Wayne State University Wayne State University Dissertations January 2018 Chchd10, A Novel Bi-Organellar Regulator Of Cellular Metabolism: Implications In Neurodegeneration Neeraja Purandare Wayne State University, [email protected] Follow this and additional works at: https://digitalcommons.wayne.edu/oa_dissertations Part of the Molecular Biology Commons Recommended Citation Purandare, Neeraja, "Chchd10, A Novel Bi-Organellar Regulator Of Cellular Metabolism: Implications In Neurodegeneration" (2018). Wayne State University Dissertations. 2125. https://digitalcommons.wayne.edu/oa_dissertations/2125 This Open Access Dissertation is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Dissertations by an authorized administrator of DigitalCommons@WayneState. CHCHD10, A NOVEL BI-ORGANELLAR REGULATOR OF CELLULAR METABOLISM: IMPLICATIONS IN NEURODEGENERATION by NEERAJA PURANDARE DISSERTATION Submitted to the Graduate School of Wayne State University, Detroit, Michigan in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY 2018 MAJOR: MOLECULAR BIOLOGY AND GENETICS Approved By: Advisor Date © COPYRIGHT BY NEERAJA PURANDARE 2018 All Rights Reserved ACKNOWLEDGEMENTS First, I would I like to express the deepest gratitude to my mentor Dr. Grossman for the advice and support and most importantly your patience. Your calm and collected approach during our discussions provided me much needed perspective towards prioritizing and planning my work and I hope to carry this composure in my future endeavors. Words cannot describe my gratefulness for the support of Dr. Siddhesh Aras. You epitomize the scientific mind. I hope that I have inculcated a small fraction of your scientific thought process and I will carry this forth not just in my career, but for everything else that I do. None of this would have been possible without your guidance and constant encouragement. I would also like to thank my graduate committee members - Dr. Russell Finley, Dr. Kezhong Zhang, and Dr. Miriram Greenberg for their insightful questions, constructive criticism, and valuable advice. I would like to acknowledge my lab members Stephanie Gladyck, Marissa Petitpas and Mohsen Mohktari for all their help and support. A special thank you to Dr. Mallika Somayajulu- Nitu for her advice and help with imaging and analysis of the microscopy data for this work. I would also like to thank Dr. Maik Hüttemann and members of his lab - Jenney Liu, Asmita Vaishnav, and Hasini Kalpage for all the help and advise both in the lab and during our shared lab meetings. I would also like to express my gratitude for the help from the support personnel of the Center for Molecular Genetics and Genomics for their help and advice. Lastly, but not the least, I would like to thank my friends and family members. I have been blessed with two sets of parents. My parents Mr. Aniruddha Purandare and Mrs. Tejaswini Purandare have always been supportive of my career. But this journey would not have been possible without my uncle; Mr. Ashutosh Kale, and my aunt; Mrs. Kalyani Kale, in the US. I consider myself fortunate indeed to be your third child. Thanks to your constant encouragement and understanding, I have never felt like I was away from home. ii TABLE OF CONTENTS Acknowledgements ......................................................................................................... ii List of Figures ....…….….………………..……….……………..............……....................... vii List of Tables ..…......….........….….…….…………………….……………........................... ix List of AbbreviAtions ...….................……………................……….….…….….................. x ChApter I: Introduction ............…...……………..............….………….....………............... 1 1. The structure and origin of mitochondria ….....………….……………......…................ 3 2. Mitochondria and their role in cellular physiology …….………..…….......................... 3 2.1. Energy production ………….…….………..........................………….................... 3 2.2. Apoptosis ……….……………...……….…......……...….…….……………...….….. 4 2.3. Generation of reactive oxygen species (ROS) ……….........…….………............ 5 2.4. Calcium homeostasis .......……….….…………......……….........…....................... 5 2.5. Lipid homeostasis ......….................……...........………...…………......…............. 6 2.6. Iron homeostasis ……....……….…..…...…….....……….………........................... 7 3. Electron Transport Chain .…….......…………..…....………....…….............................. 7 3.1. Complex I ….………….......………...…..…….…...……......................................... 8 3.2. Complex II ….………...………...……........….….....……….................................... 8 3.3. Complex III …..………………...............…….......…...………….......….................. 