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

阐明 ATP 合酶亚基 ATP5A1 在响应氧化应激时维持基因组完整性的线粒体和核功能

• 批准号: 10667513
• 负责人: Neil Thomas Pfister
• 金额: $ 25.33万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10667513
• 关键词: Adenocarcinoma Cell; Aging; Amino Acids; Antibodies; Antioxidants; Binding; Biochemical; Biological; Biology; CRISPR/Cas technology; Cell Fate Control; Cell Fractionation; Cell Nucleus; Cell Respiration; Cells; Cessation of life; Co-Immunoprecipitations; Complement; Complex; Comprehensive Cancer Center; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA lesion; DNA replication fork; DNA-PKcs; Data; Doctor of Philosophy; Electron Transport; Environment; Event; Funding; Gamma-H2AX; Genetic; Genome; Goals; Hybrids; Immunoprecipitation; Impairment; Ionizing radiation; K-Series Research Career Programs; Knock-out; LIG4 gene; Laboratories; Life; Link; Lung Adenocarcinoma; Maintenance; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Mediator; Mentors; Mentorship; Mitochondria; Mitochondrial Proton-Translocating ATPases; Molecular; N-terminal; Nuclear; Organism; Oxidative Stress; Oxygen; Pathologic; Peptides; Phenocopy; Physicians; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Production; Protein Binding Domain; Proteins; Protons; RNA; RNA helicase A; Reactive Oxygen Species; Regulation; Repression; Research; Research Technics; Research Training; Resolution; Role; Scientist; Site; Stress; Structure; System; Testing; Therapeutic; Time; Training; Universities; Variant; Work; analytical method; antibody detection; cancer initiation; cancer therapy; career; career development; cell killing; crosslink; design; genome integrity; genome-wide; hands on research; innovation; knock-down; novel; overexpression; p53-binding protein 1; pressure; prevent; recruit; response

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.

项目摘要 – 维持基因组完整性是所有细胞生命的基本功能。 生物体维持强大的抗氧化系统来防御病理性活性氧 (ROS) ROS 通过多种方式激活 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 通过诱导 ROS 介导细胞杀伤的最常见癌症治疗方法是电离。 然而，DDR 响应 ROS 以维持基因组完整性的机制是 PI 医学博士 Neil Pfister 将 ATP5A1 确定为全基因组中的热门产品。 使用 IR 作为选择压力的 CRISPR/Cas9 敲除筛选 ATP5A1 是可溶性的 α 亚基。 ATP 合成酶的 F1 亚基，末端电子传递链复合物，在存在时生成 ATP 发现 ATP5A1 的裂解形式共定位。 聚（ADP-核糖）和 γH2AX 焦点，氧化应激会增强其作用，而 PARP 抑制会抑制该作用。 切割的 ATP5A1 包含聚 (ADP-核糖) 结合所需的聚 (ADP-核糖) 相互作用结构域 和定位到 DSB 的 R 环解析蛋白 DHX9 和 hnRNPU 被确定为顶级蛋白。 相互作用因子以及 ATP5A1、DHX9 或 hnRNPU 的消耗显着增加了 R 环和 该项目检验了裂解的 ATP5A1 与自发 DSB 合作的中心假设。 聚（ADP-核糖）聚合酶可促进 R 环解析和基因组维护以响应氧化 为了检验这一假设，提出了 3 个具体目标，具体目标 1 将确定 ATP5A1 的裂解方式。 具体目标 2 将描述裂解的 ATP5A1 如何通过促进基因组维持。 与 DHX9、hnRNPU 和聚 (ADP-核糖) 的相互作用 具体目标 3 将剖析裂解的 ATP5A1 如何影响。 Pfister 博士由 David Yu 博士和 Kathy Griendling 博士指导。 Francesca Storici 博士、Xingming Deng 博士和 William Dynan 博士的额外支持。 在 NCI 指定的综合癌症中心拥有出色的研究环境，以完成 K08 职业发展奖的目标是让 Pfister 博士获得职业发展。 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，这补充了他过去的培训，以研究裂解的作用 ATP5A1 协调氧化代谢与基因组维护，这是一个对癌症至关重要的话题 拟议的研究，以及结构化的指导和培训。 计划，旨在促进菲斯特博士的长期目标，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。