The Biosynthetic Pathway of Mitochondrial Respirasomes

描述（由申请人提供）：线粒体氧化磷酸化（OXPHOS）缺陷导致大量异质性多系统疾病，并且还与神经变性和衰老相关。OXPHOS系统由酶复合物I-IV（Cl至CIV）和ATP合酶（复合物V）形成的呼吸链（MRC）组成。RC复合物被组织成超复合物或双酶体，以促进底物和电子通道，并使活性氧物质的形成最小化。作为这种组织的结果，单一复合物生物发生依赖于其他RC组件，并且一个蛋白质编码基因的改变可导致组合酶复合物缺陷。更好地了解RC生物发生是阐明这些疾病的分子基础是必不可少的。拟议研究的主要目标是使用酵母酿酒酵母和培养的人类细胞作为研究模型，研究RC超复合物组装中涉及的参与者和机制。我们的长期目标是完全了解导致生物合成的途径及其组成部分，作为开发治疗与RC缺陷相关疾病的治疗方法的先决条件。我们最近报道了人类细胞中MRC超复合物生物合成途径的首次描述。我们的数据表明，paclasome生物合成涉及一个复杂的I组装中间体作为一个支架的复合物III和IV亚基的结合，而不是起源于协会的预组装的个人全酶。该过程以复合物I NADH脱氢酶催化模块的掺入结束，这导致辅酶A酶体活化。该建议的中心假设是，虽然复合物III和IV组装成自由全酶或通过将自由亚基并入超复合物中，但复合物I组装和激活的结构单元由β-内酰胺酶体构成，从而解释了β-内酰胺酶体对RC功能的功能意义。提出了三个具体的目标来表征超复杂的生物合成途径及其在健康和疾病中的调节。目的#1-完善所提出的超复合物组装途径并测试其在转化和非转化细胞系中的普适性目的#2-研究在不存在一种全酶的情况下的超复合物组装途径目的#3-探索在诱导MRC酶亚基同种型表达的细胞应激条件下超复合物的组装

DESCRIPTION (provided by applicant): Mitochondrial oxidative phosphorylation (OXPHOS) deficiencies account for a large group of heterogeneous multisystemic disorders and have also been associated to neurodegeneration and aging. The OXPHOS system is comprised of the respiratory chain (MRC) formed by enzymatic Complexes I-IV (CI to CIV) and the ATP synthase (Complex V). The RC complexes are organized into supercomplexes or respirasomes to facilitate substrate and electron channeling and minimize formation of reactive oxygen species. As a consequence of this organization, single complex biogenesis depends on other RC components and alterations in one protein-coding gene can result in combined enzyme complex defects. A better understanding of RC biogenesis is essential for elucidating the molecular basis underlying these diseases. The main objective of the proposed research is to investigate the players and mechanisms involved in RC supercomplex assembly 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 respirasome biogenesis and their components as a prerequisite to the development of therapies for the management of disorders associated with RC deficiencies. We have recently reported the first description of the MRC supercomplexes biosynthetic pathway in human cells. Our data indicate that respirasome biogenesis involves a complex I assembly intermediate acting as a scaffold for the combined incorporation of complexes III and IV subunits, rather than originating from the association of preassembled individual holoenzymes. The process ends with the incorporation of complex I NADH dehydrogenase catalytic module, which leads to the respirasome activation. The central hypothesis of this proposal is that while complexes III and IV assemble either as free holoenzymes or by incorporation of free subunits into supercomplexes, the respirasomes constitute the structural units where complex I is assembled and activated, thus explaining the functional significance of the respirasomes for RC function. Three specific aims are proposed to characterize the supercomplex biosynthetic pathway and its regulation in health and disease. Aim # 1 - To refine the proposed supercomplex assembly pathway and test its universality in transformed and non-transformed cell lines Aim # 2 - To investigate the supercomplex assembly pathway in absence of one of the holoenzymes Aim # 3 - To explore the assembly of supercomplexes under cellular stress conditions that induce the expression of MRC enzyme subunit isoforms