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Principal Investigator: Paige Elizabeth Burrell
Organization: DUKE UNIVERSITY
Fiscal Year: 2024
Award: $41,391
Funding agency: National Cancer Institute
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.
Terms: <1-Phosphatidylinositol 3-Kinase><ATM Protein><ATM Serine/Threonine Protein Kinase><ATM Signaling Pathway><ATM activation><ATM deficiency><ATM deficient><ATM function><ATM gene><ATM kinase><ATM null mice><ATM pathway><ATM protein kinase><ATM signaling><Active Oxygen><Affect><Aging><Alleles><Allelic Loss><Allelomorphs><Antibodies><Assay><Ataxia><Ataxia Telangiectasia><Ataxia Telangiectasia Mutated><Ataxia Telangiectasia Protein><Ataxia Telangiectasia Syndrome><Ataxia-Telangiectasia Gene><Ataxia-Telangiectasia Mutated Gene><Ataxia-Telangiectasia-Mutated protein kinase><Ataxy><Autophagocytosis><Autophagosome><Autoregulation><B cell lymphoma 2><B-Cell CLL/Lymphoma 2 Gene><B-cell lymphoma/leukemia-2><BCL2><BCL2 gene><Bcl-2><Binding><Bioassay><Biological Assay><CRISPR approach><CRISPR based approach><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Calcium Ion Signaling><Calcium Signaling><Cancers><Cas nuclease technology><Cell Body><Cell Communication and Signaling><Cell Function><Cell Physiology><Cell Process><Cell Respiration><Cell Signaling><Cells><Cellular Function><Cellular Physiology><Cellular Process><Cellular Respiration><Clinical><Closure by Ligation><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Complex><Consensus><Coordination Impairment><Cytoplasm><Cytoplasmic Protein><DNA Damage><DNA Injury><Data><Data Set><Degenerative Neurologic Disorders><Development><Down-Regulation><Dysfunction><Dyssynergia><Electron Transport><Embryo><Embryonic><Endoplasmic Reticulum><Energy Expenditure><Energy Metabolism><Ensure><Ergastoplasm><Exposure to><Family><Fibroblasts><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><Functional disorder><GRP94><Genes><Genetic Alteration><Genetic Change><Genetic Diseases><Genetic defect><Genus Hippocampus><HSP-90><HSP90><Heat-Shock Proteins 90><Heterozygote><Homeostasis><Human><Immune Precipitation><Immunoprecipitation><Impairment><In Vitro><Insulin Resistance><Intracellular Communication and Signaling><Kinases><Lead><Leucine><Ligation><Loss of Heterozygosity><Louis-Bar Syndrome><Louis-Bar Syndrome Gene><Lung><Lung Respiratory System><Malignant Neoplasms><Malignant Tumor><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Mediating><Mediation><Metabolic><Mice><Mice Mammals><Mitochondria><Mitochondrial Proteins><Modern Man><Molecular><Molecular Interaction><Murine><Mus><Mutate><Mutation><Negotiating><Negotiation><Nerve Degeneration><Nervous System Degenerative Diseases><Neural Degenerative Diseases><Neural degenerative Disorders><Neurodegenerative Diseases><Neurodegenerative Disorders><Neurologic Degenerative Conditions><Neuron Degeneration><Ortholog><Orthologous Gene><Oxidation-Reduction><Oxidative Phosphorylation><Oxidative Phosphorylation Pathway><Oxygen Consumption><Oxygen Radicals><PI-3 Kinase><PI3-Kinase><PI3CG><PI3KGamma><PI3k><PIK3><PIK3CG><PIK3CG gene><Pathway interactions><Pb element><Phenotype><Phosphatidylinositol 3-Kinase><Phosphatidylinositol-3-OH Kinase><Phosphoinositide 3-Hydroxykinase><Phosphorylation><Phosphorylation Site><Phosphotransferase Gene><Phosphotransferases><Physiological Homeostasis><Physiopathology><Play><Pro-Oxidants><Protein Phosphorylation><Protein-Serine Kinase><Protein-Serine-Threonine Kinases><Protein-Threonine Kinase><Proteins><Proteomics><PtdIns 3-Kinase><Quality Control><Radiation Sensitivity><Radiation Tolerance><Radiosensitivity><Reactive Oxygen Species><Redox><Regulation><Reporting><Role><Seahorse><Serine Kinase><Serine-Threonine Kinases><Serine/Threonine Protein Kinase Gene><Signal Transduction><Signal Transduction Systems><Signaling><Site><Site-Directed Mutagenesis><Site-Specific Mutagenesis><Sterility><Stress><Subcellular Process><Symptoms><Targeted DNA Modification><Targeted Modification><Threonine Kinase><Transphosphorylases><Type I Phosphatidylinositol Kinase><Type III Phosphoinositide 3-Kinase><Yeasts><aerobic metabolism><aerobic respiration><ataxia telangiectasia mutated activation><ataxia telangiectasia mutated deficiency><ataxia telangiectasia mutated deficient><ataxia telangiectasia mutated function><ataxia telangiectasia mutated null mice><ataxia telangiectasia mutated pathway><ataxia telangiectasia mutated protein><ataxia telangiectasia mutated signaling><ataxia telangiectasia mutated signaling pathway><autophagy><bcl-2 Genes><biological signal transduction><calcium flux><calcium mobilization><cancer predisposition><ced9 homolog><cell immortalization><degenerative diseases of motor and sensory neurons><degenerative neurological diseases><developmental><electron transfer><experiment><experimental research><experimental study><experiments><flow cytophotometry><genetic condition><genetic disorder><genome mutation><heavy metal Pb><heavy metal lead><heterozygosity><hsp90 Family><hypoimmunity><immune deficiency><immunodeficiency><insulin resistant><insulin tolerance><interest><loss of function mutation><malignancy><member><mitochondrial><mitochondrial dysfunction><neoplasm/cancer><neural degeneration><neurodegeneration><neurodegenerative><neurodegenerative illness><neurological degeneration><neuronal degeneration><novel><overexpress><overexpression><oxidation reduction reaction><oxidative metabolism><pathophysiology><pathway><pharmacologic><pulmonary><radio-sensitivity><radiosensitive><release of sequestered calcium ion into cytoplasm><response><social role><stem><sterile><thymocyte><tumor>