DELTAF508-CFTR TRAFFICKING REGULATED BY 4-PHENYLBUTYRATE

囊性纤维化跨膜电导调节因子(CFTR)最常见的突变是DeltaF508-CFTR，它是一种转运突变体，保留在内质网(ER)中，至少部分通过泛素/蛋白酶体系统进行细胞内快速降解。我们之前已经证明，这种转运缺陷可以在体外修复，部分可以在体内用药物4-苯丁酸钠(4PBA)修复。然而，4PBA修复DeltaF508-CFTR转运的机制尚不清楚，4PBA是一种已知的基因转录调节因子，尽管在体外有效浓度下，它不会显著改变CFTRmRNA水平。有了这一证据，这一提议的总体假设是，4PBA通过调节一种或多种蛋白质来修复deltaF508-CFTR的细胞内转运，这种蛋白质在调节新生蛋白质的折叠和靶向错误折叠的蛋白质进行细胞内降解方面起重要作用。具体地说，我们将检验这一假设，即4PBA通过下调Hsc70的表达来修复DeltaF508-CFTR的细胞内转运。Hsc70是70 kDa热休克蛋白家族中结构性表达的成员，对于许多细胞蛋白的泛素化和蛋白酶体降解是必需的。在针对以下主要具体目标的研究中，本提案处理了这一具体假设。(1)确定除4-PBA处理外，体外对Hsc70表达的特异性调节是否导致DeltaF508-CFTR和野生型CFTR细胞内转运的改变。(2)通过检测4PBA对Hsc70蛋白和信使核糖核酸合成和降解动力学的影响，从蛋白和mRNA水平探讨4PBA对Hsc70的调控机制。

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.