Cell Biology of CFTR in Polarized Epithelia

描述（由申请人提供）：囊性纤维化（CF）是高加索人中最常见的隐性遗传性疾病。CF是由编码氯离子通道的囊性纤维化跨膜传导调节（CFTR）基因突变引起的。CFTR表达于上皮细胞的顶膜，调节盐和水的稳态。最常见的CF是由508位苯丙氨酸的缺失引起的（?F508）。突变蛋白被错误折叠并被er相关降解（ERAD）机制降解。由于发病率高，？F508 CFTR已成为蛋白质折叠疾病的流行模型。？F508 CFTR对温度敏感，它可以在27℃时被拯救到细胞表面，或者通过化学伴侣，在那里它保持一些活性。确定了三个缺陷？F508 CFTR: 1)折叠不正常，退出ER；2)获救的蛋白质在细胞表面不稳定3)氯离子通道性质改变。虽然已发表的数千篇论文描述了CFTR的各个方面(和？F508 CFTR)的转运和功能，几乎所有这些都采用非生理表达环境，如成纤维细胞系。相反，指导我们研究的基本原则是，模型的选择对于理解WT和？F508 CFTR生物学。我们能证明走私野生动物还是？气道上皮细胞中的F508 CFTR在不自然表达CFTR的细胞中不能忠实地再现。我们展示了吗？F508 CFTR在极化气道上皮细胞中迅速内化，而野生型（WT） CFTR则不然。我们的假设是，通过与细胞骨架相关的蛋白质复合物的相互作用，WT CFTR在根尖表面通常是稳定的。F508 CFTR失去了这种相互作用。因此，我们建议研究WT和？F508 CFTR在极化上皮细胞中的生物学研究。我们想要量化？F508转运缺陷，并了解突变体在质膜上不稳定的机制。根据我们的结果，先前选择的化学伴侣在营救中是无效的？在气道上皮细胞F508 CFTR中，我们发现了新的校正因子。我们正在相关模型中评估这些分子，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：验证WT和？F508 CFTR是由它们与接头复合物和功能重要的结合伙伴的替代结合而产生的。这些差异是什么造成的？F508 CFTR错误折叠和泛素依赖性降解；目的2：确定新型化学伴侣拯救和纠正的机制。F508雌性生殖道。我们的目标是了解分子机制如何识别质膜上的异常蛋白质。本研究结果对其他蛋白质折叠疾病的分析具有一定的参考价值。

DESCRIPTION (provided by applicant): 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 27oC 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. PUBLIC HEALTH RELEVANCE: Protein folding defects are responsible for a large number of human diseases including cystic fibrosis. CFTR, the protein defective in cystic fibrosis, is an excellent model to investigate whether correction of the folding defect will lead to proper function. In the proposed studies, we are examining the cell surface stability of this chloride channel and the cellular mechanisms that regulate its cell surface stability.