DeltaF508-CFTR Trafficking Regulated by 4-Phenylbutyrate

DeltaF508-CFTR 贩运受 4-苯基丁酸酯监管

• 批准号: 6865050
• 负责人: Ronald C Rubenstein
• 金额: $ 32.87万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6865050
• 关键词: RNA interference; Xenopus; chloride channels; cystic fibrosis; gel mobility shift assay; heat shock proteins; intracellular transport; luminescence; messenger RNA; molecular chaperones; phenylbutyrates; protein folding; protein transport; sodium channel; western blottings

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.

囊性纤维化跨膜电导调节器 (CFTR) 最常见的突变 deltaF508-CFTR 是一种运输突变体，它保留了一些氯离子转运功能，但保留在内质网 (ER) 中，并针对细胞内快速降解，至少部分是通过泛素/蛋白酶体系统进行的。由于 deltaF508-CFTR 保留了氯离子转运功能，我们和其他人研究了以下假设：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，例如通过上皮钠通道 (ENaC) 的钠和碳酸氢盐。由于假设 ENaC 功能亢进在 CF 气道的病理生理学中至关重要，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此类 deltaF508-CFTR 修复策略的最终功效和实用性至关重要。 DeltaF508-CFTR 的运输缺陷可以通过药剂 4-苯基丁酸钠 (4PBA)（一种已知的基因转录调节剂）在体外和部分体内修复。然而，4PBA 修复 deltaF508-CFTR 运输的机制及其对 ENaC 功能表达的影响均未知。该提议的一般假设是，4PBA 通过调节在新生蛋白质折叠中重要的一个或多个蛋白质以及靶向错误折叠蛋白质进行细胞内降解来修复 deltaF508-CFTR 的细胞内运输。我们还将测试这样的假设：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 4PBA 处理以外的方式对体外分子伴侣表达的特异性调节是否会导致 CFTR 和 deltaF508-CFTR 细胞内运输的改变。 2) 确定4PBA导致其细胞内效应的机制。 3)评估分子伴侣表达调节对ENaC细胞内运输和表达的影响。