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Duke University Dissertation Template Regulation of Ferroptosis by a novel NADPH phosphatase MESH1 by Chien-Kuang Cornelia Ding University Program in Genetics and Genomics Duke University Date:_______________________ Approved: ___________________________ Jen-Tsan Ashley Chi, Supervisor ___________________________ Kris Wood, Chair ___________________________ Pei Zhou ___________________________ Hiroaki Matsunami Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Program in Genetics and Genomics in the Graduate School of Duke University 2018 i v ABSTRACT Regulation of Ferroptosis by a novel NADPH phosphatase MESH1 by Chien-Kuang Cornelia Ding University Program in Genetics and Genomics Duke University Date:_______________________ Approved: ___________________________ Jen-Tsan Ashley Chi, Supervisor ___________________________ Kris Wood, Chair ___________________________ Pei Zhou ___________________________ Hiroaki Matsunami An abstract of a thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Program in Genetics and Genomics in the Graduate School of Duke University 2018 i v Copyright by Chien-Kuang Cornelia Ding 2018 Abstract Ferroptosis is a form of regulated cell death featured by lipid peroxidation and breakage of cell membrane. However, the molecular mediators and regulators are not fully understood. Here, we identified the metazoan homologues of ppGpp hydrolase (MESH1) is an efficient cytosolic NADPH phosphatase, an unexpected enzymatic activity that is captured by the crystal structure of the MESH1-NADPH complex. Ferroptosis elevates MESH1, whose upregulation depletes NADPH and sensitizes cells to ferroptosis. Conversely, MESH1 depletion rescues ferroptosis by sustaining the levels of NADPH and GSH and by reducing lipid peroxidation. Importantly, the ferroptotic protection by MESH1 depletion is ablated by suppression of the cytosolic NAD(H) kinase, NADK, but not its mitochondrial counterpart NADK2. MESH1 depletion also triggers extensive transcriptional changes that are distinct from the canonical integrated stress response, but show striking similarity to the bacterial stringent response. MESH1 depletion also leads to dNTP depletion and inhibition of cell proliferation in tumor cells. Finally, we established Mesh1 knockout mouse model to study the physiological relevance of MESH1. iv Contents Abstract .................................................................................................................................... iv Contents .................................................................................................................................... v List of Tables............................................................................................................................ ix List of Figures ........................................................................................................................... x Acknowledgements ................................................................................................................ xi 1. Introduction .......................................................................................................................... 1 1.1 Bacterial Stringent Response ....................................................................................... 1 1.1.1 ppGpp ...................................................................................................................... 1 1.1.2 RSH protein family ................................................................................................. 3 1.2.3 Transcriptional response in bacterial stringent response ..................................... 4 1.2 Ferroptosis: an iron-dependent oxidative cell death ................................................. 5 1.3 NADPH ........................................................................................................................ 9 1.4 Overview of chapters ................................................................................................. 10 2. The Role of MESH1 as NADPH phosphatase in Ferroptosis Survival .......................... 11 2.1 Introduction: Enzyme kinetics and structure of MESH1 as a NADPH phosphatase ........................................................................................................................................... 11 2.2 Methods ...................................................................................................................... 15 2.2.1 Cell culture and ferroptosis induction ................................................................. 15 2.2.2 Genetic depletion of MESH1 and other genes by RNAi .................................... 16 2.2.3 Overexpression of MESH1 by lentivirus or plasmid .......................................... 16 2.2.4 Quantitative real-time PCR .................................................................................. 18 v 2.2.5 Protein lysates collection and Western Blots....................................................... 18 2.2.6 Measurement of cellular NADPH phosphatase activity .................................... 19 2.2.7 NADP(H) measurements ..................................................................................... 19 2.2.8 Lipid Peroxidation measurement ........................................................................ 20 2.2.9 Cell viability and cell death assays ...................................................................... 20 2.2.10 Cell fractionation ................................................................................................. 21 2.2.11 Metabolomic profiling of MESH1 silenced cells ............................................... 22 2.3 MESH1 is a major NADPH phosphatase in human cells ....................................... 23 2.4 Regulation of MESH1 during stresses ...................................................................... 26 2.5 MESH1 depletion lead to ferroptosis protection by preserving NADPH ............. 28 2.6 MESH1 is a cytosolic NADPH phosphatase ............................................................ 35 3. The phenotypes of MESH1 depletion ............................................................................... 38 3.1 Introduction ................................................................................................................ 38 3.2 Methods ...................................................................................................................... 40 3.2.1 Cell Culture ........................................................................................................... 40 3.2.2 Cell Viability assays .............................................................................................. 40 3.2.3 Genetic depletion of MESH1 by siRNA or shRNA ............................................ 41 3.2.4 cDNA microarray analysis ................................................................................... 42 3.2.5 RNAseq analysis ................................................................................................... 43 3.2.6 dNTP quantification.............................................................................................. 43 3.2.7 Xenograft of MESH1-depleted tumors ................................................................ 44 3.2.8 Generation of transgenic Mesh1 knockout mice ................................................. 45 vi 3.2.9 Isoprotenerol challenge in mice to profile cardiac function............................... 46 3.3.10 Uropathogenic E.coli (UPEC) infection challenge ............................................. 47 3.3 Phenotypes of MESH1-depleted cells ....................................................................... 47 3.3.1 Transcriptional response ...................................................................................... 47 3.3.2 dNTP depletion in MESH1 silencing cells........................................................... 55 3.3.3 Inhibition of cell growth ....................................................................................... 57 3.4 Phenotypes of Mesh1 knockout mice ....................................................................... 59 3.4.1 Validation of knockout ......................................................................................... 59 3.4.2 General phenotypes .............................................................................................. 61 3.4.3 Response to isoproterenol challenge ................................................................... 63 3.4.4 Response to E.coli Urinary tract infection ............................................................ 65 4. Discussion ........................................................................................................................... 67 4.1 Physiological relevance of MESH1 as a NADPH phosphatase .............................. 67 4.1.1 MESH1 as the only known NADPH phosphatase ............................................. 67 4.1.2 The regulation of MESH1 expression .................................................................. 69 4.1.3 Potential MESH1 phenotypes that links to NADPH regulation ....................... 71 4.2 Potential therapeutic implications of MESH1inhibition ......................................... 72 4.2.1 Inhibition of tumor growth .................................................................................. 72 4.2.2 Resistance to ferroptosis stress or oxidative stress ............................................
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