Genetics of Bacterial Thiol Redox Proteins

项目概述：硫氧还蛋白超家族的蛋白质成员在所有生物中都大量存在。 有机体这些蛋白质进行各种反应，包括形成蛋白质二硫键， 二硫键的还原和异构化、过氧化物的破坏等。这样做的目的 该项目是对这些蛋白质以及它们在细胞中相互作用的蛋白质进行全面研究。 模式生物大肠杆菌。我们将研究这些蛋白质的折叠，它们的作用机制， 它们的底物特异性是如何确定的。这些研究将集中在蛋白质DsbA上， 蛋白质中的二硫键，在硫氧还蛋白和谷氧还蛋白上，其还原蛋白质中的二硫键， 以及一种过氧化物氧还蛋白AhpCF，它能破坏过氧化物。我们还将描述抑制基因突变 恢复生长的菌株是缺乏细胞质成员的硫氧还蛋白家族， 还原反应这些研究已经允许并将允许我们识别出其他蛋白质， 二硫键还原或与这些还原蛋白相互作用的其它生理途径。在 在DsbA的情况下，我们将确定这种酶如何识别底物蛋白中的半胱氨酸，并选择 那些它将以二硫键连接的分子。对于硫氧还蛋白和谷氧还蛋白，通过突变， 它们的特异性，我们将探索这些蛋白质如何识别其底物，并确定新的底物。 我们将开发一种新的蛋白质折叠方法，以获得大量的突变体，这些突变体干扰蛋白质的折叠。 硫氧还蛋白的折叠，并阐明这种蛋白质如何折叠成其三维结构。每个 该项目的组成部分与公共卫生有关。二硫键结合蛋白质，如肽 激素（胰岛素等），用于治疗心脏病的蛋白质（tPA）和抗体可以产生更多 通过对E.杆菌我们过去的研究已经导致了 这些蛋白质在细菌中产生的量增加。硫氧还蛋白和谷氧还蛋白 还原剂在影响疾病发展的生物过程中是重要的 疾病和癌症。目前，在大肠杆菌中对这一蛋白家族的研究已经取得了进展。大肠杆菌提供了基本的知识， 使我们更好地了解那些在高等生物中发现的家族成员，包括 人类

Project Summary: Members of the thioredoxin superfamily of proteins are found in large numbers in all organisms. These proteins perform a variety of reactions, including formation of protein disulfide bonds, reduction and isomerization of disulfide bonds, destruction of peroxides and others. The purpose of this project is to undertake a comprehensive study of these proteins and the proteins they interact with in the model organism, Escherichia coli. We will study the folding of these proteins, their mechanism of action, and how their substrate specificity is determined. The studies will focus on the protein DsbA, which makes disulfide bonds in proteins, on the thioredoxins and glutaredoxins, which reduce disulfide bonds in proteins, and on a peroxiredoxin, AhpCF, which destroys peroxides. We will also characterize suppressor mutations that restore growth to strains that are missing cytoplasmic members of the thioredoxin family which perform reductive reactions. Such studies have allowed and will allow us to identify additional proteins that carry out disulfide bond reduction or other physiological pathways that interact with these reducing proteins. In the case of DsbA, we will determine how this enzyme recognizes cysteines in substrate proteins and chooses those that it will join in a disulfide bond. For the thioredoxins and glutaredoxins, through mutations that alter their specificity, we will explore how these proteins recognize their substrates, and identify new substrates. We will exploit a novel approach to protein folding to obtain a large collection of mutants that interfere with folding of thioredoxin and shed light on how this protein folds into its three-dimensional structure. Each component of this project has relevance to public health. Disulfide-bonded proteins such as peptide hormones (insulin, etc.), proteins used to treat heart condtions (tPA) and antibodies can be produced more cheaply and in high quantities through the genetic manipulation of E. coli. Our studies in the past have led to increased amounts of these proteins being produced in the bacteria. The thioredoxin and glutaredoxin reductants are important in biological processes that influence the development of diseases such as heart disease and cancer. Already, studies on this family of proteins in E. coli has provided basic knowledge that has led to a better understanding of those family members that are found in higher organisms, including humans.