CFTR DEGRADATION IN THE ENDOPLASMIC RETICULUM

内质网中的 CFTR 降解

• 批准号: 6110238
• 负责人: ALAN Michael TARTAKOFF
• 金额: --
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6110238
• 关键词: Saccharomyces cerevisiae; chloride channels; cystic fibrosis; endoplasmic reticulum; gene expression; gene mutation; lung disorder; membrane transport proteins; molecular cloning; mutant; protein degradation

This project will test the hypothesis that some forms of CFTR are degraded in intracellular compartments. The initial experiments will be conducted in yeast, capitalizing on a series of mutants which halt processing of molecules at various stages in the endoplasmic reticulum (with or without vesicle formation), Golgi, or beyond. Wild type and mutant (deltaF508 and others) CFTR either in native form, containing a FLAG sequence for ease of immunologic identification, or fused to beta- galactosidase for ease of reporting and later genetic analysis will be followed along the processing sequence using sec, vps, erd, and kar2 mutants. The beta-galactosidase fusions will be utilized to facilitate screening for mutants which fail to process or degrade CFTR. The genes which are defective in the mutants will be cloned from yeast, and human homologs sought. The nature of the gene products will be determined and means of inhibiting them devised. These experiments will determine at which stage in protein processing CFTR fails to be properly processed and is degraded and will isolate the protein(s) responsible. If, as predicted by the work of Cheng and coworkers, a major cause of cystic fibrosis is the defective processing and degradation of mutant forms of CFTR, so that proper location at the plasma membrane is never achieved, these results might permit us to design interventions to permit processing and escape of some mutant protein to the membrane, where it may be possible to activate it and restore function.

该项目将测试的假设，某些形式的CFTR是 在细胞内降解。 最初的实验将是 在酵母中进行，利用一系列突变体， 内质网中不同阶段的分子加工 (with或没有囊泡形成）、高尔基体或更远。 野生型和 突变体（deltaF 508和其他）CFTR，其为天然形式，含有 FLAG序列，以便于免疫学鉴定，或融合到β- 半乳糖苷酶，以便于报告和以后的遗传分析， 沿着使用sec、vps、erd和kar 2的处理序列 变种人 β-半乳糖苷酶融合体将用于促进 筛选不能加工或降解CFTR的突变体。 的基因 突变体中的缺陷将从酵母中克隆出来， 寻找同类 基因产物的性质将被确定， 抑制它们的方法被设计出来。 这些实验将决定在 CFTR蛋白质加工的哪个阶段不能被正确加工， 被降解并将分离负责的蛋白质。 如果像 通过程和同事的工作预测，囊性变的一个主要原因是 纤维化是一种有缺陷的加工和降解的突变形式， CFTR，使得在质膜上的适当位置永远无法实现， 这些结果可能使我们能够设计干预措施， 一些突变蛋白质的加工和逃逸到膜上， 也许可以激活它并恢复功能。