Characterization of redox-mediated protein in mitochondria

线粒体中氧化还原介导的蛋白质的表征

• 批准号: 8208123
• 负责人: Sonya Elina Neal
• 金额: $ 3.12万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8208123
• 关键词: Alzheimer' s Disease; Amino Acids; Biochemical; Biochemical Genetics; Biochemistry; Biogenesis; Biological Assay; Biological Models; Cardiac; Cells; Chemistry; Cysteine; Cytochrome c Peroxidase; Defect; Disease; Disulfides; Doctor of Philosophy; Electrons; Environment; Fluorescence; Future; Genetic; Goals; Injury; Iron; Ischemia; Lead; Link; Lipids; Maintenance; Mammals; Mediating; Metabolic Pathway; Metabolism; Methodology; Mitochondria; Mitochondrial Myopathies; Modeling; Molecular; Myopathy; Neurodegenerative Disorders; Neuropathy; Organelles; Oxidants; Oxidation-Reduction; Oxygen; PTEN-induced putative kinase; Parkinson Disease; Pathway interactions; Perfusion; Play; Process; Production; Property; Protein Import; Proteins; Proteome; Public Health; Reaction; Role; Saccharomyces cerevisiae; Sulfhydryl Compounds; Sulfur; Temperature; Testing; Titrations; Tryptophan; Yeasts; cysteine rich protein; cytochrome c; disulfide bond; graduate student; human disease; inhibitor/antagonist; innovation; insight; kohl; mitochondrial dysfunction; mutant; novel; oxidation; periplasm; reconstitution; small molecule; sulfhydryl oxidase

DESCRIPTION (provided by applicant): The overall goal of this project is to further our understanding of the role of redox chemistry in mitochondrial biogenesis. Previous studies, including ours, have shown that the mitochondrial intermembrane space contains a novel oxidative folding pathway. A redox-regulated import pathway consisting of Mia40 was identified to mediate the import of small Tim proteins and cysteine-rich proteins in the intermembrane space. The sulfhydryl oxidase Erv1 also functions in this pathway as a putative oxidant for Mia40. Both Mia40 and Erv1 contain sets of highly conserved cysteine pairs that play an important role in the thiol/disulfide exchange required for import of a subset of cysteine-rich proteins in the intermembrane space. In addition, we have shown that cytochrome c and oxygen act as a terminal electron acceptors. However, our genetic studies suggest that other acceptors, including anaerobic acceptors, may also function in this pathway. The goal of this proposal is to use biochemical and genetic approaches to characterize this import pathway. In contrast to Erv1, the redox state of the 6 cysteine residues of Mia40 and the identity and function of its disulfide bonds in regulating import has not been determined. Thus, the first aim is to reconstitute the disulfide exchange reaction with Mia40, Erv1 and potential substrates, including biochemical characterization of the redox properties of Mia40, Erv1 and substrates. A battery of tests including monobromobimane titration, intrinsic tryptophan fluorescence, and AMS thiol-trapping will be utilized on wildtype and mutants of Erv1, Mia40, and substrates to dilineate the role that each cysteine residues play in the thiol/disulfide exchange mechanism. In addition, the second aim is to use a genetic approach to identify potential substrates and interacting factors of Erv1. In this case, a multi-copy suppressor screen using the temperature-sensitive en/1 mutants will be used for the identification of possible substrates. In addition, candidate interacting proteins will be investigated in genetic and biochemical approaches to determine how they function with Erv1. In all, characterization of this pathway will provide insight into in the redox environment of the mitochondrion. Characterization of this pathway is important for public health because mitochondrial dysfunction has been linked to a broad range of neurodegenerative and muscular diseases such as cardiac ischemia and perfusion injury, mitochondrial myopathies and neuropathies and general neurodegenerative diseases such as Parkinson's and Alzheimer's. This proposal will provide insight into fundamental pathways in mitochondrial assembly that are impaired in the disease state.

描述（由申请人提供）：本项目的总体目标是进一步了解氧化还原化学在线粒体生物发生中的作用。以前的研究，包括我们的，已经表明，线粒体膜间隙包含一个新的氧化折叠途径。一个氧化还原调节的进口途径组成的Mia 40被确定为介导的进口的小蒂姆蛋白和富含半胱氨酸的蛋白质在膜间隙。巯基氧化酶Erv 1也在该途径中作为Mia 40的推定氧化剂起作用。Mia 40和Erv 1都含有一组高度保守的半胱氨酸对，这些半胱氨酸对在膜间空间中富含半胱氨酸的蛋白质的子集的输入所需的硫醇/二硫键交换中发挥重要作用。此外，我们已经表明，细胞色素c和氧作为一个终端电子受体。然而，我们的遗传研究表明，其他受体，包括厌氧受体，也可能在这一途径中发挥作用。该提案的目标是使用生物化学和遗传学方法来表征这种输入途径。与Erv 1相反，Mia 40的6个半胱氨酸残基的氧化还原状态及其二硫键在调节输入中的身份和功能尚未确定。因此，第一个目标是重建与Mia 40，Erv 1和潜在底物的二硫键交换反应，包括Mia 40，Erv 1和底物的氧化还原性质的生物化学表征。一组测试，包括monobromobimane滴定，固有色氨酸荧光，和AMS巯基捕获将用于野生型和突变体的Erv 1，Mia 40，和底物，以dilineate的作用，每个半胱氨酸残基发挥巯基/二硫键交换机制。此外，第二个目标是使用遗传方法来确定Erv 1的潜在底物和相互作用因子。在这种情况下，使用温度敏感的en/1突变体的多拷贝抑制筛选将用于鉴定可能的底物。此外，候选相互作用蛋白将在遗传和生物化学方法进行研究，以确定它们如何与Erv 1一起发挥作用。总之，这一途径的表征将提供深入了解的氧化还原环境中的氧化还原子。该途径的表征对于公共健康是重要的，因为线粒体功能障碍与广泛的神经变性和肌肉疾病如心脏缺血和灌注损伤、线粒体肌病和神经病以及一般神经变性疾病如帕金森病和阿尔茨海默病有关。该提案将提供对疾病状态下受损的线粒体组装中的基本途径的深入了解。