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Repair of Environmentally Induced Mitochondrial DNA Damage

环境引起的线粒体 DNA 损伤的修复

基本信息

批准号：
10597039
负责人：
Aishwarya Prakash
金额：
$ 42.35万
依托单位：
UNIVERSITY OF SOUTH ALABAMA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10597039
关键词：
Acetyl Coenzyme A Acetylation Address Aging Antioxidants Base Excision Repairs Binding Proteins Biological Availability Cadmium Cardiovascular Diseases Cell Nucleus Cells Cellular Metabolic Process Chemicals Clustered Regularly Interspaced Short Palindromic Repeats Complex DNA DNA Damage DNA Repair DNA glycosylase Data Deacetylation Disease Dyslipidemias Endogenous Factors Environment Environmental Risk Factor Enzymes Exogenous Factors Exposure to Free Radicals Genes Genetic Genetic Transcription Genomic Instability Heavy Metals Herbicides Homeostasis Human Hydrogen Peroxide In Vitro Individual Inner mitochondrial membrane Ionizing radiation Knowledge Left Lesion Location Lysine Maintenance Malignant Neoplasms Mass Spectrum Analysis Measures Mediating Membrane Potentials Metabolic Metabolic Diseases Metabolic dysfunction Metabolism Mitochondria Mitochondrial DNA Mitochondrial Diseases Mitochondrial Proteins Molecular Monitor Morphology Mutagenesis Mutation Names Nerve Degeneration Neurodegenerative Disorders Nuclear Nucleosomes Obesity Oxidative Stress Oxidative Stress Induction Oxygen Consumption Paint Paraquat Pathway interactions Play Polymerase Post-Translational Regulation Potassium Predisposition Process Production Productivity Protein Acetylation Proteins Publishing Reactive Oxygen Species Recombinants Regulation Research Respiration Roentgen Rays Role SS DNA BP Scientific Advances and Accomplishments Sirtuins Site Stress Structure Testing Toxic Environmental Substances Western Blotting Work X-Ray Crystallography base cigarette smoke endonuclease VIII environmental agent enzyme activity experience experimental study factor A helicase insight knockout gene live cell imaging mitochondrial dysfunction mitochondrial genome novel nucleobase oxidation oxidative damage preference prevent protein complex protein function protein protein interaction public health relevance repaired response toxicant transcription factor treatment response

项目摘要

Project Summary/ Abstract This proposal connects metabolic dysfunction to the regulation of repair following mitochondrial DNA (mtDNA) damage caused by environmental toxicants. Environmental agents such as ionizing radiation, chemicals found in cigarette smoke, herbicides, and heavy metals as well as normal cellular metabolic processes, generate reactive oxygen species (ROS) in cells. ROS cause damage to cellular DNA, which if not properly repaired, can trigger genome instability and the progression of metabolic diseases including neurodegenerative disorders, aging, and cancer. MtDNA is more susceptible than its nuclear counterpart to oxidative stress. The base excision repair (BER) pathway mends damaged bases in both nuclear and mitochondrial compartments. Specialized enzymes called DNA glycosylases play a critical role in initializing BER by excising damaged bases and mediating other aspects of the repair process via essential protein:protein interactions. We will determine the role and regulation of two DNA glycosylases, NEIL1 and NEIL2, in the repair of mtDNA. We hypothesize that the NEIL enzymes form unique and distinct complexes with mitochondrial proteins that are responsible for mtDNA replication and transcription including mitochondrial single-stranded DNA binding protein (mtSSB), transcription factor A (TFAM), polymerase γ (Polγ), and the twinkle helicase. Our central hypothesis is that complex formation between the NEIL enzymes and mitochondrial proteins drives repair ahead of the replication/ transcription forks and is regulated via (de)acetylation. To address this hypothesis, we will examine the functional interactions between the NEIL enzymes and the named mitochondrial proteins via structure-driven analyses. Experiments using protein painting, small angle-X-ray scattering, and X-ray crystallography will be used to determine the structures of complexes formed between NEIL1, mtSSB, Polγ, and Twinkle as well as between NEIL2, TFAM, and Polγ. Next, we will test the impact of (de)acetylation on NEIL function. High levels of acetyl-coenzyme A in the mitochondrion drives chemical acetylation of proteins and our preliminary data suggests that NEIL2 is modified in this manner. Deacetylation of the NEIL proteins by the NAD+-dependent sirtuin enzymes regulates protein function and will be explored here. Cellular metabolism, mitochondrial dysfunction, and environmental toxicants that cause an increase in ROS levels adversely impact the bioavailability of key metabolites (NAD+) required for deacetylation; a pivotal aspect of our research. Lastly, we will study the localization of the NEIL enzymes under conditions of environmentally induced oxidative stress and their impact on mitochondrial respiration, membrane potential, and morphology. This will shed light on essential nuclear-mitochondrial crosstalk that results from oxidative stress. MtDNA repair is a budding field, with the NEIL enzymes placed at the forefront of the repair process by our recent discoveries. By addressing critical questions of NEIL complex formation, regulation of activity, and localization, the proposed studies will provide the molecular insight necessary for understanding how the repair of mtDNA damage caused by environmental toxicants prevents mutagenesis and offers a novel connection between mitochondrial metabolic function and mtDNA repair.

项目摘要/摘要