CYTOCHROME C OXIDASE ASSEMBLY IN HEALTH AND DISEASE

描述（由申请人提供）：大分子复合物的形成和调节提供了线粒体膜内氧化磷酸化系统产生有氧能量的支柱。我们的母基金专注于细胞色素c氧化酶（考克斯），最后一种线粒体呼吸链酶，其生物发生涉及一个复杂的动态蛋白质-蛋白质相互作用网络，我们刚刚开始披露。考克斯是由核和线粒体遗传来源的多肽形成的含血红素α-铜的多聚体酶。在父母的补助金，我们已经揭示了一个装配反馈调节机制，通过该机制的关键mtDNA编码和血红素a-含有Cox1亚基的合成调节其装配合作伙伴在酿酒酵母的可用性。通过这种方式，Cox1的合成和组装紧密协调。该调节系统的中心元件是双功能COX1 mRNA翻译激活剂和Cox1分子伴侣Mss51，其从酵母到人类都是保守的。在Cox1合成过程中，Mss51与翻译复合物和翻译后预组装含Cox1的高分子量复合物中的延伸多肽相互作用。这些复合物是由两个保守的小，单一的跨膜结构域蛋白，Cox14和新发现的Cox25稳定。在与Cox1半化和/或Cox5亚基掺入同时的点，Mss51被释放并可用于进一步的翻译。相反，Cox14和Cox25保持与Cox1和其他装配分子伴侣结合，以促进考克斯装配的晚期。 最近的观察表明，人类细胞中存在类似的机制。父母补助金探索的作用，人类MSS 51同源物在考克斯大会。现在，还鉴定了Cox14和Cox25的同源物。该修订的中心假设是存在于人类细胞中的组装依赖性调节Cox1的合成，其中MSS 51，COX 14和COX 25含有多分子伴侣复合物的作用，以协调Cox1的合成和多步考克斯组装。提出了两个具体的目标，以表征相互作用的合作伙伴的Cox14和Cox25和保护这些相互作用从酵母到人类。目的#1-表征通过使用TALENs（转录激活因子样效应物核酸酶）方法创建的MSS51、COX14和COX25敲除人细胞系。目的#2-表征涉及人MSS 51、COX 14和COX 25的大分子多分子伴侣考克斯组装复合物

DESCRIPTION (provided by applicant): The formation and regulation of macromolecular complexes provides the backbone of aerobic energy production by the oxidative phosphorylation system within the mitochondrial membranes. Our parent grant is focused on cytochrome c oxidase (COX), the last mitochondrial respiratory chain enzyme, whose biogenesis involves a sophisticated network of dynamic protein-protein interactions that we are just beginning to disclose. COX is a heme a-copper-containing multimeric enzyme formed by polypeptides of nuclear and mitochondrial genetic origins. In the parent grant, we have revealed an assembly-feedback regulatory mechanism by which the synthesis of the key mtDNA-encoded and heme a-containing Cox1 subunit is regulated by the availability of its assembly partners in Saccharomyces cerevisiae. In this way, Cox1 synthesis and assembly are tightly coordinated. The central element of this regulatory system is the bi-functional COX1 mRNA translational activator and Cox1 chaperone Mss51 which is conserved from yeast to humans. During Cox1 synthesis, Mss51 interacts with the elongating polypeptide in translational complexes and in post-translational pre-assembly Cox1- containing high molecular weight complexes. These complexes are stabilized by two conserved small, single transmembrane domain proteins, Cox14 and the newly identified Cox25. At a point concurrent with Cox1 hemylation and/or the incorporation of subunit Cox5, Mss51 is released and is available for further rounds of translation. Cox14 and Cox25 instead, remain bound to Cox1 and other assembly chaperones to promote late stages of COX assembly. Recent observations suggest a similar mechanism exists in human cells. The parent grant explores the role of the human MSS51 homologue in COX assembly. Now, homologues of Cox14 and Cox25 have been also identified. The central hypothesis of this revision is the existence in human cells of an assembly-dependent regulation of Cox1 synthesis, in which MSS51, COX14 and COX25-containing multichaperone complexes act to coordinate Cox1 synthesis and multistep COX assembly. Two specific aims are proposed to characterize interacting partners of Cox14 and Cox25 and the conservation of these interactions from yeast to humans. Aim # 1 - Characterize MSS51, COX14 and COX25 knockout human cell lines created by a using a TALENs (transcription activator-like effector nucleases) approach. Aim # 2 - Characterize macromolecular multi-chaperone COX assembly complexes involving human MSS51, COX14 and COX25