Cell Biology of CFTR in Polarized Epithelia

囊性纤维化（CF）是白种人中最常见的隐性遗传性疾病。CF由以下原因引起： 囊性纤维化跨膜传导调节因子（CFTR）基因中的突变，该基因编码氯离子， 频道CFTR表达于上皮细胞的顶膜，并调节盐和水 体内平衡CF最常见的形式是由508位的苯丙氨酸缺失引起的（<$F508）。 突变蛋白质被错误折叠并被ER相关降解（ERAD）机制降解。因为 F508 CFTR由于其发病率高，已成为蛋白质折叠疾病的流行模型。粤F508 CFTR是温度敏感的，它可以在27 ℃或通过化学伴侣被拯救到细胞表面， 在那里它保留了一些活性。已确定<$F508 CFTR的三个缺陷：1）无法正确折叠 并离开内质网; 2）拯救的蛋白质在细胞表面不稳定; 3）氯离子通道特性是 改变了虽然数千篇已发表的论文描述了CFTR（和<$F508 CFTR）的各个方面， 运输和功能，几乎所有这些都采用非生理表达环境，如成纤维细胞 细胞系相比之下，指导我们研究的基本原则是，模型的选择至关重要， 关于理解WT和<$F508 CFTR生物学。我们证明，贩运WT或 气道上皮细胞中的F508 CFTR在不表达CFTR的细胞中没有忠实地重现 自然.我们发现，<$F508 CFTR在极化的气道上皮细胞中迅速内化，而野生型CFTR在极化的气道上皮细胞中迅速内化。 类型（WT）CFTR不是。我们的假设是WT CFTR通常在顶面稳定， 与细胞骨架相关的蛋白质复合物的相互作用，并且F508 CFTR已经失去了这一点。 互动因此，我们建议在极化上皮细胞中研究WT和<$F508 CFTR生物学的关键方面。 细胞我们希望量化F508的运输缺陷，并了解突变体的机制。 在质膜上不稳定。根据我们的结果，先前选择的化学伴侣是 由于在气道上皮细胞中拯救<$F508 CFTR效率低下，我们鉴定了新的校正剂。我们正在评估 这些分子在相关模型中，如气道上皮细胞、Cftr <$F508小鼠和原代人， 支气管上皮细胞具体目的是：目的1：检验细胞表面运输 WT和Δ F508 CFTR之间的差异是由于它们与衔接子复合物的交替结合， 功能上重要的结合伙伴。这些差异是由于F508 CFTR错误折叠， 目的2：确定新的化学物质通过其降解泛素的机制。 监护人救援和纠正<$F508 CFTR。我们的目标是了解分子机制 识别质膜上的异常蛋白质。这项研究的结果与以下分析有关： 其他蛋白质折叠疾病

Cystic fibrosis (CF) is the most frequent recessive, hereditary disease in Caucasians. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes a chloride channel. CFTR is expressed in the apical membrane of epithelial cells and regulates salt and water homeostasis. The most common form of CF is caused by deletion of phenylalanine at position 508 (¿F508). The mutant protein is misfolded and degraded by the ER-associated degradative (ERAD) machinery. Because of its high degree of incidence, ¿F508 CFTR has become a popular model of protein folding diseases. ¿F508 CFTR is temperature-sensitive and it can be rescued to the cell surface at 27¿C or by chemical chaperones, where it retains some activity. Three defects have been identified for ¿F508 CFTR: 1) it fails to fold properly and exit the ER; 2) the rescued protein is unstable at the cell surface 3) the chloride channel properties are altered. While thousands of published papers have described various aspects of CFTR (and ¿F508 CFTR) trafficking and function, virtually all of these employed non-physiological expression contexts such as fibroblast cell lines. In contrast, the fundamental principle guiding our studies is that the choice of model is critical with regard to understanding both WT and ¿F508 CFTR biology. We demonstrate that trafficking of the WT or ¿F508 CFTR in airway epithelial cells is NOT faithfully recapitulated in cells that that do not express CFTR naturally. We show that ¿F508 CFTR is rapidly internalized in polarized airway epithelial cells, whereas wild type (WT) CFTR is not. Our hypothesis is that WT CFTR is normally stable at the apical surface through interactions with a protein complex associated with the cytoskeleton and that ¿F508 CFTR has lost this interaction. We therefore propose to study key aspects of WT and ¿F508 CFTR biology in polarized epithelial cells. We wish to quantify ¿F508 trafficking defects, and understand the mechanisms by which the mutant is destabilized at the plasma membrane. Based on our results that previously selected chemical chaperons are inefficient in rescuing ¿F508 CFTR in airway epithelial cells, we identified novel correctors. We are evaluating these molecules in relevant models such as airway epithelial cells, the Cftr¿F508 mouse, and primary human bronchial epithelial cells. The specific aims are: Aim 1: To test the hypothesis that the cell surface trafficking differences between WT and ¿F508 CFTR result from their alternative association with adaptor complexes and functionally significant binding partners. These differences are the result of ¿F508 CFTR misfolding and ubiquitin-dependent degradation; and Aim 2: To determine the mechanisms by which novel chemical chaperones rescue and correct ¿F508 CFTR. Our goal is to understand how the molecular machinery recognizes aberrant proteins at the plasma membrane. The results of this study are relevant in the analysis of other protein folding diseases.