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deltaF 508-CFTR，它是一种运输突变体，保留了一些氯离子转运功能，但保留在内质网（ER）中，并至少部分通过泛素/蛋白酶体系统靶向快速细胞内降解。因为deltaF 508-CFTR保留了氯离子转运功能，我们和其他人已经检验了deltaF 508-CFTR运输的恢复或修复将恢复囊性纤维化（CF）上皮的CFTR功能的假设。最初，这些研究集中在CFTR的氯离子转运功能上，但最近的证据表明，CFTR在调节其他离子的上皮转运中也具有关键功能，例如通过上皮钠通道（ENaC）和碳酸氢盐的钠。由于假设ENaC的功能亢进在CF气道的病理生理学中是关键的，因此确定修复deltaF 508-CFTR运输的试剂对ENaC功能表达的影响在评估此类deltaF 508-CFTR修复策略的最终功效和效用中是关键的。DeltaF 508-CFTR的运输缺陷可以在体外和部分在体内通过药物4-苯基丁酸钠（4PBA）（一种已知的基因转录调节剂）修复。然而，4PBA修复deltaF 508-CFTR运输的机制及其对ENaC功能表达的影响都是未知的。该提议的一般假设是，4PBA通过调节在新生蛋白质的折叠中重要的一种或多种蛋白质以及靶向错误折叠的蛋白质以进行细胞内降解来修复deltaF 508-CFTR的细胞内运输。我们还将检验这样的假设，即4PBA对蛋白质折叠和运输的调节也将调节ENaC的功能表达。本提案通过针对以下具体目标的研究来解决这些假设： 1)确定通过除4PBA处理以外的方法在体外特异性调节分子伴侣的表达是否导致CFTR和Δ F508-CFTR细胞内运输的改变。 2)探讨4PBA的细胞内效应机制。 3)评估分子伴侣表达的调节对ENaC细胞内运输和表达的影响。

The most common mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), deltaF508-CFTR, is a trafficking mutant that retains some chloride transport function but is retained in the endoplasmic reticulum (ER) and targeted for rapid intracellular degradation, at least in part by the ubiquitin/proteasome system. Because deltaF508-CFTR retains chloride transport function, we, and others have examined the hypothesis that restoration or repair of deltaF508-CFTR trafficking will restore CFTR function to Cystic Fibrosis (CF) epithelia. Initially these investigations have concentrated on CFTR's chloride transport function, but recent evidence suggests that CFTR also has critical functions in regulating epithelial transport of other ions, such as sodium via the epithelial sodium channel (ENaC) and bicarbonate. As hyperfunction of ENaC is hypothesized to be critical in the pathophysiology of the CF airway, determining the influence of agents that repair deltaF508-CFTR trafficking on ENaC functional expression is critical in evaluating the eventual efficacy and utility of such deltaF508-CFTR repair strategies. DeltaF508-CFTR's trafficking defect can be repaired in vitro and partially in vivo by the pharmaceutical agent sodium 4- phenylbutyrate (4PBA), a known regulator of gene transcription. However, neither the mechanism by which 4PBA repairs deltaF508-CFTR trafficking, nor its effects on ENaC functional expression are known. The general hypothesis of this proposal is that 4PBA repairs the intracellular trafficking of deltaF508-CFTR by regulation of a protein or proteins important in the folding of nascent proteins and targeting of misfolded proteins for intracellular degradation. We will also test the hypothesis that such regulation of protein folding and trafficking by 4PBA will also modulate the functional expression of ENaC. The present proposal addresses these hypotheses with studies directed at the following Specific Aims: 1) To determine whether specific modulation of expression of molecular chaperones in vitro by means other than 4PBA treatment results in alterations in CFTR and deltaF508-CFTR intracellular trafficking. 2) To determine the mechanism by which 4PBA leads to it intracellular effects. 3) To assess the effect of modulation of expression of molecular chaperones on ENaC intracellular trafficking and expression.