MICROINJECTION STUDIES ON PROTEIN DEGRADATION

蛋白质降解的微注射研究

• 批准号: 3274575
• 负责人: MARTIN C RECHSTEINER
• 金额: $ 23.72万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-01-01 至 1994-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3274575
• 关键词: HeLa cells; albumins; arginine; autophagy; biological signal transduction; cell bank /registry; cell growth regulation; cell nucleus; cell type; chemical conjugate; chemical stability; conformation; cysteine; cytoplasm; deficient growth media; dyes; endopeptidases; enzyme mechanism; enzyme substrate analog; globin; immunochemistry; laboratory mouse; laboratory rabbit; lysozyme; microinjections; oxyhemoglobin; phenylhydrazines; proline; protease inhibitor; protein folding; protein metabolism; protein reconstitution; protein structure; proteolysis; radiotracer; stress proteins; temperature sensitive mutant; thermostability; tissue /cell culture; transport proteins; ubiquitin; western blottings

The long term objective of this proposal os to elucidate the mechanisms by which cells selectively degrade their intracellular proteins and to discover the features of protein that promote their catabolism. Two cell systems are available in which it is possible to eliminate either proline endopeptidase or ubiquitin (Ub) conjugation. Individual, radiolabeled proteins will be injected into each cell line to determine which proteins are substrates for these pathways. This procedure has already revealed that ubiquitin and bovine serum albumin ar stabilized in cells lacking proline endopeptidase and that oxidized hemoglobin is stabilized in ts85 mouse cells which contain a labile Ub-activating enzyme. The half-lives of a series of temperature-sensitive T4 lysozymes will also be measured to test the hypothesis that thermodynamic stability correlates with metabolic stability. Surprisingly, T4 lysozyme is degraded very rapidly in HeLa cells, and we will determine whether surface cysteines or arginine-arginine pairs are responsible for its short life. In addition, specific proteins will be targeted to the nucleus to discover whether their degradation rates are affected by mislocalization. Finally, ubiquitin will be injected into ts85 mouse cells or HeLa cells and several aspects of Ub metabolism will be measured, including Ub's role in autophagy and release of injected proteins, as well as the subcellular location of Ub conjugates before and after heat-shock.

本建议的长期目标是通过以下方式阐明这些机制