The Mechanism of Elimination of the Mitochondrial DNA Replisome

线粒体DNA复制体的消除机制

• 批准号: 10582403
• 负责人: Grzegorz Leszek Ciesielski
• 金额: $ 10万
• 依托单位: AUBURN UNIVERSITY AT MONTGOMERY
• 依托单位国家: 美国
• 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/10582403
• 关键词: Adenosine Triphosphate; Affinity; Age; Aging; Alpers' Syndrome; Alzheimer' s Disease; Animal Model; Binding Sites; Biochemical; Biological Assay; Cells; Cessation of life; Chemicals; Client; Clonal Expansion; DNA; DNA Binding; DNA Maintenance; DNA biosynthesis; Defect; Degenerative Disorder; Development; Diabetes Mellitus; Disease; Enzymes; Eukaryotic Cell; Food Energy; Frequencies; Human; 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; Myocardium; Neuraxis; Organelles; Palliative Care; Parkinson Disease; Pathogenesis; Pathologic; Peptide Hydrolases; Polymerase; Population; Process; Proteins; Publications; Reporting; Role; Saccharomyces cerevisiae; Site; Students; System; Techniques; Testing; Therapeutic; Time; Tissues; Training; Universities; autism spectrum disorder; cancer type; deprivation; early childhood; in vivo; meetings; mitochondrial genome; novel; prevent; repaired; response; treatment strategy; undergraduate research; undergraduate student

The mechanism of elimination of the mitochondrial DNA replisome. Specific Aims: Mitochondria are essential organelles of eukaryotic cells that convert chemical energy from food into that of the phosphoanhydride bonds of adenosine triphosphate (ATP). The human mitochondrial genome encodes proteins critical for ATP synthesis, therefore, defects in the maintenance of mitochondrial DNA (mtDNA) result in energy deprivation and may lead to the development of degenerative disorders involving the heart, muscles, kidneys, liver and the central nervous system (1-3). For example, Alpers syndrome is characterized by intractable epilepsy, psychomotor retardation and liver failure that leads to death in early childhood (4,5). Defects of mtDNA maintenance have also been linked to other prominent disorders such as Parkinson’s and Alzheimer’s diseases, autism spectrum disorders, diabetes, as well as multiple types of cancer and aging (6-13). The mechanisms of pathogenesis of mitochondrial diseases are unknown. There is no cure for any of the mtDNA- associated diseases and only palliative treatment strategies are currently available (14). The PI proposes 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 (15-17). The mechanism of deletions formation is unknown, but studies reported to date indicate that they commonly originate from mtDNA replication stalling, which promotes breakage of DNA strands. Deletions are most likely formed in the process of DNA breaks repair (18-20). Notably, the absence of specific mitochondrial molecular chaperones and proteases promotes the destabilization of mtDNA and accumulation of deletions (21-27), which implies their role in preventing deletions formation. On the other hand, our preliminary results indicate that a stalled mitochondrial replicative polymerase remains DNA-bound for a significant extent of time, which could be deleterious and likely requires active elimination. Therefore, we infer that, in normal conditions, dysfunctional mtDNA replisomes are eliminated by specific chaperones and proteases, which in turn promotes replication restart. In pathological conditions, the increased frequency of replication stalling (e.g. due to defects of the replicative enzymes) exceeds the capacity of the putative elimination system resulting in an increase in DNA breaks frequency and the initiation of the deleterious repair mechanism (we discussed this in detail in a recent review (20)). Notably, it has been observed that the large-scale deletions accumulate in tissues with age (12,13,28) and, curiously, the activity of the related chaperones and proteases has been observed to decrease with age as well (29-31). This apparent correlation calls for the investigation of a causative relationship. In addition, the putative relationship between chaperones/proteases systems and the accumulation of deletion-bearing (Δ)mtDNA molecules recently gained significant recognition, due to reports indicating that mtUPR (unfolded protein response) warrants rapid expansion of ΔmtDNA in the mtDNA population, which in turn exacerbates the development of related disorders (32,33). The molecular basis of the clonal expansion of ΔmtDNA remains elusive. Markedly, the proteins and mechanisms that we propose to investigate appear to be central to the clonal expansion of ΔmtDNA. In summary, the project will help to understand the mechanism of ΔmtDNA formation and their clonal expansion, which are currently the major challenges in the field. Furthermore, identification and characterization of a direct relationship between the capacity of a cell to remove defective mitochondrial replisomes and the integrity of the mitochondrial genome would bring to the field a novel and exciting perspective on the development of mitochondrial disorders, with a potential for therapeutic applications. Aim: To elucidate the role of human Lon and ClpXP proteases, and the Hsp70/Hsp40 chaperone system in the elimination of the core mitochondrial DNA replisome. Prominent mtDNA replisome stalling sites (34,35) correspond with binding sites of the major mitochondrial protease, Lon (36). Previous studies from various model organisms indicated that Lon often requires the assistance of a chaperone Hsp70/40 system, which unfolds and delivers protein substrates (37). The Hsp70/40 system can also cooperate with another mitochondrial protease, ClpXP (38-40). Loss of Lon, Hsp40 and ClpX impairs mtDNA stability in vivo (21,24,27). Therefore, the PI proposes that the stalled mtDNA replisome is 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 (Figure 1). We will evaluate this hypothesis by applying a comprehensive approach combining the cutting-edge technique of biolayer interferometry for the analysis of molecular affinities and kinetic parameters, a methodical biochemical analysis entailing specialized enzymatic assays, and testing whether elevated levels of Lon, Hsp70/40 and ClpX can alleviate the formation of induced deletions in vivo using Saccharomyces cerevisiae as a model.