9 3.4. Complex V (FOF1 ATP synthase) ........………...…...…………...…….….…........... 9 4. Complex IV (Cytochrome c oxidase) ….……………...................................…....……. 10 5. Twin CX9C Proteins ….………....……..……...…......….……..........................….......... 12 6. Orthologs for CHCHD10 and MNRR1 ………......…………...………........................... 16 7. Similarities and differences between CHCHD10 and MNRR1 ................................... 18 8. MNRR1 ….…….……………........….……….......…...……........................................... 23 8.1. MNRR1 and its role in the nucleus and mitochondria …….….…...…................... 23 iii 8.2. MNRR1 and disease ..……….………....……....……...….....…............................ 28 9. CHCHD10 ………...………….…………..............……...............………....................... 32 9.1. CHCHD10’s role in the nucleus and mitochondria ...………...……..................... 32 9.2. CHCHD10 and its role in disease ……….………………..................................... 33 ChApter II: Results ……...…………………………...………....…...……............................. 41 1. Preliminary characterization of CHCHD10 localization and function ………..…........ 41 1.1. CHCHD10 is localized to the nucleus and the mitochondria ……….....……....... 41 1.2. CHCHD10 is a hypoxia-sensitive gene …...................………............................. 42 1.3. Knockdown of CHCHD10 has pleiotropic effects in cells ………............…......... 44 2. CHCHD10 regulates transcription in the nucleus …….........…………..........….......... 45 2.1. CHCHD10 functions as a repressor at the oxygen responsive element (ORE) ____ in the nucleus ...……………….………….............…….......................................……. 45 2.2. CHCHD10 functions as a repressor by interacting with the inhibitory CXXC5 at ___ the ORE ….…………...…….…………....…….………................................................. 46 3. CHCHD10’s regulates oxygen consumption in the mitochondria .............................. 48 3.1. CHCHD10 interacts with COX ............................................................................ 48 3.2. CHCHD10 stimulates oxygen consumption in the mitochondria …........……...... 50 3.3. Defective mitochondrial oxygen consumption in CHCHD10-KD cells arises via ___ defective phosphorylation of MNRR1 ....……………...……….…….....…..…............... 50 4. CHCHD10’s effects in the nucleus and mitochondria under stress .…....….............….. 54 4.1. CHCHD10 function is enhanced at 8% hypoxia ……………............................... 54 4.2. Point mutations in CHCHD10 abrogate CHCHD10’s function in the nucleus ___ 56 adds and mitochondria ...................….………….…….....…….……….…........……............. 4.2.1. Point mutations in CHCHD10 fail to repress ORE-mediated transcription _____ the nucleus ………...……….………....….………………...…...………..................... 57 4.2.2. Point mutations in CHCHD10 are defective in maintaining optimal ETC ______ in function in the mitochondria ……...…....….…........................………................. 58 iv ChApter III: Discussion …………..…...…......................………...……….......................... 62 1. CHCHD10 and MNRR1’s effects in the mitochondria ….……………..……................. 63 2. CHCHD10 and MNRR1’s effects in the nucleus …………………….....….…............... 65 3. The hypoxia sensitivity of MNRR1 and CHCHD10 ……………........….……............... 68 4. The mechanism of mitochondrial dysfunction for CHCHD10 mutations .....…..…....... 70 5. Summary ……...........................………………………………...................................... 72 6. Future Directions ……….…….…….…………….…………...…..…….......................... 73 ChApter IV: MAteriAls And Methods …………..…….....……....….…………….................. 76 1. Cell culture ……….……….…………….......…….….........…....…………..................... 76 2. Effector and reporter plasmids …….……………......……….….....…………............... 77 3. Transient transfection of HEK293 cells ……….….………...............…………............ 77 4. Real-time polymerase chain reaction ……...….….........……………….………........... 77 5. Hypoxia assays …............……….……….…...………....…………...……..................... 78 6. Luciferase reporter assays ………........…………….......…………...………................. 78 7. DNA binding assays ……..................…....……………….………...………................... 78 8. Cell proliferation assay ……...……...……........….……….……...………...................... 79 9. Cell counting assay …...………........……….…...…......…....…………..…................... 79 10. Intact cellular oxygen consumption ……….………..........…….…...………................ 79 11. Cytochrome c oxidase assay ……….…..….……......…..……………...….................. 79 12. ROS measurement …......………….......…………...……….…...…………….................. 79 13. Confocal microscopy .…………….........…………...……….…...………...…..................