Genetics of Bacterial Thiol Redox Proteins

细菌硫醇氧化还原蛋白的遗传学

• 批准号: 7917831
• 负责人: JONATHAN BECKWITH
• 金额: $ 24.23万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-18 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7917831
• 关键词: Affect; Animal Model; Antibodies; Bacteria; Behavior; Biochemical; Biological Process; Bond-It; Collection; Cysteine; Cytoplasm; Development; Disease; Electron Transport; Electrons; Enzymes; Escherichia coli; Family; Family member; Gel; Genes; Genetic; Glutathione; Growth; Grx3 protein; Heart; Heart Diseases; Human; In Vitro; Insulin; Knowledge; Light; Location; Malignant Neoplasms; Modeling; Mutagenesis; Mutation; Organism; Oxidants; Oxidation-Reduction; Pathway interactions; Peroxides; Physiological; Process; Protein Disulfide Isomerase; Protein Family; Protein translocation; Proteins; Public Health; Reaction; Reducing Agents; Replication Initiation; Ribonucleotide Reductase; Role; Specificity; Substrate Specificity; Sulfhydryl Compounds; Suppressor Mutations; Testing; Thioredoxin; Time; bacterial genetics; base; disulfide bond; disulfide bond reduction; genetic manipulation; genetic selection; glutaredoxin; in vivo; member; mutant; novel; novel strategies; oxidation; peptide hormone; periplasm; peroxiredoxin; preference; protein complex; protein folding; research study; thioredoxin reductase; three dimensional structure; two-dimensional

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.

项目摘要：蛋白质的硫氧还蛋白超家族的成员在所有人中都大量发现 有机体。这些蛋白质会执行多种反应，包括形成蛋白质二硫键， 二硫键的减少和异构化，过氧化物和其他人的破坏。这个目的 项目将对这些蛋白质及其在其中相互作用的蛋白质进行全面研究 模型有机体，大肠杆菌。我们将研究这些蛋白质的折叠，它们的作用机理以及 如何确定其底物特异性。研究将重点介绍蛋白质DSBA，这使得 蛋白质中的二硫键，硫氧还蛋白和谷蛋白上的二硫键，从而减少蛋白质中的二硫键，， 在过氧化物毒素AHPCF上，会破坏过氧化物。我们还将表征抑制剂突变 将生长恢复为缺失硫氧还蛋白家族的细胞质成员的菌株 还原反应。这样的研究允许，并且将使我们能够识别执行的其他蛋白质 与这些还原蛋白相互作用的二硫键还原或其他生理途径。在 DSBA病例，我们将确定该酶如何识别底物蛋白中的半胱氨酸并选择 那些将加入二硫键。对于硫氧还蛋白和谷蛋白，通过改变的突变 它们的特异性，我们将探讨这些蛋白质如何识别其底物并识别新底物。 我们将利用一种新型的蛋白质折叠方法，以获得大量干扰的突变体 硫氧还蛋白的折叠并阐明了该蛋白如何折叠成三维结构。每个 该项目的组成部分与公共卫生有关。二硫键蛋白（如肽） 激素（胰岛素等），用于治疗心脏条件（TPA）和抗体的蛋白质可以产生更多 通过大肠杆菌的遗传操纵，便宜，大量的。过去的研究导致了 这些蛋白质在细菌中产生的量增加。硫氧还蛋白和谷毒素 还原剂在影响心脏等疾病发展的生物过程中很重要 疾病和癌症。大肠杆菌中有关该蛋白质家族的研究已经提供了基础知识 已经使人们更好地了解了在高等生物中发现的那些家庭成员，包括 人类。