Disulfide Bond Formation: Mechanisms for Isomerization and Novel Pathways

二硫键的形成：异构化机制和新途径

• 批准号: 7173909
• 负责人: JONATHAN BECKWITH
• 金额: $ 49.54万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7173909
• 关键词: Alkylation; Alteplase; Amino Acid Motifs; Amino Acids; Antibodies; Bacteria; Binding; Biochemical; Biochemical Genetics; Cadmium; Cell membrane; Cells; Communities; Complement; Complex; Cysteine; Cytoplasm; Cytoplasmic Protein; DNA Sequence Rearrangement; Defect; Detection; Development; Disruption; Disulfides; Electron Transport; Engineering; Enzymes; Escherichia coli; Genes; Genetic; Genetic Engineering; Genetic Screening; Grant; Growth; Homologous Gene; Human; Immunoglobulins; Insulin; Isomerase; Laboratories; Lead; Medical; Membrane; Membrane Proteins; Methods; Molecular Conformation; Mutation; Natural regeneration; Numbers; Object Attachment; Organism; Oxidation-Reduction; Pathway interactions; Peptides; Physiology; Procedures; Process; Production; Protein Disulfide Isomerase; Proteins; Proteolysis; Purpose; Rate; Research; Research Personnel; Resistance; Somatropin; Structural Protein; Structure; Substrate Specificity; Sulfur; Suppressor Mutations; System; Techniques; Technology; Testing; Thioredoxin; Work; X-Ray Crystallography; base; catalyst; cell motility; chemical bond; design; disulfide bond; disulfide bond reduction; enzyme substrate; gain of function; genetic selection; glutaredoxin; human tissue; insight; mutant; novel; oxidation; peptide hormone; periplasm; programs; therapeutic protein; three dimensional structure; tool

DESCRIPTION (provided by applicant): Project Summary: Proteins containing disulfide bonds are found in all organisms. These bonds are important for the folding of such proteins into its tertiary structure. Organisms have evolved protein disulfide isomerases which insure that the correct disulfide bonds are found in the final product. In the bacterium E. coli, the DsbC protein corrects incorrectly formed disulfide bonds. DsbC is regenerated as an active enzyme by the membrane protein DsbD. We will study the mechanisms of action of DsbC and DsbD using genetic, biochemical and structural approaches. Isolation and characterization of mutants of dsbC, genetic engineering of it and studies on its interaction with misoxidized substrates should yield information on the amino acids required for its functioning, structural features important for it to work properly and what aspects of disulfide bond formation necessitate the existence of such isomerases. DsbD transfers electrons across the cytoplasmic membrane from thioredoxin to the periplasmic DsbC by a disulfide bond reduction cascade. Isolation and characterization of mutants of the dsbD gene combined with biochemical studies on mutant proteins and structural analysis should illuminate the unusual electron transfer mechanism involving the membrane-embedded domain of DsbD. We will develop strains altered either in their cytoplasmic redox state or in components of the periplasm that influence disulfide bond formation. Strains obtained provide means of producing higher yields of disulfide-bonded proteins than normal bacterial strains. Medically important proteins- e.g. insulin, human growth hormone and antibodies- contain disulfide bonds essential for their activity. Understanding features of disulfide bond formation may lead to insights into the disruption of these proteins' activities in cases of malfunction. Understanding mechanisms involved in disulfide bond formation can enhance the ability to efficiently produce some of these proteins for medical purposes. Relevance: Many proteins important to human growth and physiology contain chemical bonds between the sulfurs of two cysteines. These disulfide-bonded proteins include a large number of peptide hormones (e.g. insulin) and immunoglobulins (antibodies). Understanding how disulfide bonds are formed correctly gives information allowing efficient production of these proteins for medical purposes.

项目简介：含二硫键的蛋白质存在于所有生物体中。这些键对于这些蛋白质折叠成三级结构很重要。生物体已经进化出蛋白质二硫异构酶，它确保在最终产物中找到正确的二硫键。在大肠杆菌中，DsbC蛋白纠正不正确形成的二硫键。DsbC被膜蛋白DsbD再生为一种活性酶。我们将从遗传、生化和结构等方面研究DsbC和DsbD的作用机制。分离和鉴定dsbC突变体，对其进行基因工程，研究其与误氧化底物的相互作用，将有助于了解其功能所需的氨基酸，对其正常工作至关重要的结构特征，以及二硫键形成的哪些方面需要这种异构酶的存在。DsbD通过二硫键还原级联将电子从硫氧还蛋白转移到质周DsbC。dsbD基因突变体的分离和鉴定，结合突变体蛋白的生化研究和结构分析，将有助于阐明dsbD不同寻常的膜内电子传递机制。我们将开发改变细胞质氧化还原状态或影响二硫键形成的周质成分的菌株。获得的菌株提供了比正常菌株产生更高产量的二硫结合蛋白的手段。医学上重要的蛋白质，如胰岛素、人类生长激素和抗体，都含有对其活性至关重要的二硫键。了解二硫键形成的特征可能会导致洞察这些蛋白质在故障情况下的活动破坏。了解二硫键形成的机制可以提高有效生产一些用于医疗目的的这些蛋白质的能力。相关性：许多对人体生长和生理很重要的蛋白质含有两个半胱氨酸硫之间的化学键。这些二硫结合蛋白包括大量肽激素（如胰岛素）和免疫球蛋白（抗体）。了解二硫键是如何正确形成的，可以提供信息，从而有效地生产用于医疗目的的这些蛋白质。