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: 8403576
• 负责人: Sonya Elina Neal
• 金额: $ 2.02万
• 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 2013-06-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8403576
• 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）：该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。先前的研究，包括我们的研究表明，线粒体膜间空间包含一种新型的氧化折叠途径。确定了由MIA40组成的氧化还原调节的导入途径，以介导膜间空间中小型TIM蛋白和富含半胱氨酸的蛋白的进口。硫酰氧化酶ERV1在该途径中也起作用，是MIA40的推定氧化剂。 MIA40和ERV1都包含一组高度保守的半胱氨酸对，这些对在膜间空间中进口富含半胱氨酸的蛋白所需的硫醇/二硫化物交换起着重要作用。此外，我们已经表明细胞色素C和氧充当末端电子受体。但是，我们的遗传研究表明，包括厌氧受体在内的其他受体也可能在该途径中起作用。该建议的目的是使用生化和遗传方法来表征这种进口途径。与ERV1相反，尚未确定MIA40 6半胱氨酸残基的氧化还原状态以及其在调节进口中的二硫键的身份和功能。因此，第一个目的是重建与MIA40，ERV1和潜在底物的二硫化物交换反应，包括MIA40，ERV1和底物的氧化还原特性的生化表征。一系列测试将用于野生型和ERV1，MIA40和底物的固有色氨酸荧光和AMS硫醇捕获的测试，并将用于抑制每种半胱氨酸残留物在硫醇/二硫化物交换机构中都起着每种半胱氨酸残留物的作用。此外，第二个目的是使用遗传方法来识别ERV1的潜在底物和相互作用因子。在这种情况下，使用对温度敏感的EN/1突变体的多拷贝抑制屏幕将用于识别可能的底物。此外，将在遗传和生化方法中研究候选蛋白质的相互作用，以确定它们如何与ERV1发挥作用。总的来说，该途径的表征将在线粒体的氧化还原环境中提供深入的了解。该途径的特征对于公共卫生很重要，因为线粒体功能障碍与多种神经退行性和肌肉疾病有关，例如心脏缺血和灌注损伤，线粒体肌病和神经病以及一般的神经性疾病，以及一般的神经减毒性疾病，例如帕克森森和帕克森氏症。该提案将提供有关疾病状态受损的线粒体组件中基本途径的见解。