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The Mitochondrial Protein Import System UCLA UCLA Electronic Theses and Dissertations Title A small molecule dissociates the PAM motor from the TIM23 channel Permalink https://escholarship.org/uc/item/9kh6586x Author Cheung, Tsz Mei Jesmine Publication Date 2017 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA Los Angeles A small molecule dissociates the PAM motor from the TIM23 channel A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biochemistry and Molecular Biology by Tsz Mei Jesmine Cheung 2017 © Copyright by Tsz Mei Jesmine Cheung 2017 ABSTRACT OF THE DISSERTATION A small molecule dissociates the PAM motor from the TIM23 channel by Tsz Mei Jesmine Cheung Doctor of Philosophy in Biochemistry and Molecular Biology University of California, Los Angeles, 2017 Professor Carla Marie Koehler, Chair Mitochondrial dysfunction is a contributing factor in various neural and muscular degenerative diseases. Modulation of mitochondrial protein pathways can have regulatory effects on mitochondrial function. Conventional methods using yeast genetics and RNAi approaches are powerful, but limited because RNAi requires several days and during this time, cells can alter metabolism. Therefore, we have developed several approaches to identify small molecule modulators for mitochondrial protein translocation. This study utilizes a high-throughput small molecule screening approach to identify modulators of the TIM23 import pathway, which transports proteins across the inner membrane and into the matrix. This strategy will be useful in generating a toolbox of small molecule modulators for mitochondrial translocation to understand how defects in mitochondrial assembly contribute to disease. ii To this end, we have identified and characterized a small molecule probe MitoBloCK-20 (MB-20) that binds specifically to Tim17 in the TIM23 complex. MB-20 inhibits the import of substrates that use the TIM23 translocon, but not the TIM22 translocon. MB-20 seems to target Tim17, because Tim17p, but not Tim23p and the PAM complex, was resistant to protease in the presence of MB-20. In pull-down experiments with His-tagged components, the interaction between Tim17 and Tim23 was stabilized but Tim44, Pam18 and Pam16 of the motor precipitated, suggesting the interaction between Tim17/Tim23 and the PAM complex was disrupted. Based upon biochemical analysis, MB-20 seems to target Tim17 and suggests that Tim17 plays a critical role in coordinating activities between the channel and the import motor. This modulator also showed a critical role for mitochondria in development of the zebrafish neural system. iii The dissertation of Tsz Mei Jesmine Cheung is approved. Catherine F. Clarke Alexander M. Van der Bliek Carla Marie Koehler, Committee Chair University of California, Los Angeles 2017 iv TABLE OF CONTENTS List of Figures Acknowledgements Vita Chapter 1. Introduction – High-throughput screening methods to develop small molecule probes targeting mitochondria ................................................................. 1 Chapter 2. Discovery of MB-20, a small molecule modulator of the Tim23 channel ........... 39 Chapter 3. MB-20 reveals the role of Tim17 in stabilizing the TIM23/PAM complex .........69 v LIST OF FIGURES Chapter 1. Figure 1-1. The diverse roles of mitochondria .................................................................... 8 Figure 1-2. Integration of different organelles in cells ........................................................ 9 Figure 1-3. Main components of the mitochondrial import system .................................. 10 Figure 1-4. The TIM23 and TIM22 import pathways ....................................................... 11 Figure 1-5. Design of the small molecule screen targeting the TIM23 pathway .............. 12 Figure 1-6. Hits from the screen grouped by similar scaffold on the structure ................. 13 Chapter 2 Figure 2-1. Respiration assay of the hits ...........................................................................48 Figure 2-2. Disc3(5) assay of the hits................................................................................. 49 Figure 2-3. Membrane permeability assay of the hits .......................................................50 Figure 2-4. Illustration of the rationale of using in vitro import to identify the target complex of the small molecules ...................................................................... 51 Figure 2-5. Examples of the results obtained from the in vitro import assay with different small molecules ................................................................................ 52 Figure 2-6. In vitro import assay with mitochondria and mitoplast .................................. 