Characterization of redox-mediated protein in mitochondria

描述(由申请人提供)：本项目的总体目标是加深我们对氧化还原化学在线粒体生物发生中的作用的理解。以前的研究，包括我们的研究，都表明线粒体膜间间隙包含一种新的氧化折叠途径。由Mia40组成的氧化还原调节的输入途径被确定为介导小TIM蛋白和富含半胱氨酸的蛋白在膜间隙的输入。在这个途径中，巯基氧化酶Erv1也可能作为Mia40的氧化剂发挥作用。Mia40和Erv1都包含一组高度保守的半胱氨酸对，它们在膜间隙中输入富含半胱氨酸的蛋白质子集所需的硫醇/二硫键交换中发挥重要作用。此外，我们还证明了细胞色素c和氧作为末端电子受体。然而，我们的遗传学研究表明，其他受体，包括厌氧受体，也可能在这一途径中发挥作用。这项提议的目标是使用生化和遗传方法来表征这一输入途径。与Erv1不同的是，Mia40的6个半胱氨酸残基的氧化还原状态及其二硫键在调节进口中的身份和功能尚未确定。因此，第一个目标是重建与Mia40、Erv1和潜在底物的二硫键交换反应，包括对Mia40、Erv1和底物的氧化还原性质进行生化表征。在Erv1、Mia40和底物的野生型和突变型上，将利用一系列测试，包括单溴莫比胺滴定、本征色氨酸荧光和AMS硫醇捕获，来描述每个半胱氨酸残基在硫醇/二硫键交换机制中所起的作用。此外，第二个目标是使用遗传方法来确定Erv1的潜在底物和相互作用因素。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子筛选将用于鉴定可能的底物。此外，候选相互作用蛋白将通过遗传和生化方法进行研究，以确定它们如何与Erv1一起发挥作用。总而言之，这一途径的特征将提供对线粒体氧化还原环境的洞察。这一途径的特征对公众健康很重要，因为线粒体功能障碍与多种神经退行性疾病和肌肉疾病有关，如心肌缺血和灌注损伤，线粒体肌病和神经疾病，以及普通神经退行性疾病如帕金森氏症和阿尔茨海默氏症。这一建议将为深入了解在疾病状态下受损的线粒体组装的基本途径提供帮助。

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.