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O-Glcnac Regulation of Mitochondrial Function and Energy Metabolism

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O-GlcNAc Regulation of Mitochondrial Function and Energy Metabolism © 2017 Ee Phie Tan B.Sc., University of Iowa, 2011 Submitted to the graduate degree program in Biochemistry and Molecular Biology and the Graduate Faculty of the University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Co-Chairperson: Russell Swerdlow, M.D. Co-Chairperson: Chad Slawson, Ph.D. Gerald Carlson, Ph.D. Antonio Artigues, Ph.D. Hao Zhu, Ph.D. Date Defended: 18 April 2017 ii The dissertation committee for Ee Phie Tan certifies that this is the approved version of the following dissertation: O-GlcNAc Regulation of Mitochondrial Function and Energy Metabolism Co-Chairperson: Russell Swerdlow, M.D. Co-Chairperson: Chad Slawson, Ph.D. Date Approved: 18 April 2017 iii Abstract O-GlcNAc is a post-translational modification (PTM) of a single N-acetylglucosamine sugar attachment on serine or threonine residues of nuclear, cytoplasmic, and mitochondrial proteins. Two opposing enzymes facilitate the modification; O-GlcNAc transferase (OGT) adds the modification, while O-GlcNAcase (OGA) removes it. The addition and the removal of O- GlcNAc, termed O-GlcNAc cycling, is often a dynamic process sensitive to changes in the cellular environment. Disruptions in O-GlcNAcylation contribute to diseases such as diabetes, cancer, and neurodegeneration. Accumulative chronic dysfunctional mitochondria also lead to the development of disease; and importantly, O-GlcNAcylation regulates mitochondrial function. In order to test our first hypothesis that disruptions in O-GlcNAc cycling affect mitochondrial function by changing the mitochondrial proteome, we employed a proteomics screen using SH-SY5Y neuroblastoma cells. We found that OGT and OGA overexpression severely disrupted the mitochondrial proteome, including proteins involved in the respiratory chain and TCA cycle. Furthermore, mitochondrial morphology in the over-expressing cells had disorganized cristae and altered shape and size. Both cellular respiration and glycolysis is impaired. These data support that O-GlcNAc cycling was essential for the proper regulation of mitochondrial function. We next investigated how sustained elevations in cellular O-GlcNAc levels would alter the metabolic profile of the cell. We elevated cellular O-GlcNAc levels by either treating SH- SY5Y cells with low levels of glucosamine (GlcN), the metabolic substrate of OGT, or the OGA inhibitor Thiamet-G (TMG). We found cellular respiration was altered and ATP levels were lower in these cells with sustained elevated O-GlcNAc. Additionally, these cells produce significantly less reactive oxygen species (ROS). Both GlcN and TMG treated cells have iv elongated mitochondria, while mitochondrial fusion/fission protein expressions were decreased. RNA-sequencing analysis showed that the transcriptome is reprogrammed and NRF2 anti- oxidant response is down-regulated. Importantly, sustained O-GlcNAcylation in mice brain and liver validated the metabolic phenotypes seen in cells, whereas liver OGT knockdown elevated ROS levels, impaired mitochondrial respiration, and increased NRF2 anti-oxidant response. Furthermore, we discovered from an indirect calorimetric study that sustained elevated O- GlcNAc promoted weight loss and lowered respiration, skewing mice toward using carbohydrates as their main energy source. Here, our results demonstrated that sustained elevation in O-GlcNAcylation, coupled with increased OGA expression, reprograms energy metabolism and can potentially impact the development of metabolic diseases. Altogether, these studies provide new evidence supporting the role of O-GlcNAc as a critical regulator of mitochondrial function and energy metabolism. v Acknowledgment I would like to take this opportunity to thank my mentor, Dr. Chad Slawson, who has supported me unconditionally throughout my graduate career. I have been able to advance my scientific ability tremendously during these five ‘painful’ graduate school years, truly because of him. His confidence and faith in me, and his generosity and forgiveness towards me are the reasons I used to push myself to continuously be fearless in science and to be my best. The second person that I would like to thank is Dr. Christopher Stipp from the University of Iowa, who was the first person that raised my interests in science. His patience and scientific wisdom inspired me to want to pursue science. I would especially like to thank Dr. Gerald Carlson, who is also one of my committee members, for his encouragement when I encountered some bumpy roads during my graduate school years, and his wise advice on navigating myself to be a better person. Certainly, his advice was proven to be useful! Furthermore, I