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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

基本信息

批准号：
10640088
负责人：
Paige Elizabeth Burrell
金额：
$ 3.99万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
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/10640088
关键词：
ATM Signaling Pathway ATM activation ATM deficient ATM function ATM gene ATM null mice Affect Aging Alleles Antibodies Ataxia Ataxia Telangiectasia Autophagocytosis Autophagosome BCL2 gene Binding Biological Assay CRISPR/Cas technology Calcium Signaling Cell Respiration Cell physiology Cells Clinical Complex Consensus Cytoplasm 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 PIK3CG gene Pathway interactions 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 pharmacologic release of sequestered calcium ion into cytoplasm response stem thymocyte tumor

项目摘要

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.