Cell Biology of CFTR in Polarized Epithelia

极化上皮细胞 CFTR 的细胞生物学

• 批准号: 7745379
• 负责人: JAMES F. COLLAWN
• 金额: $ 34.2万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-12-20 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7745379
• 关键词: Actins; Apical; Binding; Biochemical; Biogenesis; Biology; Caucasians; Caucasoid Race; Cell Line; Cell membrane; Cell surface; Cells; Cellular biology; Chemicals; Chloride Channels; Complex; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytoskeleton; Data; Defect; Development; Disease; Endocytosis; Endoplasmic Reticulum; Epithelial Cells; Epithelium; Fibroblasts; Goals; Hereditary Disease; Homeostasis; Human; Incidence; Intervention; Lead; Measures; Mediating; Messenger RNA; Methods; Modeling; Molecular; Molecular Chaperones; Mus; Mutation; Nose; Paper; Phenylalanine; Physiological; Physiology; Positioning Attribute; Process; Property; Protein-Folding Disease; Proteins; Publications; Publishing; Quality Control; Regulation; Regulator Genes; Role; Series; Small Interfering RNA; Sodium Chloride; Surface; TFAP2A gene; Temperature; Testing; Transcription Factor AP-2 Alpha; Ubiquitin; Ubiquitination; Water; airway epithelium; apical membrane; base; cell type; cold temperature; cystic fibrosis patients; design; disease-causing mutation; experience; high throughput screening; human disease; in vivo; mouse model; mutant; novel; protein complex; protein folding; protein misfolding; public health relevance; research study; trafficking; ubiquitin-protein ligase

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.

描述（由申请人提供）：囊性纤维化（CF）是白种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电导调节因子 (CFTR) 基因突变引起的。 CFTR 在上皮细胞的顶膜中表达，调节盐和水的稳态。最常见的 CF 形式是由 508 位苯丙氨酸 (?F508) 缺失引起的。突变蛋白被 ER 相关降解 (ERAD) 机制错误折叠和降解。由于其高发病率，?F508 CFTR 已成为蛋白质折叠疾病的流行模型。 ?F508 CFTR 对温度敏感，可以在 27°C 或化学伴侣的作用下被拯救到细胞表面，并保留一些活性。已发现 ?F508 CFTR 存在三个缺陷：1）它无法正确折叠并退出急诊室； 2) 被拯救的蛋白质在细胞表面不稳定 3) 氯离子通道特性发生改变。虽然数千篇已发表的论文描述了 CFTR（和 ?F508 CFTR）运输和功能的各个方面，但实际上所有这些论文都采用了非生理表达环境，例如成纤维细胞系。相比之下，指导我们研究的基本原则是模型的选择对于理解 WT 和 ?F508 CFTR 生物学至关重要。我们证明，气道上皮细胞中 WT 或 ?F508 CFTR 的运输并未在不自然表达 CFTR 的细胞中忠实地重现。我们发现 ?F508 CFTR 在极化气道上皮细胞中快速内化，而野生型 (WT) CFTR 则不然。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相互作用而在顶端表面保持稳定，而 ?F508 CFTR 已失去这种相互作用。因此，我们建议研究极化上皮细胞中 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处不稳定的机制。基于我们的结果，即先前选择的化学伴侣在拯救气道上皮细胞中的?F508 CFTR方面效率低下，我们确定了新的校正剂。我们正在相关模型（例如气道上皮细胞、Cftr?F508 小鼠和原代人支气管上皮细胞）中评估这些分子。具体目标是： 目标 1：检验以下假设：WT 和 ?F508 CFTR 之间的细胞表面运输差异是由于它们与衔接子复合物和功能上重要的结合配偶体的选择性关联造成的。这些差异是 ?F508 CFTR 错误折叠和泛素依赖性降解的结果；目标 2：确定新型化学伴侣拯救和纠正 ?F508 CFTR 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。这项研究的结果与其他蛋白质折叠疾病的分析相关。 公共健康相关性：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 是囊性纤维化中的缺陷蛋白，是研究纠正折叠缺陷是否会导致正常功能的绝佳模型。在拟议的研究中，我们正在检查该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。