Non-canonical roles for ATM kinase in regulating mitochondrial function and redox homeostasis

ATM 激酶在调节线粒体功能和氧化还原稳态中的非典型作用

• 批准号: 10461498
• 负责人: Paige Elizabeth Burrell
• 金额: $ 4.49万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10461498
• 关键词: 1-Phosphatidylinositol 3-Kinase; ATM activation; ATM deficient; ATM function; ATM gene; ATM null mice; Affect; Aging; Alleles; Ally; Antibodies; Ataxia; Ataxia Telangiectasia; Autophagocytosis; Autophagosome; BCL2 gene; Binding; Biological Assay; CRISPR/Cas technology; Calcium Signaling; Cell Respiration; Cell physiology; Cells; Clinical; Complex; Consensus; Cytoplasmic Protein; DNA Damage; Data; Data Set; Development; Down-Regulation; Electron Transport; Embryo; Endoplasmic Reticulum; Energy Metabolism; Ensure; Exposure to; Family; Fibroblasts; Flow Cytometry; Functional disorder; GRP94; Genes; Genetic Diseases; Genus Hippocampus; Heat-Shock Proteins 90; Heterozygote; Homeostasis; Human; Immune; Immunologic Deficiency Syndromes; Immunoprecipitation; Impairment; In Vitro; Insulin Resistance; Lead; Leucine; Ligation; Loss of Heterozygosity; Lung; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mediation; Metabolic; Mitochondria; Mitochondrial Proteins; Molecular; Mus; Mutate; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Orthologous Gene; Oxidation-Reduction; Oxidative Phosphorylation; Oxygen Consumption; Pathway interactions; Pharmacology; Phenotype; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Protein-Serine-Threonine Kinases; Proteins; Proteomics; Quality Control; Radiation Tolerance; Reactive Oxygen Species; Regulation; Reporting; Role; Signal Transduction; Site; Site-Directed Mutagenesis; Sterility; Stress; Symptoms; Yeasts; ataxia telangiectasia mutated protein; cancer predisposition; cell immortalization; experimental study; interest; loss of function mutation; member; mitochondrial dysfunction; novel; overexpression; release of sequestered calcium ion into cytoplasm; response; stem; thymocyte; tumor

PROJECT SUMMARY. Ataxia-telangiectasia (A-T) is a pleiotropic genetic disorder caused by bi-allelic mutations in the Ataxia- telangiectasia, mutated (ATM) gene. A-T leads to numerous clinical symptoms, including radiosensitivity, sterility, immunodeficiency, insulin resistance, neurodegeneration, and progressive pulmonary dysfunction. Many of these symptoms are attributed to dysfunction in DNA damage signaling, ATM’s canonical function. However, several of the hallmark symptoms of A-T align with the clinical presentation of mitochondrial dysfunction. Mitochondria regulate a variety of cellular functions, including cellular respiration and calcium signaling. Due to their role in oxidative phosphorylation, mitochondria are exposed to reactive oxygen species (ROS)-mediated damage. The specific autophagic degradation of mitochondria, mitophagy, is essential for the degradation of damaged mitochondria. Impaired mitophagy causes increased cellular ROS, leading to cellular dysfunction that eventually results in aging, cancer development, and neurodegeneration. The Kastan lab has previously characterized mitochondrial dysfunction in primary ATM-/- murine cells, indicating that ATM regulates mitochondrial, metabolic, and redox homeostasis. Additionally, the Kastan lab has reported that mono-allelic deletion of the autophagy regulating protein Beclin-1 rescues many aspects of mitochondrial dysfunction in ATM- /- cells, although the relationship between ATM and Beclin-1 remains undefined. Preliminary data outlined in this proposal indicates that CRISPR/Cas9 deletion or pharmacological inhibition of ATM causes mitochondrial dysfunction in immortalized cells, including increased mitochondrial mass and ROS. I have also demonstrated that mitochondrial stress leads to activation of ATM and its downstream effector kinase Chk2. In order to further elucidate the molecular pathways by which ATM regulates mitochondrial function, we have performed unbiased proteomic screens to identify interactors of both ATM and Beclin-1. These screens identified GRP94 and LRPPRC as putative ATM and Beclin-1 interactors. Specifically, the endoplasmic reticulum protein GRP94 is reported to regulate autophagy, calcium flux, and cellular response to ROS; overexpression of GRP94 is implicated in tumor development and neurodegenerative disease. LRPPRC is a mitochondrial protein involved in the regulation of mitochondrial ROS and electron transport chain (ETC) activity. Mutations in LRPPRC lead to ataxia and neurodegeneration, similar to A-T. The experiments proposed herein will validate the interactions between ATM and its putative interactors, GRP94 and LRPPRC, and determine whether these proteins are direct substrates of ATM. Then, I will determine the functional impact of these relationships on mitochondrial/metabolic function and redox homeostasis, including mitochondrial mass, mitochondrial ROS, ETC activity, and oxygen consumption rate will be further characterized. Additionally, the manner by which ATM affects binding within the Beclin-1 interactome, including the putative interactors GRP94 and LRPPRC, to modulate Beclin-1-mediated autophagosome formation, autophagosome maturation, and mitophagy flux will be thoroughly assessed.

