A Systems Approach to Discover Sensors and Regulators of the Mitochondrial Genome

摘要 线粒体从细菌内共生体进化而来，经过15亿年的进化， 在几乎所有的真核生物中，线粒体DNA都是能量代谢所必需的。人类mtdna 编码呼吸链中的13个核心蛋白，在血液中的拷贝数为100， 在未受精卵中，每个细胞都保持着一个内在的、最佳的设定点， 机制等该设定点的下降会导致罕见但严重的疾病，没有经过验证的治疗方法， 常见的与年龄相关的线粒体功能障碍与帕金森病和阿尔茨海默病有关。 在我的K99阶段，我建议解决临床和基础线粒体生物学中两个主要未满足的需求 - 克服线粒体DNA丢失的有害后果，并了解线粒体DNA的稳态调节 副本编号。酵母、锥虫的遗传学研究，以及我发表的人类细胞的研究，揭示了一种 膜电位和线粒体DNA丢失耐受性之间的保守联系。在目标1中，我将确定 通过提高膜电位来挽救mtDNA耗尽细胞的生长的机制。我的完成 线粒体DNA缺失和补充状态的多组学分析已经确定了少量候选效应子 我将使用一种针对压力信号的药物来测试，这种药物是一种代谢物， 外源性补充，以及由我导师的实验室开创的用于氧化还原操纵的蛋白质假体。在 目的2，我将采取更公正的方法，系统地确定人类mtDNA拷贝的核调节因子 第一次使用基于FACS的全基因组CRISPR敲除筛选， 在我早期的博士后工作中发展起来的。虽然我的K99专注于线粒体DNA的传感和调节， 线粒体基质，我的R00（目的3）检查线粒体DNA的途径和免疫原性后果 泄漏到细胞溶质，这与自身免疫性疾病高度相关。因此，我的独立研究 该计划的目的是在线粒体系统生物学和博士博士后培训的协同高潮 先天免疫方面的专业知识 通过我的K99目标，我将掌握生物能量学的理论和实验，并动手 高通量遗传学培训，从筛选设计到计算分析。我的导师，瓦姆西博士 Mootha是一位世界领先者，在我所建议的培训领域拥有超过20年的经验- 线粒体系统生物学-并推动了十几名学员进入蓬勃发展的学术研究 careers.我有一个多元化的咨询委员会，由与我的建议相关的当地和国家专家组成，包括 我系的一名初级教员和一名免疫学家在R00过渡期提供具体指导。 因此，有了NIH独立之路奖，我将朝着我的长期目标进行训练， 实验室和发现线粒体和自身免疫性疾病的基本分子机制。

Abstract Over their ~1.5-billion-year evolution from a bacterial endosymbiont, mitochondria have retained genes essential for energy metabolism on their own distinct genome (mtDNA) in almost all eukaryotes. Human mtDNA encodes 13 core proteins in the respiratory chain and varies up to 10,000-fold in copy number from ~100 in blood to ~half a million in the unfertilized egg, with each cell maintaining an intrinsic, optimal setpoint via yet unknown mechanisms. A decline in this setpoint causes rare but severe disorders with no proven therapies and underlies the common age-related mitochondrial dysfunction with relevance to Parkinson’s and Alzheimer’s Disease. In my K99 phase, I propose to address two major unmet needs in clinical and basic mitochondrial biology – to overcome the detrimental consequences of mtDNA loss and to understand homeostatic regulation of mtDNA copy number. Genetic studies in yeast, trypanosomes, and my published work in human cells, reveal a conserved link between membrane potential and tolerance to mtDNA loss. In aim 1, I will determine the mechanism by which boosting membrane potential rescues the growth of mtDNA-depleted cells. My completed multi-omic profiling of mtDNA depletion and repletion states has identified a small number of candidate effectors of membrane potential which I will test using a drug that targets stress signaling, a metabolite that can be supplemented exogenously, and a protein prosthetic for redox manipulation pioneered by my mentor’s lab. In aim 2, I will take a more unbiased approach and systematically identify nuclear regulators of human mtDNA copy number for the first time using a FACS-based genome-wide CRISPR knockout screen, which I conceptualized and developed in my early postdoctoral work. While my K99 focuses on mtDNA sensing and regulation within the mitochondrial matrix, my R00 (aim 3) examines the pathways and immunogenic consequences of mtDNA leakage to the cytosol which is highly relevant to autoimmune disorders. Thus, my independent research program aims at a synergistic culmination of postdoctoral training in mitochondrial systems biology and PhD expertise in innate immunity. Through my K99 goals I will master the theory and experimentation of bioenergetics and get hands-on training in high-throughput genetics, from screen design to computational analysis. My mentor, Dr. Vamsi Mootha, is a world leader with >20 years of experience in precisely the field of my proposed training – mitochondrial systems biology – and has propelled over a dozen trainees into thriving academic research careers. I have a diverse advising committee with local and national experts relevant to my proposal, including one junior faculty member in my department and one immunologist for specific guidance at the R00 transition. Thus, with the NIH Pathway to Independence award, I will train towards my long-term goal of launching my own laboratory and discovering basic molecular mechanisms underlying mitochondrial and autoimmune diseases.