DELTAF508-CFTR TRAFFICKING REGULATED BY 4-PHENYLBUTYRATE

囊性纤维化跨膜传导调节因子（CFTR）最常见的突变是deltaF508-CFTR，它是一种运输突变，保留在内质网（ER）中，至少部分被泛素/蛋白酶体系统靶向快速细胞内降解。我们之前已经证明，这种运输缺陷可以通过药物剂4-苯基丁酸钠（4PBA）在体外和体内部分修复。然而，4PBA修复deltaF508-CFTR运输的机制尚不清楚，4PBA是一种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。鉴于这一证据，该提议的总体假设是，4PBA通过调节一种或多种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起重要作用的蛋白质来修复deltaF508-CFTR的细胞内运输。具体来说，我们将验证4PBA通过下调Hsc70的表达来修复deltaF508-CFTR细胞内运输的假设，Hsc70是70 kDa热休克蛋白家族的组成性表达成员，是许多细胞蛋白泛素化和蛋白酶体降解所必需的。在本建议中，针对以下主要具体目标的研究讨论了这一具体假设。(1)确定通过4PBA处理以外的其他方式特异性调节体外Hsc70表达是否会导致deltaF508-CFTR和野生型CFTR细胞内运输的改变。(2)通过检测4PBA对Hsc70蛋白和mRNA合成和降解动力学的影响，确定4PBA在蛋白和mRNA水平调控Hsc70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.