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：硫氧还蛋白超家族的成员在所有 有机体。这些蛋白质执行各种反应，包括形成蛋白质二硫键， 二硫键的还原和异构化，过氧化氢的破坏等。这样做的目的是 该项目是对这些蛋白质及其与之相互作用的蛋白质进行全面的研究。 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，以及 它们的底物专一性是如何确定的。这些研究将集中在蛋白质DsbA上，这种蛋白质可以使 蛋白质中的二硫键，硫氧还蛋白和谷氧还蛋白上的二硫键，它们减少蛋白质中的二硫键， 另一种是过氧化还蛋白AhpCF，它可以破坏过氧化物质。我们还将描述抑制子突变 使缺失硫氧还蛋白家族细胞质成员的菌株恢复生长 还原反应。这样的研究已经并将使我们能够识别更多的蛋白质，这些蛋白质进行 二硫键还原或与这些还原蛋白相互作用的其他生理途径。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 那些它将以二硫键形式加入的分子。对于硫氧还蛋白和谷氧还蛋白，通过突变改变 我们将探索这些蛋白质如何识别它们的底物，并识别新的底物。 我们将利用一种新的蛋白质折叠方法来获得大量干扰 硫氧还蛋白的折叠，并揭示了这种蛋白质如何折叠成其三维结构。每个人 该项目的组成部分与公共卫生有关。二硫键蛋白质，如多肽 激素(胰岛素等)、用于治疗心脏疾病的蛋白质(TPA)和抗体可以产生更多 通过对大肠杆菌的基因操作，廉价和大量地生产。我们过去的研究导致了 细菌中产生的这些蛋白质的数量增加。硫氧还蛋白和谷氧还蛋白 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。对大肠杆菌中这一蛋白质家族的研究已经提供了基本的知识 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。