DELTAF508-CFTR TRAFFICKING REGULATED BY 4-PHENYLBUTYRATE

囊性纤维化跨膜传导调节因子（CFTR）最常见的突变，deltaF 508-CFTR，是一种运输突变体，其保留在内质网（ER）中并靶向快速细胞内降解，至少部分通过泛素/蛋白酶体系统。 我们以前已经证明，这种运输缺陷可以在体外修复，部分在体内，由药剂4-苯基丁酸钠（4PBA）。 然而，4PBA修复deltaF 508-CFTR运输的机制尚不清楚，4PBA是已知的基因转录调节剂，尽管在体外有效浓度下，它不会显著改变CFTR mRNA水平。 考虑到这一证据，该提议的总体假设是4PBA通过调节在调节新生蛋白质折叠和靶向错误折叠的蛋白质以进行细胞内降解中重要的一种或多种蛋白质来修复deltaF 508-CFTR的细胞内运输。 具体来说，我们将测试的假设，4PBA修复deltaF 508-CFTR细胞内运输下调Hsc 70的表达，组成型表达的70 kDa的热休克蛋白家族的成员，是必要的泛素化和蛋白酶体降解的细胞蛋白。 本提案在针对以下主要具体目标的研究中探讨了这一具体假设。(1)为了确定通过除4PBA处理以外的手段体外特异性调节Hsc 70表达是否导致deltaF 508-CFTR和野生型CFTR细胞内运输的改变。(2)通过检测4-PBA对Hsc 70蛋白和mRNA合成和降解动力学的影响，从蛋白和mRNA水平探讨4-PBA调控Hsc 70的机制。

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.