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: 8762078
• 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/8762078
• 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; Disease Association; 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）：新出现的证据支持线粒体呼吸链通过称为超复合物的有组织多复合物结构发挥功能的主张。复合物I组装在超复合物组装中起着至关重要的作用。然而，我们对复合体I的功能及其调节的理解是不完整的。复合体I的组装过程，特别是涉及mtDNA编码亚基的细节，在很大程度上还不清楚。特别是，只有非常有限数量的组装因素已被确定。我们的长期目标是了解线粒体呼吸机制的动力学，包括它们的组装和周转过程。本申请的目的是了解复合体I组装中几个关键参与者的作用，包括几个mtDNA编码的亚基：ND 4、ND 5和ND 6以及推定的复合体I组装因子DsbA-L和HSP 60。自传统的S模型以来，复杂I装配的研究一直是一个难点。酿酒酵母没有复合体I，几乎不可能在哺乳动物mtDNA中诱导特异性突变;因此，在编码复合体I亚基的基因中携带mtDNA突变的突变细胞是罕见的。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，并产生了几个细胞模型与复杂的I组装缺陷，然后用于启动对这个复杂的全面研究。然后，我们分离了几个细胞系，来自这些携带mtDNA突变的突变系，恢复了复合体I的组装。采用分子和蛋白质组学方法对这些细胞系进行进一步表征，表明分子伴侣HSP 60和DsbA-L参与了复合物I和超复合物组装。该应用的中心假设是呼吸复合物I的组装是一个精细调节的过程，其中mtDNA编码的亚基ND 4、ND 5和ND 6以及组装因子DsbA-L和HSP 60发挥不同的作用。为了验证这一假设，我们提出了以下三个具体目标：1） 确定mtDNA编码的亚基ND 4、ND 5和ND 6在复合物I动力学中的作用;通过结合脉冲追踪和BNG分析，我们将跟踪复合物I和超复合物的逐步组装。2)表征HSP 60在复合体I和超复合体组装中的作用;我们将使用过表达方法测试HSP 60抑制某些复合体I组装缺陷的能力。3)利用我们合作者建立的动物模型，表征DsbA-L在复合物I组装中的作用，并表征神经元特异性敲除DsbA-L的小鼠模型。该方法是创新的，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相结合，以了解呼吸复合物组装过程的复杂性。这项研究意义重大，因为阐明这些机制可以为线粒体复合物I缺乏引起的疾病的发病机制提供新的见解。此外，我们预计识别与线粒体功能障碍相关的人类疾病相关的新风险基因。