associated UCH37 specific Nbs ....... 81 Characterization of Nanobodies ................................................................... 87 Nanobodies as intrabodies for proteasome-associated UCH37 .................. 94 Discussion and Future directions .................................................................... 97 Materials and Methods .................................................................................. 100 viii Page References ...................................................................................................