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

项目摘要--保持基因组的完整性是所有细胞生命的基本功能。呼吸 生物体保持强大的抗氧化系统，以抵御病理性的活性氧物种(ROS)。 这是对基因组完整性的威胁。ROS通过几种途径激活DNA损伤反应(DDR) 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)。最常见的癌症疗法是电离疗法，它通过诱导ROS来介导细胞杀伤。 辐射(IR)。然而，DDR响应ROS以维持基因组完整性的机制是 不是很清楚。PI，Neil Pfister，MD，PhD，发现ATP5A1是全基因组中最热门的 以IR为选择压力的CRISPR/CAS9基因敲除筛。ATP5A1是可溶性 三磷酸腺苷合成酶的F1亚基，末端电子传递链复合体，在存在的情况下产生三磷酸腺苷 电化学质子梯度和分子氧。发现裂解形式的ATP5A1共定位 对聚腺苷二磷酸核糖和γH_2AX焦点，氧化应激增强，而PARP抑制。 裂解的ATP5A1含有聚(ADP-核糖)相互作用结构域，这是聚(ADP-核糖)结合所必需的 和本地化到DSB。R-环分辨蛋白DHX9和hnRNPU被鉴定为顶端蛋白 相互作用因子，以及ATP5A1、DHX9或hnRNPU的耗尽显著增加R-LOOP和 自发的DSB。该项目检验了裂解的ATP5A1与 多聚(ADP-核糖)聚合酶促进R-环的分解和氧化反应中的基因组维持 压力。为了验证这一假设，本文提出了三个具体目标。特定目标1将决定ATP5A1如何被切割 是受监管的。特定目标2将描绘切割的ATP5A1如何通过 与DHX9、hnRNPU和聚(ADP-核糖)相互作用。具体目标3将剖析被切割的ATP5A1如何影响 氧化应激后的细胞命运。Pfister博士由David Yu博士和Kathy Griendling博士指导， 弗朗西丝卡·斯托里奇博士、邓兴明博士和威廉·戴南博士的补充支持。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。K08职业发展奖的目标是让Pfister博士获得职业生涯 在基因组维护和细胞命运、ROS和氧化代谢、线粒体方面的指导和培训 生物学和R环生物学，这是对他过去的培训的补充，目的是研究裂解的作用 ATP5A1在协调氧化代谢与基因组维持中的作用，这是一个对癌症至关重要的话题 启蒙和癌症治疗。拟议的研究与结构化的指导和培训相结合 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， 研究细胞如何对IR和ROS作出反应，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.