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Cell Biology of CFTR in Polarized Epithelia

极化上皮细胞 CFTR 的细胞生物学

基本信息

批准号：
8460501
负责人：
JAMES F. COLLAWN
金额：
$ 30.14万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-12-20 至 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8460501
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 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 research study trafficking ubiquitin-protein ligase

项目摘要

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.

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无法有效拯救气道上皮细胞中的µ F508 CFTR，我们发现了新型纠正剂。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

项目成果

期刊论文数量（16）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Regulation of angiogenesis by hypoxia: the role of microRNA.

DOI：
10.2478/s11658-012-0037-0
发表时间：
2013-03
期刊：
Cellular & molecular biology letters
影响因子：
8.3
作者：
Madanecki P;Kapoor N;Bebok Z;Ochocka R;Collawn JF;Bartoszewski R
通讯作者：
Bartoszewski R

Unlocking the mysteries of Na+-K+-ATPase endocytosis: phosphorylation is the key.

解开Na-K-ATP酶内吞作用的奥秘：磷酸化是关键。

DOI：
10.1165/rcmb.f317
发表时间：
2006
期刊：
American journal of respiratory cell and molecular biology
影响因子：
6.4
作者：
Collawn,JamesF
通讯作者：
Collawn,JamesF

Regulation of the unfolded protein response by microRNAs.

DOI：
10.2478/s11658-013-0106-z
发表时间：
2013-12
期刊：
Cellular & molecular biology letters
影响因子：
8.3
作者：
Bartoszewska S;Kochan K;Madanecki P;Piotrowski A;Ochocka R;Collawn JF;Bartoszewski R
通讯作者：
Bartoszewski R

miR-429 regulates the transition between Hypoxia-Inducible Factor (HIF)1A and HIF3A expression in human endothelial cells.

DOI：
10.1038/srep22775
发表时间：
2016-03-08
期刊：
Scientific reports
影响因子：
4.6
作者：
Janaszak-Jasiecka A;Bartoszewska S;Kochan K;Piotrowski A;Kalinowski L;Kamysz W;Ochocka RJ;Bartoszewski R;Collawn JF
通讯作者：
Collawn JF

Targets for cystic fibrosis therapy: proteomic analysis and correction of mutant cystic fibrosis transmembrane conductance regulator.