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

摘要 在从细菌内共生体到线粒体大约15亿年的进化过程中，线粒体保留了基因 在几乎所有真核生物中，它们各自不同的基因组(MtDNA)对能量代谢是必不可少的。人类线粒体DNA 编码呼吸链中的13个核心蛋白，拷贝数从血液中的~100个不等，最多为10,000倍 到~50万的未受精卵，每个细胞通过未知的方式保持一个内在的、最佳的设定点 机械装置。这一设定值的下降会导致罕见但严重的疾病，但没有得到证实的治疗方法和基础。 常见的与年龄相关的线粒体功能障碍与帕金森氏症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未得到满足的主要需求 -克服线粒体DNA丢失的有害后果，了解线粒体DNA的稳态调节 复印号。对酵母、锥虫的遗传学研究，以及我在人类细胞中发表的工作，揭示了 膜电位与线粒体DNA丢失耐受性之间的保守联系。在目标1中，我将确定 提高膜电位挽救线粒体DNA缺失细胞生长的机制。我的已完成 线粒体DNA耗竭和重复状态的多组谱分析已经确定了少量候选效应因子 我将使用一种针对应激信号的药物进行测试，这种代谢物可以 外源性补充，以及我导师实验室首创的用于氧化还原操作的蛋白质假体。在……里面 目标2，我将采取更公正的方法，系统地识别人类mtDNA拷贝的核调控因子 第一次使用基于FACS的全基因组CRISPR基因敲除屏幕进行编号，我将其概念化 并在我早期的博士后工作中发展起来。而我的K99专注于线粒体DNA传感和调节 线粒体基质My R00(Aim 3)检测mtDNA的途径和免疫原性 渗漏到胞浆，这与自身免疫性疾病高度相关。因此，我的独立研究 该计划旨在使线粒体系统生物学和博士学位博士后培训达到协同顶峰 先天免疫力方面的专业知识。 通过我的K99目标，我将掌握生物能量学的理论和实验，并亲身实践 高通量遗传学培训，从屏幕设计到计算分析。我的导师，Vamsi博士 穆萨是一位世界领导者，在我提议的培训领域拥有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 事业。我有一个多元化的咨询委员会，成员包括与我的建议相关的当地和国家专家，包括 我系的一名初级教员和一名免疫学家在R00过渡时提供具体指导。 因此，有了NIH独立之路奖，我将朝着推出自己的长期目标进行培训 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.