A Genomic and Biochemical Analysis of Mitochondrial Respiratory Chain Biogenesis

描述（由申请人提供）：尽管线粒体呼吸链（MRC）在细胞能量产生和许多心血管、神经退行性和遗传性代谢紊乱中起着重要作用，但MRC形成所需的许多因素目前尚不清楚。事实上，在大约1000种已知的人类线粒体蛋白中，几乎有20%仍然完全没有特征。在这里，我们建议解决差距在我们的理解MRC的形成系统地识别和表征新的MRC生物成因因素。我们已经开发了一个综合的基因组策略的基础上的线索，从进化史，高通量基因表达和蛋白质相互作用的研究，以发现新的MRC基因。我们的两个优先基因，C1 orf 31和C6 orf 57的实验工作，已经显示了它们的要求MRC复合体IV和II的生物发生，分别。值得注意的是，最近的一项测序研究发现了一名线粒体疾病患者的C1 orf 31突变。由于C1 orf 31与人类健康的直接相关性，我们专注于在酵母模型中表征这种蛋白质的功能，在该模型中，我们证明了铜的补充可以挽救线粒体呼吸缺陷。在目前的提案中，我们的目标是：（1）确定C1 orf 31在MRC复合物IV组装中的作用;（2）研究C1 orf 31在线粒体、细胞和生物体水平上丢失的病理后果，并确定患者突变的致病性;（3）使用我们的基于RNAi的“营养物敏化”新测定法鉴定其他MRC生物发生因子，该测定法利用了MRC复合物IV组装中的差异生长。 在葡萄糖或半乳糖中的呼吸缺陷人类细胞以询问线粒体呼吸。 我们将对纯化的C1 orf 31进行体外生化实验和体内酵母遗传实验，以确定C1 orf 31在MRC复合物IV组装中的精确功能。我们将利用我们在人类细胞系和斑马鱼胚胎中的C1 orf 31敲低模型，同时揭示线粒体，细胞和生物生理学中缺乏C1 orf 31的病理后果，并测试这些缺陷可以通过补充铜来治愈的假设。最后，我们将通过实验测试我们的计算预测的MRC生物发生基因候选者，包括C6 orf 57，使用我们的营养物敏化测定法来确定它们在细胞呼吸中的作用，并将命中分配给MRC生物发生途径中的特定步骤。因此，我们工作的影响既有基础性的（阐明MRC形成的基本机制），也有医学性的（为孤儿线粒体疾病的分子诊断提供基础，为C1 orf 31突变患者提供可能的治疗选择）。

DESCRIPTION (provided by applicant): Despite the fundamental roles of the mitochondrial respiratory chain (MRC) in both cellular energy production and a number of cardiovascular, neurodegenerative and inherited metabolic disorders, many factors required for MRC formation are currently unknown. In fact, almost 20% of the approximately 1000 known human mitochondrial proteins remain completely uncharacterized. Here we propose to address the gap in our understanding of MRC formation by systematically identifying and characterizing novel MRC biogenesis factors. We have developed an integrative genomic strategy based on clues from evolutionary history, high- throughput gene expression and protein interaction studies to discover novel MRC genes. Experimental work on two of our prioritized genes, C1orf31 and C6orf57, has shown their requirement for MRC complex IV and II biogenesis, respectively. Remarkably, a recent sequencing study identified mutations in C1orf31 in a mitochondrial disease patient. Due to the immediate relevance of C1orf31 to human health, we focused on characterizing the function of this protein in a yeast model where we demonstrated that copper supplementation rescued mitochondrial respiratory defects. In the current proposal we aim to: (1) Determine the role of C1orf31 in MRC complex IV assembly; (2) Investigate the pathological consequences of the loss of C1orf31 at the mitochondrial, cellular, and organismal level and determine the pathogenicity of patient mutations; and (3) Identify additional MRC biogenesis factors using our novel RNAi-based "nutrient-sensitized" assay that utilizes differential growth of respiratory deficient human cells in glucose or galactose to interrogate mitochondrial respiration. We will perform in vitro biochemical experiments on purified C1orf31 and in vivo yeast genetic experiments to define the precise function of C1orf31 in MRC complex IV assembly. We will exploit our C1orf31 knockdown models in human cell lines and zebrafish embryos to simultaneously unravel the pathological consequences of lack of C1orf31 in mitochondrial, cellular, and organismal physiology, as well as test the hypothesis that these defects could be cured by copper supplementation. Finally, we will experimentally test our computationally predicted MRC biogenesis gene candidates, including C6orf57, for their role in cellular respiration using our nutrient-sensitized assay and assign hits to specific steps in the MRC biogenesis pathway. Thus, the impact of our work is both fundamental (elucidating basic mechanisms of MRC formation) and medical (providing the basis for molecular diagnosis of orphan mitochondrial disorders and a possible therapeutic option for patients with C1orf31 mutations).