消除线粒体DNA复制体的机制。 具体目的：线粒体是真核细胞的必不可少的细胞器，可从食物转化化学能 进入三磷酸腺苷（ATP）的磷酸氢键。人线粒体基因组 编码对ATP合成至关重要的蛋白质，因此，维持线粒体DNA（mtDNA）的缺陷 导致能量剥夺，并可能导致退化性疾病的发展涉及心脏， 肌肉，肾脏，肝脏和中枢神经系统（1-3）。例如，Alpers综合征的特征是 顽固性癫痫，心理运动迟缓和肝衰竭导致幼儿死亡（4,5）。缺陷 MTDNA维护也与其他突出的疾病有关，例如帕金森氏症和阿尔茨海默氏症 疾病，自闭症谱系疾病，糖尿病以及多种类型的癌症和衰老（6-13）。 线粒体疾病的发病机理尚不清楚。任何mtDNA-无法治愈 目前可用相关的疾病和仅姑息治疗策略（14）。 PI的提议研究了一种推定的机制，以防止形成大规模删除 在mtDNA中，这是线粒体基因组的最常见（从头）缺陷（15-17）。机制 缺失的形成尚不清楚，但迄今为止报告的研究通常起源于mtDNA 复制失速，促进DNA链的破裂。删除很可能是在此过程中形成的 DNA断裂修复（18-20）。值得注意的是，缺乏特定的线粒体分子伴侣和蛋白酶 促进mtDNA的不稳定和缺失的积累（21-27），这意味着它们在 防止缺失形成。另一方面，我们的初步结果表明一个停滞的线粒体 复制性聚合酶在很大的时间内保持了DNA结合，这可能是有害的，并且可能是可能的 需要主动消除。因此，我们推断，在正常情况下，功能失调的mtDNA复制品是 被特定的伴侣和蛋白酶消除，从而促进复制重新启动。在病理中 条件，复制失速的频率增加（例如，由于复制酶的缺陷）超过 假定的消除系统的容量导致DNA断裂频率和启动的增加 有害修复机制（我们在最近的评论（20）中详细讨论了这一点）。值得注意的是，已经 观察到大规模缺失随着年龄的年龄（12,13,28）而积聚在组织中，奇怪的是 已经观察到相关的伴侣和蛋白酶也随着年龄的增长而降低（29-31）。这个外观 相关要求建立一种因果关系的投资。此外， 伴侣/蛋白酶系统以及最近获得的缺失（δ）mtDNA分子的积累 由于报告表明MTUPR（未折叠的蛋白质反应）​​警告快速，因此很大的认可 mtDNA种群中ΔmtDNA的扩展，这反过来加剧了相关疾病的发展 （32,33）。 ΔmtDNA克隆膨胀的分子基础仍然难以捉摸。明显地，蛋白质和 我们建议研究外观的机制对于ΔmtDNA的克隆膨胀至关重要。总之， 该项目将有助于了解ΔmtDNA形成的机理及其克隆扩张的机理，这是 目前，该领域面临的主要挑战。此外，直接关系的识别和表征 在细胞消除有缺陷的线粒体重新分裂的能力和线粒体的完整性之间 基因组将使该领域有关于线粒体疾病发展的小说而令人兴奋的观点， 具有治疗应用的潜力。 目的：阐明人LON和CLPXP蛋白酶的作用，以及HSP70/HSP40伴侣系统 在消除核心线粒体DNA复制体中。 突出的mtDNA复制体档位位点（34,35）与主要的结合位点相对应 线粒体蛋白酶，LON（36）。来自各种模型生物的先前研究表明，LON经常 需要伴侣HSP70/40系统的协助，该系统展开并提供蛋白质底物（37）。 HSP70/40系统还可以与另一种线粒体蛋白酶CLPXP（38-40）进行协调。失去LON， HSP40和CLPX会损害体内mtDNA稳定性（21,24,27）。因此，pi停滞的mtDNA提议 复制体被两种接合LON或CLPXP蛋白酶的替代机制消除。此外， HSP70/40伴侣系统可能有助于拆卸复制体，并将其组件传递到 客户蛋白酶（图1）。我们将通过采用全面的方法来评估这一假设 分子亲和力分析的生物层干扰的尖端技术和动力学 参数，有条不紊的生化分析需要专门的酶学测定，并测试是否 LON，HSP70/40和CLPX的水平升高可以减轻使用体内诱导缺失的形成 酿酒酵母作为模型。

项目成果

Implications of DNA Polymerase Gamma in the Repair of the Mitochondrial Genome.

DNA 聚合酶 Gamma 在线粒体基因组修复中的意义。

• 发表时间: 2022
• 期刊: FASEB journal : official publication of the Federation of American Societies for Experimental Biology
• 作者: deBoviPontes,Carolina;Jefferys,Cody;Bedwan,Muhamad;Ciesielska,ElenaJ;Ciesielski,GrzegorzL
• 通讯作者: Ciesielski,GrzegorzL

Mitochondrial DNA maintenance in Drosophila melanogaster.

• DOI: 10.1042/bsr20211693
• 发表时间: 2022-11-30
• 期刊: Bioscience reports
• 影响因子: 4

Effects of Antiviral Nucleoside Analogues on the Maintenance of the Mitochondrial Genome.

抗病毒核苷类似物对维持线粒体基因组的影响。

• 发表时间: 2022
• 期刊: FASEB journal : official publication of the Federation of American Societies for Experimental Biology
• 作者: Bisimwa,Hyacintha-GhislaineM;Ciesielska,ElenaJ;Kim,Noelle;Oliveira,MarcosT;Ciesielski,GrzegorzL
• 通讯作者: Ciesielski,GrzegorzL