Regulation of mitochondrial respiratory complex I dynamics

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体的有组织的多复合体结构起作用的主张。配合物I的组装在超配合物的组装中起着至关重要的作用。然而，我们对复合体I的功能及其调控的了解尚不完整。复合体I的组装过程，特别是涉及mtdna编码亚基的细节，在很大程度上是不清楚的。特别是，只确定了数量非常有限的装配因素。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。这个特殊应用的目的是了解几个关键参与者在Complex I组装中的作用，包括几个mtdna编码的亚基：ND4、ND5和ND6，以及假定的Complex I组装因子DsbA-L和HSP60。复合体I组装的研究一直很困难，因为传统的酿酒酵母模型不具有复合体I，并且几乎不可能诱导哺乳动物mtDNA的特异性突变；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。我们之前已经建立了一种有效的方法来分离携带mtDNA突变的细胞，并生成了几个具有Complex I组装缺陷的细胞模型，然后用于启动对该复合体的全面研究。然后，我们从这些携带mtDNA突变的突变细胞系中分离出几株恢复了复合体I组装的细胞系。利用分子组学和蛋白质组学方法对这些细胞系的进一步表征表明，分子伴侣HSP60和DsbA-L参与了复合体I和超复合体的组装。该应用的核心假设是，呼吸复合体I的组装是一个精细调控的过程，其中mtdna编码的亚基ND4、ND5和ND6以及组装因子DsbA-L和HSP60发挥着不同的作用。为了验证这一假设，我们建议追求以下三个具体目标：1)

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.