DeltaF508-CFTR Trafficking Regulated by 4-Phenylbutyrate

描述(申请人提供)：囊性纤维化跨膜电导调节剂(CFTR)是一种多功能蛋白质，既能通过上皮细胞的顶端质膜转运氯离子，又能调节其他蛋白质的离子转运，如上皮钠通道(ENaC)。囊性纤维化(CF)的一个基本特征是通过ENaC在呼吸道上皮细胞中的Na转运过度活跃，尽管这是由于CFTR缺失而导致的机制尚不清楚。对突变的CFTR功能进行药理修复的努力，例如使用4-苯丁酸钠(4PBA)来纠正最常见的突变CFTRF508-CFTR的转运，主要集中在评估突变CFTR的氯离子转运功能的恢复。这些研究往往忽视了4PBA或其他CFTR“校正者”对ENaC贩运和/或功能的影响。我们建议的研究将致力于药物修复或纠正F508-CFTR的贩运是否将促进在CF气道中对ENaC的适当调节。这是实施药理学策略以改善F508 CFTR的贩运和功能的关键问题。我们的假设是，4PBA诱导上皮细胞分子伴侣表达的调节，导致CFTR和ENaC在细胞内的转运和功能表达的改变。由于4PBA还改善了一些突变蛋白在除CF以外的疾病中的细胞内转运，这一假说也可能与开发其他一些蛋白质构象疾病的药物治疗密切相关。4PBA引起胞浆伴侣蛋白Hsc70和Hsp70以及一种新的29 kDa内质网蛋白ERp29的表达改变。我们的数据表明，这些伴侣蛋白的特异性表达改变调节了CFTR、F508和ENaC的运输和功能表达。本提案将建立在这些初步数据的基础上，并通过针对以下特定目标的研究来验证这一假说：特定目标1：确定Hsc70和Hsp70调控CFTR、F508和ENaC在上皮细胞中运输的机制。特异性目的2：探讨新型4-苯基丁酸酯调控的腔内内质网状蛋白ERp29调控CFTR、F508和ENaC在上皮细胞转运的机制。 与公共健康相关：肺和呼吸道保护自身免受环境影响的主要机制依赖于呼吸道上皮细胞中适当的离子运输。这种离子转运在囊性纤维化中是异常的，导致显著的发病率和死亡率。这些数据将促进更好地了解囊性纤维化和其他呼吸道疾病中负责离子运输的通道的调节，并为囊性纤维化的新的、基于机制的治疗方法的开发提供信息。

DESCRIPTION (provided by applicant): The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a multifunctional protein that both transports Cl- across the apical plasma membrane of epithelial cells and regulates ion transport by other proteins, such as the Epithelial Sodium Channel, ENaC. A cardinal feature of Cystic Fibrosis (CF) is hyperactivity of Na+ transport via ENaC in the airway epithelia, although the mechanism by which this results from the absence of CFTR is not known. Efforts at pharmacologic repair of mutant CFTR function, such as the use of Sodium 4- Phenylbutyrate (4PBA) to correct trafficking of the most common mutant CFTR, ?F508-CFTR, have concentrated on assessing restoration of a mutant CFTR's Cl- transport function. These studies have often ignored the influence of 4PBA or other CFTR "correctors" on ENaC trafficking and/or function. Our proposed studies will address whether pharmacologic repair, or correction of ?F508-CFTR trafficking will promote appropriate regulation of ENaC in the CF airway. This is a key issue in the implementation of pharmacologic strategies to improve ?F508 CFTR trafficking and function. Our hypothesis is that 4PBA induces modulations of molecular chaperone expression in epithelial cells that result in altered intracellular trafficking and functional expression of CFTR and ENaC. Because 4PBA also improves the intracellular trafficking of a number of mutant proteins in diseases besides CF, this hypothesis may also be germane to developing pharmacologic therapies for a number of other protein conformational diseases. 4PBA causes altered expression of the cytosolic chaperones Hsc70 and Hsp70, as well as a novel luminal endoplasmic reticular protein of 29 kDa, ERp29. Our data suggest that specifically altered expression of these chaperones modulates CFTR, ?F508 and ENaC trafficking and functional expression. The present proposal will build on these preliminary data and test this hypothesis with studies directed at the following Specific Aims: Specific Aim 1: To determine the mechanism by which Hsc70 and Hsp70 modulate CFTR, ?F508 and ENaC trafficking in epithelial cells. Specific Aim 2: To determine the mechanism by which ERp29, a novel 4-phenylbutyrate-regulated luminal endoplasmic reticular protein, regulates the trafficking of CFTR, ?F508 and ENaC in epithelial cells. PUBLIC HEALTH RELEVANCE: The major mechanim by which the lung and airway defends itself from the environment depends on proper ion transport in the respiratory epithelia. Such ion transport is aberrant in Cystic Fibrosis, leading to significant morbidity and mortality. These data will promote better understanding of the regulation of channels responsible for ion transport in Cystic Fibrosis and other diseases of the airway, and inform development of novel, mechanism-based therapies for Cystic Fibrosis.