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.

项目摘要。 共济失调毛细血管扩张症（A-T）是一种多效性遗传性疾病，由共济失调毛细血管扩张症的双等位基因突变引起。 毛细血管扩张症（ATM）基因突变会导致许多临床症状，包括放射敏感性、不育、 免疫缺陷、胰岛素抵抗、神经变性和进行性肺功能障碍等。 这些症状归因于 DNA 损伤信号传导功能障碍，这是 ATM 的典型功能。 A-T 的一些标志性症状与线粒体功能障碍的临床表现相符。 线粒体调节多种细胞功能，包括细胞呼吸和钙信号传导。 它们在氧化磷酸化中的作用，线粒体暴露于活性氧（ROS）介导的 线粒体的特异性自噬降解（线粒体自噬）对于线粒体的降解至关重要。 线粒体自噬受损会导致细胞活性氧增加，从而导致细胞功能障碍。 最终导致衰老、癌症发展和神经退行性变。 原代 ATM-/- 鼠细胞中线粒体功能障碍的特征，表明 ATM 调节 此外，卡斯坦实验室还报告了单等位基因。 自噬调节蛋白 Beclin-1 的缺失可以挽救 ATM 线粒体功能障碍的许多方面 /- 细胞，尽管 ATM 和 Beclin-1 之间的关系仍未定义。 提案表明 CRISPR/Cas9 缺失或 ATM 的药理抑制会导致线粒体 我还证明了永生化细胞的功能障碍，包括线粒体质量和活性氧的增加。 线粒体应激导致 ATM 及其下游效应激酶 Chk2 的激活。 为了阐明 ATM 调节线粒体功能的分子途径，我们进行了公正的研究 用于鉴定 ATM 和 Beclin-1 相互作用因子的蛋白质组筛选 这些筛选鉴定了 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 的过度表达可调节自噬、钙流和细胞对 ROS 的反应； LRPPRC 是一种参与肿瘤发展和神经退行性疾病的线粒体蛋白。 LRPPRC 的突变导致线粒体 ROS 和电子传递链 (ETC) 活性的调节。 共济失调和神经变性，类似于 A-T。本文提出的实验将验证相互作用。 ATM 与其假定的相互作用蛋白 GRP94 和 LRPPRC 之间的关系，并确定这些蛋白是否是直接的 然后，我将确定这些关系对线粒体/代谢的功能影响。 功能和氧化还原稳态，包括线粒体质量、线粒体 ROS、ETC 活性和氧 另外，ATM 影响绑定的方式将进一步表征。 Beclin-1 相互作用组，包括假定的相互作用因子 GRP94 和 LRPPRC，用于调节 Beclin-1 介导 将彻底评估自噬体的形成、自噬体的成熟和线粒体自噬通量。