DeltaF508-CFTR Trafficking Regulated by 4-Phenylbutyrate

DeltaF508-CFTR 贩运受 4-苯基丁酸酯监管

• 批准号: 7154118
• 负责人: Ronald C Rubenstein
• 金额: $ 31.16万
• 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/7154118
• 关键词: 3' Untranslated Regions; Address; Bicarbonates; Chloride Ion; Chlorides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Defect; Endoplasmic Reticulum; Epithelial; Epithelium; Functional disorder; Genetic Transcription; Heat shock proteins; In Vitro; Investigation; Ions; Lead; Messenger RNA; Molecular Chaperones; Mutation; Numbers; Pharmacologic Substance; Protein Family; Proteins; Regulation; Sodium; Sodium Channel; Sodium phenylbutyrate; Stress; System; Testing; Transcription Factor 3; Ubiquitin; Ubiquitination; cystic fibrosis airway; deltaF508-CFTR protein; epithelial Na+ channel; improved; in vivo; mRNA Stability; member; multicatalytic endopeptidase complex; mutant; novel; phenylbutyrate; protein folding; protein misfolding; repaired; response; restoration; trafficking

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剂（CFTR）的最常见突变，Deltaf508-CFTR是一种运输突变体，保留了某些氯化物的运输功能，但在内质网（ER）中保留，并靶向快速的细胞内脱落，至少在ubiquiquipItin/protipin/proticememe protipin/protipin protipin/protipin protins/prot。由于Deltaf508-CFTR保留了氯化物的运输功能，因此我们和其他人研究了以下假设：Deltaf508-CFTR运输的恢复或修复将使CFTR功能恢复为囊性纤维化（CF）上皮。最初，这些研究集中在CFTR的氯化物运输功能上，但最近的证据表明，CFTR在调节其他离子的上皮运输方面也具有关键功能，例如通过上皮钠通道（ENAC）和二氯酸钠和碳酸氢盐。由于认为ENAC的过度功能在CF气道的病理生理中至关重要，因此确定了修复Deltaf508-CFTR运输对ENAC功能表达的影响的影响对于评估这种Deltaf508-CFTR修复策略的最终功效和实用性至关重要。 Deltaf508-CFTR的运输缺陷可以在体外修复，并由已知的基因转录调节剂4-苯基丁酸钠（4PBA）在体内进行部分修复。但是，既不知道4PBA维修Deltaf508-CFTR运输的机制，也不知道其对ENAC功能表达的影响。该提议的一般假设是，通过调节对折叠新生蛋白的折叠和靶向错误折叠的蛋白质以使细胞内降解的蛋白质或蛋白质对deltaf508-CFTR的细胞内运输进行修复。我们还将检验以下假设：这种调节蛋白质折叠和4PBA运输的调节还将调节ENAC的功能表达。本提案通过针对以下特定目的的研究来解决这些假设： 1）确定分子伴侣在体外表达的特异性调节是否通过4PBA治疗以外的其他方式导致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。