DELTAF508-CFTR TRAFFICKING REGULATED BY 4-PHENYLBUTYRATE

DELTAF508-CFTR 贩运受 4-苯基丁酸酯管制

• 批准号: 6885741
• 负责人: Ronald C Rubenstein
• 金额: $ 7.24万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2004-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6885741
• 关键词: Xenopus oocyte; chloride channels; endoplasmic reticulum; heat shock proteins; intracellular transport; phenylbutyrates; protein degradation; protein folding; protein protein interaction; protein structure function; protein transport

The most common mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), deltaF508-CFTR, is a trafficking mutant that is retained in the endoplasmic reticulum (ER) and targeted for rapid intracellular degradation, at least in part by the ubiquitin/proteasome system. We have previously demonstrated that this trafficking defect can be repaired in vitro, and partially in vivo, by the pharmaceutical agent Sodium 4- Phenylbutyrate (4PBA). However, the mechanism by which 4PBA repairs deltaF508-CFTR trafficking is not known, 4PBA is a known regulator of gene transcription, although, at effective concentrations in vitro, it does not significantly alter CFTR mRNA levels. Given this evidence, the overall hypothesis of this proposal is that 4PBA repairs the intracellular trafficking of deltaF508-CFTR by regulation of a protein or proteins important in the regulation of folding of nascent proteins and targeting of misfolded proteins for intracellular degradation. Specifically, we will test the hypothesis that 4PBA repairs deltaF508-CFTR intracellular trafficking by down-regulating the expression of Hsc70, the constitutively expressed member of the 70 kDa heat shock protein family that is necessary for the ubiquitination and proteasome degradation of a number of cellular proteins. This specific hypothesis is addressed in the present proposal in the studies directed at the following principal specific aims. (1) To determine whether specific modulation of Hsc70 expression in vitro by means other than 4PBA treatment results in alterations of deltaF508-CFTR and wild type CFTR intracellular trafficking. (2) To determine the mechanism by which 4PBA regulates Hsc70 at the protein and mRNA level by examining the influence of 4PBA on the kinetics of synthesis and degradation of Hsc70 protein and mRNA.

囊性纤维化跨膜电导调节剂（CFTR）的最常见突变，Deltaf508-CFTR是一种运输突变体，保留在内质网中（ER），靶向快速的细胞内降解，至少是由泛素/蛋白酶体系统组成的部分。 。 我们以前已经证明，这种运输缺陷可以在体外修复，并由4-苯基丁酸钠（4PBA）在体外修复，部分在体内修复。 然而，尚不清楚4PBA修复Deltaf508-CFTR运输的机制，4PBA是基因转录的已知调节剂，尽管在有效的体外浓度下，它并没有显着改变CFTR mRNA水平。 鉴于这一证据，该提案的总体假设是4PBA通过调节对新生蛋白质折叠的蛋白质或蛋白质的调节来修复Deltaf508-CFTR的细胞内运输，并靶向错误折叠的蛋白质用于内细胞内降解。 具体而言，我们将检验以下假设：4PBA维修Deltaf508-CFTR细胞内贩运通过下调HSC70的表达，HSC70的表达是70 kDa热休克蛋白家族的组成型成员，这对于泛素化和泛质量降解了许多细胞的蛋白质蛋白质是必不可少的蛋白质。 本提案中针对以下主要特定目的的研究中解决了这一具体假设。 （1）确定除4PBA治疗以外的其他方式中HSC70表达的特定调节是否会导致Deltaf508-CFTR和野生型CFTR细胞内运输的改变。 （2）确定4PBA通过检查4PBA对HSC70蛋白质和mRNA的合成和降解动力学的影响来调节蛋白质和mRNA水平的HSC70的机制。