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

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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.