Molecular Mechanisms of Copper Delivery to Mitochondrial Cytochrome c Oxidase

项目摘要 尽管线粒体呼吸链（MRC）在细胞能量产生和细胞内代谢中起着重要作用， 以及一些心血管、神经退行性和遗传性代谢紊乱， MRC的形成目前尚不清楚。事实上，在大约1000名已知的人类中， 线粒体蛋白质仍然完全没有特征。在这里，我们建议解决我们的差距， 通过系统地识别和表征新的MRC生物成因来理解MRC的形成 因素我们已经开发了一种基于进化历史线索的综合基因组策略，高- 通过基因表达和蛋白质相互作用研究发现新的MRC基因。实验工作 在我们的两个优先基因，C1 orf 31和C6 orf 57，已经显示了它们对MRC复合体IV和II的需求 生物学，分别。值得注意的是，最近的一项测序研究发现了C1 orf 31的突变， 线粒体疾病患者。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，我们证明铜 补充剂挽救了线粒体呼吸缺陷。在本提案中，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缺失的病理后果。 C1 orf 31在线粒体、细胞和生物体水平上的作用，并决定患者的致病性 突变;和（3）使用我们的新的基于RNAi的“营养敏化” 利用呼吸缺陷人细胞在葡萄糖或半乳糖中的差异生长来询问 线粒体呼吸我们将对纯化的C1 orf 31进行体外生化实验，并在体内进行体外生物化学实验。 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， 在线粒体、细胞和生物体生理学中缺乏C1 orf 31的病理后果，以及 作为测试的假设，这些缺陷可以治愈铜补充。最后我们将 实验测试我们计算预测的MRC生物发生基因候选者，包括C6 orf 57， 它们在细胞呼吸中的作用，使用我们的营养素敏化测定，并指定命中MRC中的特定步骤 生物发生途径因此，我们工作的影响是根本性的（阐明MRC的基本机制 形成）和医学（为孤儿线粒体疾病的分子诊断提供基础， C1 orf 31突变患者的可能治疗选择）。

PROJECT SUMMARY 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).