项目总结。 共济失调-毛细血管扩张症(A-T)是一种由共济失调-毛细血管扩张症的双等位基因突变引起的多效性遗传病。 毛细血管扩张，突变(ATM)基因。A-T导致许多临床症状，包括辐射敏感、不孕不育、 免疫缺陷、胰岛素抵抗、神经变性和进行性肺功能障碍。许多. 这些症状被归因于DNA损伤信号的功能障碍，这是ATM的规范功能。然而， A-T的几个标志性症状与线粒体功能障碍的临床表现一致。 线粒体调节多种细胞功能，包括细胞呼吸和钙信号。由于 它们在氧化磷酸化中的作用，线粒体暴露在活性氧(ROS)介导的环境中 损坏。线粒体的特殊自噬降解，即有丝分裂，是降解线粒体的关键 线粒体受损。吞丝分裂功能受损会导致细胞ROS增加，从而导致细胞功能障碍 最终导致衰老、癌症发展和神经退化。卡斯坦实验室之前已经 在原代ATM-/-小鼠细胞中表征线粒体功能障碍，表明ATM调节 线粒体、代谢和氧化还原动态平衡。此外，卡斯坦实验室报告说，单等位基因 自噬调节蛋白Beclin-1的缺失挽救了ATM线粒体功能障碍的多个方面 /-cell，尽管ATM和Beclin-1之间的关系仍未定义。本文件概述的初步数据 研究表明，ATM的CRISPR/Cas9缺失或药物抑制导致线粒体 永生化细胞的功能障碍，包括线粒体质量和ROS增加。我还展示了 线粒体应激导致ATM及其下游效应蛋白激酶Chk2的激活。为了进一步 阐明ATM调节线粒体功能的分子途径，我们已经无偏见地进行了 蛋白质组筛选以确定ATM和Beclin-1的相互作用。这些屏幕识别出GRP94和 LRPPRC作为假定的ATM和Beclin-1相互作用因子。具体来说，内质网蛋白GRP94是 据报道，GRP94调节自噬、钙流和细胞对ROS的反应；GRP94的过表达是 与肿瘤发展和神经退行性疾病有关。LRPPRC是一种线粒体蛋白 在线粒体ROS和电子传输链(ETC)活性的调节中。LRPPRC基因突变导致 共济失调和神经变性，类似于A-T。这里提出的实验将验证相互作用。 ATM与其可能的相互作用因子GRP94和LRPPRC之间的相互作用，并确定这些蛋白是否直接 自动柜员机的衬底。然后，我将确定这些关系对线粒体/代谢的功能影响 功能和氧化还原动态平衡，包括线粒体质量、线粒体ROS等活性和氧 消费率将进一步表征。此外，自动柜员机影响 Beclin-1相互作用体，包括可能的相互作用因子GRP94和LRPPRC，以调节Beclin-1介导的 将对自噬小体的形成、自噬小体成熟和有丝分裂通量进行彻底的评估。