Cell Biology of CFTR in Polarized Epithelia

描述(申请人提供)：囊性纤维化(CF)是高加索人最常见的隐性遗传性疾病。囊性纤维化是由编码氯离子通道的囊性纤维化跨膜传导调节基因突变引起的。CFTR表达于上皮细胞的顶膜，调节盐分和水分的动态平衡。最常见的CF是由位置508(？F508)的苯丙氨酸缺失引起的。突变蛋白被内质网相关降解(ERAD)机制错误折叠和降解。由于其高发病率，？F508CFTR已成为蛋白质折叠疾病的流行模型。？F508CFTR对温度敏感，可在27℃或通过化学伴侣将其拯救到细胞表面，在那里它保持一定的活性。F508CFTR有三个缺陷：1)不能正确折叠并退出内质网；2)被拯救的蛋白在细胞表面不稳定；3)氯通道特性改变。虽然已发表的数千篇论文描述了CFTR(和F508 CFTR)运输和功能的各个方面，但几乎所有这些都使用了非生理性表达环境，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生物学至关重要。我们证明了WT或？F508 CFTR在呼吸道上皮细胞中的运输不能在自然不表达CFTR的细胞中忠实地重述。我们发现？F508CFTR能在极化的呼吸道上皮细胞中迅速内化，而野生型(WT)CFTR则不能。我们的假设是，WT CFTR通常通过与与细胞骨架相关的蛋白质复合体的相互作用在根尖表面保持稳定，而F508 CFTR已经失去了这种相互作用。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。我们希望量化？F508运输缺陷，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拯救呼吸道上皮细胞中的？F508 CFTR方面效率低下，我们确定了新的校正子。我们正在相关的模型中评估这些分子，如呼吸道上皮细胞、CFTRF508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F508CFTR的细胞表面转运差异是由于它们与接头复合体和功能显著的结合伙伴的交替结合所致的假说。这些差异是F508CFTR错误折叠和泛素依赖的降解的结果；目标2：确定新的化学伴侣拯救和纠正F508CFTR的机制。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。这项研究的结果对其他蛋白质折叠疾病的分析也是有意义的。 公共卫生相关性：蛋白质折叠缺陷是包括囊性纤维化在内的大量人类疾病的罪魁祸首。囊性纤维化中的蛋白质缺陷CFTR是研究折叠缺陷的纠正是否会导致正常功能的一个很好的模型。在拟议的研究中，我们正在检查这种氯离子通道的细胞表面稳定性，以及调节其细胞表面稳定性的细胞机制。

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.