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.

线粒体DNA复制体的消除机制。 具体目的：线粒体是真核细胞的重要细胞器，可以从食物中转化化学能。 转化为三磷酸腺苷(ATP)的磷酸酸酐键。人类线粒体基因组 编码对atp合成至关重要的蛋白质，因此线粒体dna(Mtdna)的维持缺陷 导致能量缺乏，并可能导致涉及心脏的退行性疾病的发展， 肌肉、肾脏、肝脏和中枢神经系统(1-3)。例如，阿尔珀斯综合征的特征是 导致儿童早期死亡的顽固性癫痫、精神运动迟缓和肝功能衰竭(4，5)。缺陷 线粒体DNA的维护也与其他突出的疾病有关，如帕金森氏症和阿尔茨海默氏症 疾病、自闭症谱系障碍、糖尿病以及多种癌症和衰老(6-13岁)。这个 线粒体疾病的发病机制尚不清楚。任何一种线粒体DNA都无法治愈- 目前只有相关疾病和姑息治疗策略可用(14)。 PI建议调查一种假定的机制，以防止形成大规模缺失 线粒体DNA，这是线粒体基因组最常见的(从头)缺陷(15-17)。这一机制 缺失形成的原因尚不清楚，但迄今报道的研究表明，它们通常起源于mtdna。 复制停滞，促进DNA链的断裂。删除很可能是在过程中形成的 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 促进线粒体DNA的不稳定和缺失的积累(21-27)，这意味着它们在 防止缺失的形成。另一方面，我们的初步结果表明，停滞的线粒体 复制聚合酶在相当长的一段时间内仍与DNA结合，这可能是有害的，也可能是 需要主动排除。因此，我们推断，在正常情况下，功能失调的mtDNA复制体是 被特定的伴侣和蛋白酶消除，这反过来又促进复制重新开始。在病理上 条件下，复制停滞的增加频率(例如，由于复制酶的缺陷)超过 假定消除系统的能力导致DNA断裂频率和起始时间的增加 有害修复机制(我们在最近的一篇综述中详细讨论了这一点[20])。值得注意的是，它一直是 观察到随着年龄的增长(12，13，28)，大规模的缺失在组织中积累，奇怪的是， 相关的伴侣和蛋白水解酶也随着年龄的增长而减少(29-31岁)。这是显而易见的 相关性要求对因果关系进行调查。此外，假设两者之间的关系 伴侣/蛋白水解酶系统与缺失(Δ)线粒体DNA分子的积累 由于有报告表明mtUPR(未折叠蛋白反应)需要快速进行，因此得到了显著的认可 Δ线粒体DNA在线粒体DNA人群中的扩张，进而加剧相关疾病的发展 (32，33)。Δ线粒体DNA克隆性扩增的分子基础仍不清楚。值得注意的是，蛋白质和 我们建议研究的机制似乎是Δ线粒体DNA克隆扩增的中心。总而言之， 该项目将有助于了解Δ线粒体DNA的形成和克隆扩增的机制，这些机制包括 目前，该领域面临的主要挑战。此外，直接关系的识别和表征 细胞移除有缺陷的线粒体复制体的能力与线粒体的完整性 基因组将为该领域带来关于线粒体疾病发展的新的和令人兴奋的视角， 具有治疗应用的潜力。 目的：阐明人Lon和ClpXP蛋白以及Hsp70/Hsp40分子伴侣系统的作用。 在消除核心线粒体DNA复制体方面。 显著的mtDNA复制体停滞位点(34，35)与主要 线粒体蛋白酶，Lon(36)。之前对各种模式生物的研究表明，Lon经常 需要伴侣热休克蛋白70/40系统的协助，该系统可展开并输送蛋白质底物(37)。 Hsp70/40系统还可以与另一种线粒体蛋白酶ClpXP(38-40)协同工作。失去朗， Hsp40和ClpX在体内损害mtDNA的稳定性(21，24，27)。因此，PI认为停滞不前的线粒体DNA 复制体可以通过两种不同的机制来消除，这两种机制要么是Lon，要么是ClpXP蛋白酶。此外, Hsp70/40分子伴侣系统可用于分解复制体并将其组件运送到 客户端蛋白酶(图1)。我们将通过应用一种综合的方法来评估这一假设 生物分子层干涉技术在分子亲和力和动力学分析中的应用 参数，需要专门的酶分析的系统的生化分析，并测试 Lon、Hsp70/40和ClpX水平的升高可以减轻体内诱导缺失的形成 以酿酒酵母为模型。

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