DELTAF508-CFTR TRAFFICKING REGULATED BY 4-PHENYLBUTYRATE

DELTAF508-CFTR 贩运受 4-苯基丁酸酯监管

• 批准号: 6524276
• 负责人: Ronald C Rubenstein
• 金额: $ 25.5万
• 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 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6524276
• 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器 (CFTR) 最常见的突变 deltaF508-CFTR 是一种运输突变体，保留在内质网 (ER) 中，并针对细胞内快速降解，至少部分是通过泛素/蛋白酶体系统进行的。 我们之前已经证明，这种运输缺陷可以通过药剂 4-苯基丁酸钠 (4PBA) 在体外修复，并部分在体内修复。 然而，4PBA 修复 deltaF508-CFTR 运输的机制尚不清楚，4PBA 是已知的基因转录调节因子，尽管在体外有效浓度下，它不会显着改变 CFTR mRNA 水平。 鉴于这一证据，该提议的总体假设是4PBA通过调节在调节新生蛋白折叠和靶向错误折叠蛋白进行细胞内降解中重要的一个或多个蛋白来修复deltaF508-CFTR的细胞内运输。 具体来说，我们将测试以下假设：4PBA 通过下调 Hsc70 的表达来修复 deltaF508-CFTR 细胞内运输，Hsc70 是 70 kDa 热休克蛋白家族的组成型表达成员，对于许多细胞的泛素化和蛋白酶体降解是必需的。蛋白质。 这一具体假设在针对以下主要具体目标的研究中的当前提案中得到解决。 (1) 确定通过 4PBA 处理以外的方式体外特异性调节 Hsc70 表达是否会导致 deltaF508-CFTR 和野生型 CFTR 细胞内运输的改变。 (2)通过考察4PBA对Hsc70蛋白和mRNA合成和降解动力学的影响，探讨4PBA在蛋白和mRNA水平调控Hsc70的机制。