53 Figure 2-7. Results obtained from the in vitro import assay of Cytb2(167)-DHFR and Cytb2(167)A63P-DHFR with EN30, A01 and LS59 ...................................... 54 Chapter 3. Figure 3-1. Structure and MIC50 of MB-20 and MB-20.1 ................................................. 99 Figure 3-2. MB-20 targets the TIM23 complex ...............................................................101 Figure 3-3. MB-20 targets Tim17 specifically ................................................................ 102 Figure 3-4. MB-20 disrupts the Tim44-Tim17 interaction and destabilizes the PAM motor ............................................................................................................. 103 Figure 3-5. MB-20 does not induce mitophagy ............................................................... 105 Figure 3-6. MB-20 treatment induces LC3 lipidation and LC3 tubules formation ......... 108 Figure 3-7. MB-20 treatment in zebrafish showed aberrant accumulation of neuronal phenotype ...................................................................................................... 110 Figure S3-1 MB-20 does not impair general mitochondrial functions ............................. 112 Figure S3-2 MB-20 does not affect mitochondrial membrane potential in HeLa cells .... 114 Figure S3-3 Mulan KO cells exhibit the same phenotype as WT ..................................... 115 Figure S3-4 LC3 does not localize to the mitochondria or to the ER ............................... 117 Figure S3-5 LC3 tubules formation is also observed in MEF cells and MEF cells stably expressing Parkin-flag ........................................................................ 118 Figure S3-6 MB-20 does not affect mitophagy ................................................................ 119 vi LIST OF TABLES Chapter 1 Table 1-1. Small molecules purchased for secondary screen ........................................... 31 Chapter 2 Table 2-1. Summary of the results from the respiration assay ......................................... 54 Table 2-2. List of candidates remained after the secondary screen .................................. 55 Table 2-3. Summary of the in vitro import assay of the hits ............................................ 56 vii ACKNOWLEDGEMENTS It was truly an amazing and challenging journey. Not only did I receive an incredible scientific training at a renowned university, I have also made a lot of friendship that would last a lifetime. First, I would like to thank my advisor, Dr. Carla Koehler, for the opportunity to work in a lab. Carla was a great mentor and I am very grateful for her support for all these years. I would also like to extend my gratitude to my committee members, Dr. Alex van der Bliek, Dr. Michael A Teitell, Dr. Steven G Clarke, and Dr. Catherine Clarke for their advice and guidance over the years. Thank you past and present members of the Koehler lab: Non Miyata, Meghan Johnson, Colin Douglas, Eric Torres, Eriko Shimada, Vivian Zhang, Jennifer Ngo, Gary Schmorgan, Deepa Dabir, Jisoo Han, Christina Jayson, Thao Vi, Michael Conti, Matthew Maland and Janos Steffen. Thank you all for the scientific discussions, random discussions, whining, teasing, happy hours, basically everything that happened for the last couple years. Graduate school would not have been the same without any of you. Last but not least, I would not have survived my graduate career without my family. Aunt Anna and Uncle Steve, without your generous support, I would have been able to come to the States for my education, and I would not be able to achieve what I achieved today. There is no word that can describe my gratitude. Mom and Dad, thank you so much for everything you have done for me. I am not very good at expressing my feelings, but I love both of you more than I could ever tell you and I hope you are proud of me. My little brother Jeff, thank you for taking care of mom and dad when I can’t and I am very grateful that I can do what I wanted to do because I know you are there taking care of them. Finally, I would like to thank my husband (a viii recent upgrade), Ramsay Macdonald, for being there with me through this journey. Thank you for putting up with me all these years and more! ix Chapter 1 describes the logistic of the high-throughput screening for the identification of small molecule modulators in the TIM23 protein import pathway. Chapter 2 highlights the characterization of the screening hits. From the 64 potential hits, I narrowed the list down using different approaches and focused on one small molecule, named MitoBloCK-20 (MB-20). Chapter 3 is a version of a manuscript in preparation to be authored by myself, Christina Jayson, Thao Vi, and Koehler CM.
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