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，它是一种运输突变体，保留了一些氯化物运输功能，但保留在内质网（ER）中，至少部分被泛素/蛋白酶体系统快速降解。由于deltaF508-CFTR保留了氯离子转运功能，我们和其他人已经验证了恢复或修复deltaF508-CFTR转运将恢复囊性纤维化（CF）上皮的CFTR功能的假设。最初，这些研究主要集中在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（如钠通过上皮钠通道（ENaC）和碳酸氢盐）的上皮转运中也具有关键功能。由于ENaC功能异常被认为是CF气道病理生理的关键，因此确定修复deltaF508-CFTR运输的药物对ENaC功能表达的影响对于评估这种deltaF508-CFTR修复策略的最终疗效和效用至关重要。DeltaF508-CFTR的运输缺陷可以在体外和体内通过药物剂4-苯基丁酸钠（4PBA）修复，这是一种已知的基因转录调节剂。然而，4PBA修复deltaF508-CFTR运输的机制及其对ENaC功能表达的影响尚不清楚。该提议的一般假设是，4PBA通过调节一个或多个在新生蛋白折叠中重要的蛋白，以及靶向错误折叠蛋白进行细胞内降解，来修复deltaF508-CFTR的细胞内运输。我们还将验证这样的假设，即4PBA对蛋白质折叠和运输的调节也会调节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：

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.