Elucidating the Mitochondrial and Nuclear functions of ATP Synthase Subunit ATP5A1 that Maintain Genome Integrity in Response to Oxidative Stress

维持基因组的完整性是所有细胞生命的基本功能。呼吸性 生物体保持强大的抗氧化系统，以抵御病理性活性氧（ROS） 这对基因组完整性构成了威胁。ROS激活DNA损伤反应（DDR），通过几个 DNA损伤的类型包括RNA和DNA之间的交联以及DNA双链的诱导 中断（DSB）。通过诱导ROS介导细胞杀伤的最常见的癌症治疗是电离 辐射（IR）。然而，DDR响应ROS以维持基因组完整性的机制是 没有很好地理解。PI，Neil Pfister，MD，PhD，将ATP 5A 1确定为全基因组中的热门基因。 使用IR作为选择压力的CRISPR/Cas9敲除筛选。ATP 5A 1是可溶性ATP的α亚基。 ATP合酶的F1亚基，在存在ATP的情况下产生ATP的末端电子传递链复合物 电化学质子梯度和分子氧的结合ATP 5A 1的切割形式被发现共定位于 聚（ADP-核糖）和γ H2 AX灶，这是由氧化应激增强和抑制PARP抑制。 切割的ATP 5A 1含有聚（ADP-核糖）结合所需的聚（ADP-核糖）相互作用结构域 和本地化到DSB。R环分解蛋白DHX 9和hnRNPU被鉴定为最高蛋白 相互作用，以及ATP 5A 1，DHX 9或hnRNPU的耗竭显著增加了R环的水平， 自发性DSB。该项目研究了切割的ATP 5A 1与ATP酶协同作用的中心假设。 聚（ADP-核糖）聚合酶以促进R环解析和基因组维持， 应力为了验证这一假设，提出了3个具体目标。具体目标1将决定如何切割ATP 5A 1 是受管制的。具体目标2将描述切割的ATP 5A 1如何通过以下途径促进基因组维持： 与DHX 9、hnRNPU和聚（ADP-核糖）的相互作用。具体目标3将剖析切割的ATP 5A 1如何影响 氧化应激后的细胞命运。Pfister博士由大卫余博士和凯西格林德林博士指导， 来自Francesca Storici博士、Xingming Deng博士和William Dynan博士的额外支持。埃默里大学 在NCI指定的综合癌症中心拥有出色的研究环境， 建议的研究。K 08职业发展奖的目标是让Pfister博士获得职业发展机会， 在基因组维护和细胞命运，ROS和氧化代谢，线粒体 生物学，和R-环生物学，这补充了他过去的训练，以调查切割的作用， ATP 5A 1在协调氧化代谢与基因组维护中的作用，这是一个对癌症至关重要的主题 启动和癌症治疗。拟议的研究，结合结构化的指导和培训， 该计划旨在促进Pfister博士的长期目标，即监督一个独立资助的实验室， 研究细胞如何响应IR和ROS，以确定癌症治疗的新机会。

PROJECT SUMMARY – Maintenance of genome integrity is a fundamental function of all cellular life. Respiring organisms maintain robust antioxidant systems to defend against pathologic reactive oxygen species (ROS) which are a threat to genome integrity. ROS activates the DNA damage response (DDR) by virtue of several types of DNA damage including crosslinks between RNA and DNA as well as induction of DNA double stranded breaks (DSBs). The most common cancer therapeutic that mediates cell kill through induction of ROS is ionizing radiation (IR). However, the mechanisms by which the DDR responds to ROS to maintain genome integrity are not well understood. The PI, Neil Pfister, MD, PhD, identified ATP5A1 as a top hit in a genome-wide CRISPR/Cas9 knockout screen using IR as the selective pressure. ATP5A1 is the alpha subunit of the soluble F1 subunit of ATP synthase, the terminal electron transport chain complex that generates ATP in the presence of an electrochemical proton gradient and molecular oxygen. A cleaved form of ATP5A1 was found to co-localize to poly(ADP-ribose) and γH2AX foci, which is enhanced by oxidative stress and inhibited by PARP inhibition. Cleaved ATP5A1 contains a poly(ADP-ribose) interaction domain that is required for poly(ADP-ribose) binding and localization to DSBs. R-loop resolution proteins DHX9 and hnRNPU were identified as top protein interactors, and depletion of ATP5A1, DHX9, or hnRNPU significantly increased levels of R-loops and spontaneous DSBs. This project examines the central hypothesis that cleaved ATP5A1 cooperates with poly(ADP-ribose) polymerases to facilitate R-loop resolution and genome maintenance in response to oxidative stress. To test this hypothesis, 3 specific aims are proposed. Specific Aim 1 will determine how cleaved ATP5A1 is regulated. Specific Aim 2 will delineate how cleaved ATP5A1 promotes genome maintenance through interaction with DHX9, hnRNPU, and poly(ADP-ribose). Specific Aim 3 will dissect how cleaved ATP5A1 impacts cell fate following oxidative stress. Dr. Pfister is mentored by Dr. David Yu and Dr. Kathy Griendling with additional support from Dr. Francesca Storici, Dr. Xingming Deng, and Dr. William Dynan. Emory University boasts an outstanding research environment at an NCI-designated Comprehensive Cancer Center to complete the proposed research. The goal of the K08 career development award is for Dr. Pfister to receive career mentorship and training in genome maintenance and cell fate, ROS and oxidative metabolism, mitochondrial biology, and R-loop biology, which complements his past training in order to investigate the role of cleaved ATP5A1 in coordinating oxidative metabolism with genome maintenance, a topic of critical importance to cancer initiation and cancer treatment. The proposed research, in combination with a structured mentoring and training plan, is designed to facilitate Dr. Pfister's long-term goal to supervise an independently funded laboratory that investigates how cells respond to IR and ROS in order to identify new opportunities for cancer therapy.