am grateful to all of my committee members, including Drs. Russell Swerdlow, Antonio Artigues and Hao Zhu for constantly challenging me to become a better scientist during the course of my graduate career. I am thankful for all the faculty, staff, and students in the Biochemistry and Molecular Biology department as my scientific community. Most importantly, I am eternally grateful to my parents and siblings, who always believed in me and cheered me on as I endured all the challenges I encountered. They are certainly my role models to look up to and have helped cultivate my grit in life. I would like to dedicate this dissertation to my parents David Tan and Wan Yiong Yeong, and my sister Sofia Tan and brother Philip Tan. I would like to thank all those friends who made my journey through graduate school an invaluable experience, and also acknowledge the network of individuals who have given me vi support throughout my graduate career, especially Drs. Francesca Duncan (Northwestern University), Maite Villar, Mike Werle, Aric Rogers (MDI Biological Laboratory), Sadie Wignall (Northwestern University), Johnathan Labbadia (Northwestern University), Pedro Reis Rodrigues (The Scripps Research Institute), Sornakala Ganeshkumar, Chooi Ying Sim, Melody Chambers, Steven McGreal, Robert Tessman, Lezi E, Jane Lu, Zhen Zhang, Miranda Machacek, Anish Potnis, Kellyann Jones-Jamtgaard, Pedro Rodrigues, Jeremy Hollis, Nikita Divekar, Timothy Mullen, Amanda Cara, Renee Brielmann, Rachel Gomez, and Gabriel Warner. vii Table of Contents Abstract ......................................................................................................................................... iii! Acknowledgment ........................................................................................................................... v! Chapter 1: Introduction ............................................................................................................... 1! 1.1 Overview ............................................................................................................................................. 1! 1.2 Mitochondria: Metabolic Factories ................................................................................................... 2! 1.3 O-GlcNAc: A Nutrient/Stress Sensing Modification ........................................................................... 3! 1.4 O-GlcNAc Regulates Metabolism ....................................................................................................... 4! Chapter 2: O-linked β-N-Acetylglucosamine Cycling is Important in Regulating Mitochondrial Function ............................................................................................................... 7! 2.1 Introduction ........................................................................................................................................ 7! 2.2 Methods ............................................................................................................................................... 9! 2.2.1 Antibodies .................................................................................................................................... 9! 2.2.2 Cell Culture ................................................................................................................................. 9! 2.2.3 Mitochondrial Isolation ............................................................................................................. 10! 2.2.4 Peptide Identification and Computational Analysis .................................................................. 10! 2.2.5 Electron Microscopy ................................................................................................................. 11! 2.2.6 Cellular Respiration and Glycolysis Measurement ................................................................... 12! 2.2.7 Statistical Analysis .................................................................................................................... 13! 2.3 Results ............................................................................................................................................... 13! 2.3.1 OGT/OGA Overexpression Alters Mitochondrial O-GlcNAcylation and Protein Expression . 13! 2.3.2 OGT/OGA Overexpression Impacts Electron Transport Chain and TCA Protein Expression 22! 2.3.3 Altered O-GlcNAc Cycling Disrupts Mitochondrial Morphology ............................................ 37! viii 2.3.4 OGT/OGA Overexpression Impairs Cellular Respiration and Glycolysis ............................... 39! 2.4 Discussion ......................................................................................................................................... 44! Chapter 3: O-GlcNAc is a
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