Regulation of mitochondrial respiratory complex I dynamics

线粒体呼吸复合物 I 动力学的调节

• 批准号: 8898851
• 负责人: Yidong Bai
• 金额: $ 28.41万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8898851
• 关键词: Animal Model; Architecture; Attention; Candidate Disease Gene; Cell Line; Cell model; Cell physiology; Cells; Complex; Cytochrome c Reductase; Defect; Degenerative Disorder; Disease; Electron Transport Complex III; Exhibits; Functional disorder; Genes; Genetic; Goals; Human; Investigation; Knock-out; Knockout Mice; Knowledge; Light; Literature; Medical; Metabolic Diseases; Methods; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Modeling; Molecular; Molecular Chaperones; Mutation; NADH dehydrogenase (ubiquinone); Nerve Degeneration; Neuroblastoma; Neurons; Nuclear; Parkinson Disease; Pathogenesis; Pathway interactions; Phenotype; Physiologic pulse; Play; Process; Proteomics; Public Health; Publishing; Regulation; Reporting; Research; Resolution; Respiration; Respiratory Chain; Roentgen Rays; Role; Saccharomyces cerevisiae; Structure; Testing; Up-Regulation; Work; Yeasts; enzyme mechanism; human disease; innovation; insight; interest; mitochondrial DNA mutation; mitochondrial dysfunction; mouse model; mutant; nervous system disorder; novel; protein complex; public health relevance; respiratory; restoration; risk variant

DESCRIPTION (provided by applicant): The emerging evidence supports the proposition that the mitochondrial respiratory chain functions via organized multicomplex structures called supercomplexes. Complex I assembly plays a paramount role in the assembly of supercomplexes. However, our understanding of Complex I function and its regulation is incomplete. The Complex I assembly process, especially the details involving mtDNA-encoded subunits, is largely unclear. In particular, only a very limited number of assembly factors have been identified. Our long term goal is to understand the dynamics of mitochondrial respiratory machinery, including their assembly and turnover processes. The objective of this particular application is to understand the role of several key players in Complex I assembly including several mtDNA-encoded subunits: ND4, ND5 and ND6 and putative Complex I assembly factors DsbA-L and HSP60. The study of Complex I assembly has been difficult since the conventional model S. cerevisiae does not have Complex I and it is almost impossible to induce specific mutations in mammalian mtDNA; thus, mutant cells carrying mtDNA mutations in genes encoding Complex I subunits are rare. We have previously established an efficient method to isolate cells carrying mtDNA mutations and generated several cell models with Complex I assembly deficiency which were then used to initiate comprehensive studies on this complex. We then isolated several cell lines, derived from these mutant lines carrying mtDNA mutations, that had restored Complex I assembly. Further characterizations of these cell lines employing both molecular and proteomics approaches have implicated molecular chaperones HSP60 and DsbA-L in Complex I and supercomplex assembly. The central hypothesis for this application is that the assembly of respiratory Complex I is a delicately regulated process in which the mtDNA-encoded subunits ND4, ND5 and ND6, and assembly factors DsbA-L and HSP60 play distinct roles. To test this hypothesis, we propose to pursue the following three specific aims: 1) Determine the role of mtDNA-encoded subunits ND4, ND5 and ND6 in Complex I dynamics; by combining pulse-chase and BNG analysis, we will follow the step- wise assembly of Complex I and supercomplexes. 2) Characterize the role of HSP60 in Complex I and supercomplex assembly; we will test the ability of HSP60 to suppress some Complex I assembly defects using over-expression approach. 3) Characterize the role of DsbA-L in Complex I assembly and characterize the mouse model with neuronal specific knockout DsbA-L by taking advantage of our collaborators' established animal model. The approach is innovative, because it combines our unique cell and animal models with newly developed analytic methods to understand the complexity of the respiratory complex assembly process. The research is significant, because elucidating these mechanisms could provide new insight into the pathogenesis of diseases resulting from mitochondrial Complex I deficiency. In addition, we anticipate identification of novel risk genes involved in human diseases associated with mitochondrial dysfunction.

描述（由申请人提供）：新兴证据支持以下主张：线粒体呼吸链通过有组织的多重复合结构称为超复合物。复合体I组装在超级复合物的组装中起着至关重要的作用。但是，我们对复杂I功能及其调节的理解是不完整的。复杂的I组装过程，尤其是涉及mtDNA编码亚基的细节，在很大程度上不清楚。特别是，仅确定了非常有限的装配因子。我们的长期目标是了解线粒体呼吸机械的动态，包括它们的组装和周转过程。该特定应用的目的是了解几个关键参与者在复杂I组装中的作用，包括几个MTDNA编码的亚基：ND4，ND5和ND6以及推定的复合物I组装因子DSBA-L和HSP60。自常规模型S.酿酒酵母没有复杂的I，对复杂I组装的研究一直很困难，几乎不可能在哺乳动物mtDNA中诱导特定突变。因此，在编码复合物I亚基的基因中携带mtDNA突变的突变细胞很少见。我们以前已经建立了一种有效的方法来分离携带mtDNA突变的细胞，并生成了具有复杂I组装缺陷的几个细胞模型，然后用来启动有关该复合物的全面研究。然后，我们分离了几个细胞系，这些细胞系从携带mtDNA突变的这些突变线得出，这些突变恢复了复合物I组装。这些细胞系采用分子和蛋白质组学方法的进一步表征，与分子伴侣HSP60和DSBA-L有关复合物I和超复合组装。该应用的中心假设是呼吸复合物I的组装是一个细致的调节过程，其中mtDNA编码的亚基ND4，ND5和ND6以及组装因子DSBA-L和HSP60起独特的作用。为了检验这一假设，我们建议追求以下三个特定目标：1） 确定mtDNA编码的亚基ND4，ND5和ND6在复杂I动力学中的作用；通过结合脉冲追踪和BNG分析，我们将遵循复杂I和超复合物的得分组装。 2）表征Hsp60在复合物I和超复合组件中的作用；我们将测试HSP60使用过表达方法抑制某些复杂I组装缺陷的能力。 3）表征了DSBA-L在复合I组装中的作用，并通过利用我们的合作者的既定动物模型来表征使用神经元特异性敲除DSBA-L的小鼠模型。这种方法具有创新性，因为它将我们独特的细胞和动物模型与新开发的分析方法结合在一起，以了解呼吸复合物组装过程的复杂性。这项研究很重要，因为阐明这些机制可以为线粒体复合物I缺乏症引起的疾病发病机理提供新的见解。此外，我们预计会鉴定出与线粒体功能障碍相关的人类疾病涉及的新风险基因。