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:
- 金额:$ 20.99万
- 依托单位:
- 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:
- 关键词:Academic Research Enhancement AwardsAdenosine TriphosphateAffinityAgeAgingAlpers&apos SyndromeAlzheimer&aposs DiseaseAnimal ModelBinding SitesBiochemicalBiological AssayCatalytic DomainCellsCessation of lifeChemicalsClientClonal ExpansionComplexDNADNA BindingDNA MaintenanceDNA biosynthesisDNA strand breakDefectDegenerative DisorderDevelopmentDiabetes MellitusDiseaseEnzymesEukaryotic CellFood EnergyFrequenciesGoalsHumanHuman bodyHydrolaseImpairmentInterferometryIntractable EpilepsyInvestigationKidneyKineticsLeadLinkLiverLiver FailureMaintenanceMethodsMitochondriaMitochondrial DNAMitochondrial DiseasesModelingMolecularMolecular AnalysisMolecular ChaperonesMutationMyocardiumNeuraxisOrganellesPalliative CareParkinson DiseasePathogenesisPathologicPeptide HydrolasesPolymerasePopulationProcessProteinsPublic HealthPublicationsReplication-Associated ProcessReportingRoleSaccharomyces cerevisiaeSiteStudentsSystemTechniquesTestingTherapeuticTimeTissuesTrainingUnited States National Institutes of HealthUniversitiesautism spectrum disorderbasebody systemcancer typedeprivationearly childhoodendopeptidase Lain vivomeetingsmitochondrial genomenovelpreventprospectiverepairedresponsetreatment strategyundergraduate researchundergraduate 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.
摘要
人类线粒体DNA(MtDNA)复制过程中的缺陷导致
引起多种退行性疾病的突变,涉及多个器官和系统
人体。在这个项目中,我们建议研究一种可能的机制,以防止形成
线粒体DNA的大规模缺失,这是线粒体基因组最常见的(从头)缺陷。
缺失的形成机制尚不清楚,但迄今报道的研究表明,它们起源于
线粒体DNA复制机制(复制体)停滞,导致DNA断裂
思特斯。值得注意的是,显著的mtdna复制停滞位点与主要的
线粒体蛋白水解酶,Lon,这表明它可能参与消除停滞的复制体。在……里面
此外,已发现人类线粒体Hsp70/40伴侣系统的组件共沉淀
与线粒体复制聚合酶的催化亚单位有关,这表明这些可能与
在线粒体复制体的组装或拆卸中。事实上,在模式生物中,Hsp70/40系统具有
已被发现通过展开和运送蛋白质底物来辅助Lon蛋白酶。另一方面,Hsp70/40
系统还被发现与另一种蛋白酶ClpXP协同作用,这一缺陷导致
线粒体基因组不稳定。
这项建议旨在评估停滞的线粒体DNA复制体被两个
使用Lon或ClpXP蛋白酶的替代机制。此外,Hsp70/40分子伴侣系统
可用于分解复制体并将其组分传递给客户的蛋白酶。确认和
细胞去除有缺陷线粒体的能力之间的直接关系的特征
复制体和线粒体基因组中积累的损伤,将带来一种新颖而令人兴奋的
线粒体DNA缺失相关人类疾病的发展前景,潜在地确定了
前瞻性治疗策略。为此,我们将采取综合方法,将切割-
分析分子亲和力和动力学参数的生物分子层干涉法边缘技术
需要专门的酶分析的方法性生化分析,并测试假定的相关性
Lon、Hsp70/40和ClpX的细胞水平与体内mtDNA缺失的发展之间的关系
以酿酒酵母为模式生物。到目前为止,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,但仅在没有剩余亚基的情况下
聚合酶亚基。这一发现证实了我们最初的假设,并证明了研究
HSP70/40和/或ClpX在复制体复合体的解体中的作用,以使催化核心降解
作者:Lon Protease。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Grzegorz Leszek Ciesielski其他文献
Grzegorz Leszek Ciesielski的其他文献
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{{ truncateString('Grzegorz Leszek Ciesielski', 18)}}的其他基金
The mechanism of elimination of the mitochondrial DNA replisome
线粒体DNA复制体的消除机制
- 批准号:
10880042 - 财政年份:2021
- 资助金额:
$ 20.99万 - 项目类别:
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 - 财政年份:2021
- 资助金额:
$ 20.99万 - 项目类别:
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