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：
10371212
负责人：
Aishwarya Prakash
金额：
$ 43.12万
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 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10371212
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 biosynthesis DNA copy number DNA glycosylase DNA metabolism Data Deacetylation Disease Dyslipidemias Endogenous Factors 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 Light 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 Oxides Oxygen Consumption Paraquat Pathway interactions Play Polymerase Post-Translational Regulation Potassium Process Production 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 Transcriptional Regulation 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/摘要该建议将代谢功能障碍与线粒体DNA后修复的调节联系起来（mtDNA）环境有毒物质造成的损害。环境药物，例如电离辐射，在香烟烟雾，除草剂和重金属以及正常的细胞代谢中发现的化学物质过程，在细胞中产生活性氧（ROS）。 ROS会损害细胞DNA，如果不是经过适当维修，可以触发基因组不稳定性和代谢性疾病的发展神经退行性疾病，衰老和癌症。 mtDNA比其核对方更容易受到影响氧化应激。基本的惊喜维修（BER）途径修补了核和线粒体隔室。称为DNA糖基酶的专用酶在初始化中起关键作用通过切除损坏的基地并通过必需品进行修复过程的其他方面蛋白质：蛋白质相互作用。我们将确定两种DNA糖基酶Neil1和Neil2在修复中的作用和调节 mtDNA。我们假设尼尔酶与线粒体形成独特而独特的复合物负责mtDNA复制和转录的蛋白质，包括线粒体单链 DNA结合蛋白（MTSSB），转录因子A（TFAM），聚合酶γ（POLγ）和闪烁解旋酶。我们的中心假设是尼尔酶和线粒体蛋白之间的复杂形成修复复制/转录叉之前，并通过（DE）乙酰化调节。解决这个问题假设，我们将检查尼尔酶与命名的功能相互作用线粒体蛋白通过结构驱动的分析。使用蛋白质绘画的实验，小角度X射线散射和X射线晶体学将用于确定在之间形成的复合物的结构 Neil1，MTSSB，Polγ和Twinkle以及Neil2，TFAM和POLγ之间。接下来，我们将测试（DE）乙酰化尼尔功能。线粒体中的高水平乙酰辅酶A驱动化学蛋白质的乙酰化和我们的初步数据表明，尼尔2以这种方式改变了。脱乙酰化 NAD+依赖性Sirtuin酶的Neil蛋白质调节蛋白质功能，并将探索这里。细胞代谢，线粒体功能障碍和环境有毒物质会导致增加 ROS水平对脱乙酰化所需的关键代谢产物（NAD+）的生物利用度有不利影响；关键我们的研究方面。最后，我们将研究尼尔酶的定位环境引起的氧化应激及其对线粒体呼吸，膜电位的影响，和形态。这将揭示由氧化作用的必需核核串扰。压力。 mtDNA修复是一个萌芽场，尼尔酶放置在修复过程的最前沿我们最近的发现。通过解决尼尔复合物形成，活动调节和本地化，拟议的研究将提供理解修复的分子见解由环境有毒物质造成的mtDNA损害可防止诱变并提供新的联系线粒体代谢功能和mtDNA修复之间。