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，它是一个运输突变体，它保留了一些氯的运输功能，但保留在内质网(ER)中，并至少部分地被泛素/蛋白酶体系统用于细胞内的快速降解。因为deltaF508-CFTR保留了氯的转运功能，我们和其他人检验了这样的假设，即恢复或修复deltaF508-CFTR运输将恢复囊性纤维化(CF)上皮的CFTR功能。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在调节其他离子的上皮转运方面也具有关键功能，如通过上皮钠通道(ENaC)和碳酸氢盐的钠。由于ENaC功能亢进被认为在CF气道的病理生理学中起关键作用，因此确定修复deltaF508-CFTR转运的药物对ENaC功能表达的影响对于评估这种deltaF508-CFTR修复策略的最终有效性和实用性至关重要。DeltaF508-CFTR的转运缺陷可以通过药物4-苯丁酸钠(4PBA)在体外和体内部分修复，4PBA是一种已知的基因转录调节因子。然而，4PBA修复deltaF508-CFTR转运的机制及其对ENaC功能表达的影响尚不清楚。这一提议的一般假设是，4PBA通过调节一种或多种在新生蛋白折叠中起重要作用的蛋白质和靶向错误折叠的蛋白质进行细胞内降解，修复DeltaF508-CFTR的细胞内转运。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。本提案针对以下具体目标进行研究，以解决这些假设： 1)确定除4PBA处理外，体外对分子伴侣蛋白表达的特异性调控是否导致CFTR和deltaF508-CFTR细胞内转运的改变。 2)确定4PBA导致其细胞内效应的机制。 3)探讨分子伴侣的表达调控对ENaC细胞内转运和表达的影响。

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.