Cytochrome c Oxidase Assembly in Health and Disease

描述（由申请人提供）：细胞色素c氧化酶（COX）缺乏是人类线粒体脑肌病的最常见原因，也与神经变性和衰老有关。更好地了解COX的生物发生对于阐明这些疾病群的分子基础至关重要。本研究的主要目的是利用酿酒酵母和培养的人类细胞作为研究模型，探讨COX生物发生的参与者和机制。我们的长期目标是全面了解导致COX组装及其组成的途径，作为开发与COX缺乏相关的疾病管理疗法的先决条件。在我们的资助的前一个任期，我们在理解如何调节COX生物发生方面取得了重大进展。真核生物COX是一种由核和线粒体双重遗传来源的多肽组成的多聚酶，其组装涉及大量核编码的辅助因子。COX显示其组成亚基的协同积累。未组装的亚基产生活性氧的风险很高，它们的积累受到翻译后降解的限制。我们揭示了COX生物发生过程中亚基的化学计量积累的另一个贡献。我们的数据支持一种调节机制的存在，通过这种机制，mtdna编码的COX亚基1 （Cox1）的合成受到其在酿酒酵母中组装伙伴的可用性的调节。因此，本提案的中心假设是存在COX组装依赖于COX 1合成的调节，其独特的性质反过来又提供了催化多亚基组装的手段。在酿酒酵母中，调控系统包括特定的COX1 mRNA翻译激活因子、特定的COX1伴侣和组装因子以及一般伴侣在复杂的相互作用中共同作用，我们刚刚开始描述这种相互作用。调控系统的核心元件是双功能的COX1 mRNA翻译激活子和COX1伴侣蛋白Mss51，该蛋白从酵母到人类都是保守的。本文提出了三个具体目标，以表征最近发现的新的Mss51相互作用参与者（即Ssc1和Cox25）和复杂性水平（即Mss51是一种血红素结合蛋白），这些复杂性涉及酿酒酵母的翻译调控系统及其从酵母到人类的保存。目的1 -揭示Mss51结合血红素的机制和作用；目的2 -研究Mss51与线粒体伴侣蛋白相互作用调节Cox1合成的机制；目的3 -确定酵母和人Mss51的功能等效性

DESCRIPTION (provided by applicant): Cytochrome c oxidase (COX) deficiency is the most frequent cause of mitochondrial encephalomyopathies in humans and has also been associated to neurodegeneration and aging. A better understanding of COX biogenesis is essential for elucidating the molecular basis underlying these groups of diseases. The main objective of the proposed research is to investigate the players and mechanisms involved in COX biogenesis using the yeast Saccharomyces cerevisiae and cultured human cells as research models. Our long-term goal is to attain a complete understanding of the pathways leading to COX assembly and their components as a prerequisite to the development of therapies for the management of disorders associated with COX deficiencies. In the previous term of our grant we made significant progress in the understanding of how COX biogenesis is regulated. Eukaryotic COX is a multimeric enzyme formed by polypeptides of dual genetic origin (nuclear and mitochondrial) which assembly involves a large number of nuclear-encoded auxiliary factors. COX display a concerted accumulation of its constitutive subunits. Unassembled subunits bear a high risk to produce reactive oxygen species and their accumulation is limited by posttranslational degradation. We have revealed another contribution to the stoichiometric accumulation of subunits during COX biogenesis. Our data support the existence of a regulatory mechanism by which the synthesis of mtDNA-encoded COX subunit 1 (Cox1) is regulated by the availability of its assembly partners in the yeast Saccharomyces cerevisiae. Thus, the central hypothesis of this proposal is the existence of a COX assembly dependent regulation of Cox1 synthesis, which unique properties, in turn, offer a means to catalyze multiple- subunit assembly. In S. cerevisiae, the regulatory system involves specific COX1 mRNA translational activators, specific Cox1 chaperones and assembly factors as well as general chaperones acting together in a sophisticated interplay that we are just beginning to characterize. The central element of the regulatory system is the bi-functional COX1 mRNA translational activator and Cox1 chaperone Mss51 which is conserved from yeast to humans. Three specific aims are proposed to characterize recently identified new Mss51-interacting players (i.e. Ssc1 and Cox25) and levels of complexity (i.e. Mss51 is a heme binding protein) concerning the translational regulatory system in S. cerevisiae and its conservation from yeast to humans. Aim # 1 - Disclose the mechanism and role of heme binding by Mss51 Aim #2 - Investigate the mechanism/s by which the interactions of Mss51 with mitochondrial chaperones regulate Cox1 synthesis Aim # 3 - Determine the functional equivalence of yeast and human Mss51 PUBLIC HEALTH RELEVANCE: Cytochrome c oxidase (COX) deficiency is the most frequent cause of mitochondrial encephalomyopathies in humans and has also been associated to neurodegeneration and aging. We will use the yeast Saccharomyces cerevisiae and human cultured cells to study the function of Mss51, a protein that plays dual functions in the synthesis of COX subunit 1 and in the assembly of this subunit into the COX holoenzyme. To gain knowledge on the function of this protein and the pathways in which it operates to regulate COX biogenesis is of great importance from a biological point of view and is expected to have an impact on our understanding of the pathogenic mechanisms underlying the above-mentioned kind of disorders.