How are Disulfides transported across membranes?

二硫化物如何跨膜运输？

• 批准号: 6576423
• 负责人: JAMES BARDWELL
• 金额: $ 19.44万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6576423
• 关键词: biological transport; disulfide bond; electron transport; enzyme mechanism; enzyme substrate; iron; isomerase; membrane transport proteins; oxidoreductase; sulfides

DESCRIPTION (provided by applicant): The correct formation of disulfide bonds is vital for the proper folding of proteins that possess multiple disulfides, including many proteins of pharmacological importance. Great progress has been made in the last few years in understanding the mechanism of disulfide oxidation in vivo, but the mechanism of reduction and isomerization of incorrectly formed disulfides is less clear. We have recently succeeded in the reconstitution of the isomerization/reduction pathway in vitro. This reconstitution opens up the way to extensive biochemical and mechanistic analysis of the process of disulfide correction. DsbC is a periplasmic protein that is thought to isomerize misfolded proteins. In order to be active, DsbC must be kept reduced in the very oxidizing periplasmic environment. DsbD reduces DsbC. DsbD in turn is reduced by the cytoplasmic thioredoxin. We aim to solve the interesting and long-standing topological puzzle of how disulfide bonds are transported through membranes. We have succeeded in defining the direction of electron flow between the individual domains of DsbD, and between DsbD, DsbC, and thioredoxin. Now we must determine how disulfides actually are transported through the membrane. This transport process can be thought of as an outward flow of electrons or an inward flow of disulfides. We will test two models for DsbD action, one where DsbD works via simple disulfide exchange reactions, and the second where cofactor dependent electron transport is also involved. To do this we focus on examining electron flow through the beta domain of DsbD which is the domain that spans the membrane. This domain appears to contain an iron cofactor. We will examine the role of this iron in DsbD catalyzed electron transport. Efforts to define in vivo substrates for DsbC and DsbG will give us tools to eventually address the long-standing question of how disulfide isomerization actually occurs in vivo. Overall, we aim to understand in detail the function of the DsbC-DsbD disulfide isomerase/reductase machine and in particular how disulfides are transported across membranes by the DsbD protein.

描述（由申请人提供）：二硫键的正确形成对于具有多个二硫的蛋白质的正确折叠至关重要，包括许多具有药理重要性的蛋白质。近年来，人们对体内二硫化物氧化机制的研究取得了很大进展，但对不正确形成的二硫化物的还原和异构化机制尚不清楚。我们最近成功地在体外重构了异构化/还原途径。这种重构为对二硫修正过程进行广泛的生化和机理分析开辟了道路。DsbC是一种被认为能使错误折叠的蛋白质异构化的质周蛋白。为了保持活性，DsbC必须在氧化性很强的质周环境中保持还原。DsbD降低DsbC。DsbD又被细胞质硫氧还蛋白还原。我们的目标是解决二硫键如何通过膜运输的有趣和长期存在的拓扑难题。我们成功地确定了DsbD各结构域之间以及DsbD、DsbC和硫氧还蛋白之间的电子流方向。现在我们必须确定二硫化物是如何通过细胞膜运输的。这种输运过程可以被认为是电子向外流动或二硫化物向内流动。我们将测试DsbD作用的两个模型，其中DsbD通过简单的二硫交换反应起作用，第二个模型也涉及辅因子依赖的电子传递。为了做到这一点，我们将重点放在检查电子流通过DsbD的区域，也就是横跨膜的区域。这个结构域似乎包含一个铁辅因子。我们将研究这种铁在DsbD催化电子传递中的作用。努力确定DsbC和DsbG的体内底物将为我们最终解决二硫异构化如何在体内实际发生这一长期存在的问题提供工具。总的来说，我们的目标是详细了解DsbC-DsbD二硫化物异构酶/还原酶机器的功能，特别是DsbD蛋白如何跨膜运输二硫化物。