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因子（CFTR）基因突变引起的。CFTR在上皮细胞的顶膜中表达，并调节盐和水的稳态。最常见的CF形式是由508位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。由于其发病率高，？F508 CFTR已成为蛋白质折叠疾病的流行模型。? F508 CFTR是温度敏感的，它可以在27 ℃或通过化学分子伴侣被拯救到细胞表面，在那里它保留了一些活性。已识别出3个缺陷？F508 CFTR：1）它不能正确折叠并离开ER; 2）拯救的蛋白质在细胞表面不稳定; 3）氯离子通道特性改变。虽然数千篇已发表的论文描述了CFTR的各个方面（和？F508 CFTR）运输和功能，实际上所有这些都采用非生理表达环境，如成纤维细胞系。相比之下，指导我们研究的基本原则是，模型的选择是至关重要的理解WT和？F508 CFTR生物学。我们证明，贩运野生型或？气道上皮细胞中的F508 CFTR在天然不表达CFTR的细胞中没有忠实地重现。我们展示了吗？F508 CFTR在极化的气道上皮细胞中迅速内化，而野生型（WT）CFTR不是。我们的假设是WT CFTR通常通过与细胞骨架相关的蛋白质复合物的相互作用在顶端表面稳定，并且？F508 CFTR失去了这种相互作用。因此，我们建议研究WT和？极化上皮细胞中的F508 CFTR生物学。我们要量化？F508运输缺陷，并了解突变体在质膜上不稳定的机制。根据我们的研究结果，以前选择的化学伴侣是无效的救援？F508 CFTR在气道上皮细胞，我们确定了新的校正。我们正在评估这些分子在相关模型，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WT和？F508 CFTR由它们与衔接子复合物和功能上重要的结合配偶体的替代性缔合产生。这些差异是由于？F508 CFTR错误折叠和泛素依赖性降解;目的2：确定新的化学分子伴侣拯救和纠正？F508 CFTR。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。本研究的结果在其他蛋白质折叠疾病的分析中是相关的。 公共卫生相关性：蛋白质折叠缺陷是导致包括囊性纤维化在内的大量人类疾病的原因。CFTR是囊性纤维化中的蛋白缺陷，是研究折叠缺陷的纠正是否会导致适当功能的极好模型。在拟议的研究中，我们正在研究这种氯离子通道的细胞表面稳定性和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.