The Mechanism of Elimination of the Mitochondrial DNA Replisome

线粒体DNA复制体的消除机制

基本信息

批准号：
10291978
负责人：
Grzegorz Leszek Ciesielski
金额：
$ 20.99万
依托单位：
AUBURN UNIVERSITY AT MONTGOMERY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10291978
关键词：
Academic Research Enhancement Awards Adenosine Triphosphate Affinity Age Aging Alpers&apos Syndrome Alzheimer&apos s Disease Animal Model Binding Sites Biochemical Biological Assay Catalytic Domain Cells Cessation of life Chemicals Client Clonal Expansion Complex DNA DNA Binding DNA Maintenance DNA biosynthesis DNA strand break Defect Degenerative Disorder Development Diabetes Mellitus Disease Enzymes Eukaryotic Cell Food Energy Frequencies Goals Human Human body Hydrolase Impairment Interferometry Intractable Epilepsy Investigation Kidney Kinetics Lead Link Liver Liver Failure Maintenance Methods Mitochondria Mitochondrial DNA Mitochondrial Diseases Modeling Molecular Molecular Analysis Molecular Chaperones Mutation Myocardium Neuraxis Organelles Palliative Care Parkinson Disease Pathogenesis Pathologic Peptide Hydrolases Polymerase Population Process Proteins Public Health Publications Replication-Associated Process Reporting Role Saccharomyces cerevisiae Site Students System Techniques Testing Therapeutic Time Tissues Training United States National Institutes of Health Universities autism spectrum disorder base body system cancer type deprivation early childhood endopeptidase La in vivo meetings mitochondrial genome novel prevent prospective repaired response treatment strategy undergraduate research undergraduate student

项目摘要

Abstract Defects in the human mitochondrial DNA (mtDNA) replication process result in the accumulation of mutations that give rise to a broad spectrum of degenerative disorders involving multiple organs and systems of the human body. In this project, we propose to investigate a putative mechanism that prevents the formation of large-scale deletions in mtDNA, which are the most common (de novo) defects of the mitochondrial genome. The mechanism of deletion formation is unknown, but studies reported to date indicate that they originate from stalling of the mitochondrial DNA replication machinery (the replisome), which leads to the breaking of DNA strands. Notably, prominent mtDNA replication stalling sites correspond with binding sites of the major mitochondrial protease, Lon, which suggests that it might be involved in the elimination of halted replisomes. In addition, the components of human mitochondrial Hsp70/40 chaperone system have been found to co-precipitate with the catalytic subunit of the mitochondrial replicative polymerase, which suggests that these may be involved in the assembly or disassembly of mitochondrial replisomes. In fact, in model organisms, Hsp70/40 systems have been found to assist Lon protease by unfolding and delivering protein substrates. On the other hand, Hsp70/40 systems have also been found to cooperate with another protease, ClpXP, which deficiency results in mitochondrial genome destabilization. This proposal aims to evaluate the hypothesis that stalled mtDNA replisomes are eliminated by two alternative mechanisms that engage either Lon or ClpXP protease. In addition, the Hsp70/40 chaperone system may serve to disassemble the replisome and deliver its components to the client protease. Confirmation and characterization of a direct relationship between the capacity of a cell to remove defective mitochondrial replisomes and the accumulation of damage in the mitochondrial genome, would bring a novel and exciting perspective on the development of mtDNA deletions-linked human disorders, potentially identifying targets for prospective therapeutic strategies. To this end, we will apply a comprehensive approach combining the cutting- edge technique of biolayer interferometry for the analysis of molecular affinities and kinetic parameters, a methodical biochemical analysis that entails specialized enzymatic assays, and testing the putative correlation between cellular levels of Lon, Hsp70/40 and ClpX and the development of mtDNA deletions in vivo, using Saccharomyces cerevisiae as the model organism. To date, we have demonstrated that Lon protease hydrolases the catalytic subunit of the mitochondrial replicative polymerase, but only in the absence of the remaining polymerase subunits. This finding confirms our initial hypothesis and warrants the investigation of the role of Hsp70/40 and/or ClpX in the disassembly of the replisome complex to enable degradation of the catalytic core by Lon protease.

抽象的人线粒体DNA（mtDNA）复制过程的缺陷导致积累引起广泛的变性疾病的突变，涉及多个器官和系统人体。在这个项目中，我们建议调查一种推定的机制，以防止形成 mtDNA中的大规模缺失，这是线粒体基因组最常见（从头）的缺陷。缺失形成的机制尚不清楚，但迄今为止报告的研究表明它们起源于线粒体DNA复制机制（重新质体）的停滞，导致DNA破裂链。值得注意的是，突出的mtDNA复制失速位点与主要的结合位点相对应线粒体蛋白酶，LON，这表明它可能参与消除停止的重塑。在此外，已经发现人线粒体HSP70/40伴侣系统的组件已被发现是为了脱离的用线粒体复制性聚合酶的催化亚基，这表明这些可能涉及在线粒体重新分裂的组装或拆卸中。实际上，在模型生物中，HSP70/40系统具有发现通过展开和递送蛋白质底物来帮助LON蛋白酶。另一方面，HSP70/40 还发现系统与另一种蛋白酶CLPXP合作，这不足导致线粒体基因组不稳定。该提案旨在评估一个假说，即失落的mtDNA重新分裂被两个被消除吸收LON或CLPXP蛋白酶的替代机制。此外，HSP70/40伴侣系统可以用来拆卸重置体并将其组件传递给客户蛋白酶。确认和细胞能力去除有缺陷的线粒体的能力之间的直接关系表征补充和线粒体基因组中损伤的积累，将带来一种新颖而令人兴奋的关于MTDNA缺失与人类疾病的发展的观点，有可能识别目标潜在的治疗策略。为此，我们将采用一种综合方法，结合了剪裁 - 分子亲和力和动力学参数分析的生物层干涉测量法的边缘技术，A 有条不紊的生化分析需要专门的酶学测定并测试推定的相关性在LON的细胞水平，Hsp70/40和Clpx之间以及使用体内mtDNA缺失的发展之间酿酒酵母作为模型生物。迄今为止，我们已经证明了LON蛋白酶水解酶线粒体复制聚合酶的催化亚基，但仅在没有其余的情况下聚合酶亚基。这一发现证实了我们的最初假设，并保证调查 HSP70/40和/或CLPX在重新组合复合体的拆卸中以降解催化核心由LON Protease。