CFTR Biogenesis and Function in Epithelia

CFTR 上皮细胞的生物发生和功能

• 批准号: 7652912
• 负责人: ZSUZSA BEBOK
• 金额: $ 36.57万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7652912
• 关键词: 5' Untranslated Regions; Affect; Amino Acid Sequence; Apical; Binding; Biogenesis; Cell physiology; Cell surface; Cellular Stress Response; Chloride Channels; Chronic Obstructive Airway Disease; Complex; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; DNA; Defect; Degradation Pathway; Disease; Endoplasmic Reticulum; Epithelial; Epithelial Cells; Epithelium; Exhibits; Family suidae; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Hereditary Disease; Histone Deacetylation; Human; Hypermethylation; Lead; Lung; Messenger RNA; Modeling; Molecular; Molecular Chaperones; Molecular Profiling; Mus; Mutation; Nucleotides; Other Genetics; Pathogenesis; Pathology; Pathway interactions; Point Mutation; Process; Proteins; Regulation; Reporting; Repression; Role; Stress; Structure; Surface; Symptoms; Testing; Translations; Variant; Work; activating transcription factor; base; biological adaptation to stress; cigarette smoking; endoplasmic reticulum stress; gene repression; improved; mutant; novel; promoter; protein folding; protein misfolding; public health relevance; response

DESCRIPTION (provided by applicant): The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel expressed on the apical surface of epithelial cells and is defective in cystic fibrosis (CF). Our previous project concentrated on the biogenesis of the wild type (WT) and the most common disease causing mutant, ?F508 CFTR. Based on compelling preliminary results, this renewal application focuses on two major topics: 1) the molecular mechanisms by which the unfolded protein response (UPR) regulates gene expression; and 2) the consequences of mRNA secondary structure alterations generated by point mutations using CFTR as our model. We found that the UPR suppresses endogenous WT CFTR transcription, translation, maturation efficiency and function. We have localized the UPR-associated transcriptional repression to the minimal promoter of CFTR. The mechanism of repression involves DNA hypermethylation, histone deacetylation and the binding of ATF6, the UPR-activated transcription factor, to the CFTR minimal promoter. Interestingly, the UPR-associated transcriptional repression affects only a limited number of genes. Regarding the mechanism of decreased CFTR maturation during the UPR, we show that the expression of Hsp90, a pro-folding chaperone of CFTR, is decreased during the UPR. In contrast, the expression of pro-degradation factors increases under the same conditions. Furthermore, the importance of mRNA structure in disease pathogenesis has only recently been recognized. Thus, a very significant finding in our preliminary studies is that the popular model of protein folding disorders, ?F508 CFTR, also exhibits mRNA structural defects. Specifically, the 3- base deletion creates two enlarged hairpin loops in the NBD1 encoding region that slow down translation. This provides the intriguing possibility that F508 CFTR folding, at least to some extent, is compromised because of decreased translation rate caused by mRNA "misfolding". This aspect of ?F508 CFTR biogenesis has not been examined previously, and has potentially far-reaching implications in other genetic diseases. Our hypotheses are: 1) ER stress activates the UPR which selectively represses the transcription of genes through a promoter-specific mechanism; 2) the UPR enhances endoplasmic reticulum-associated degradation (ERAD) by decreasing the expression of pro-folding chaperones and enhancing the level of pro-degradation factors; 3) The ?F508 mutation results in mRNA structure defects that decrease translation rate and may promote protein misfolding. The specific aims are: 1) To identify the mechanisms responsible for transcriptional repression of CFTR during ER stress; 2) to understand the mechanisms by which the UPR decreases CFTR maturation efficiency; and 3) to elucidate the role of ?F508 CFTR mRNA secondary structure alterations in protein misfolding and ERAD. These studies will provide novel information regarding the role of the UPR on gene expression regulation and how mRNA structure may contribute to the pathogenesis of genetic disorders. PUBLIC HEALTH RELEVANCE: The goal of the proposed studies is to examine how cellular stress responses affect the expression of a protein that is critical for normal epithelial function, the cystic fibrosis transmembrane conductance regulator, CFTR. Environmental stress such as cigarette smoke causes endoplasmic reticulum stress and activates the unfolded protein response, and the effects of this cellular response on the gene expression profiles of a number of critical genes including CFTR are being examined in these studies. Understanding these processes are important in understanding the cellular basis for a number of lung pathologies including cystic fibrosis and chronic obstructive pulmonary disease (COPD).

描述（申请人提供）：囊性纤维化跨膜电导调节因子（CFTR）是上皮细胞顶端表面表达的氯离子通道，在囊性纤维化（CF）中存在缺陷。我们之前的项目集中于野生型 (WT) 和最常见的致病突变体 ?F508 CFTR 的生物发生。基于令人信服的初步结果，该更新申请重点关注两个主要主题：1）未折叠蛋白反应（UPR）调节基因表达的分子机制； 2) 使用 CFTR 作为我们的模型，点突变产生的 mRNA 二级结构改变的后果。我们发现 UPR 抑制内源性 WT CFTR 转录、翻译、成熟效率和功能。我们将 UPR 相关的转录抑制定位于 CFTR 的最小启动子。抑制机制涉及 DNA 高甲基化、组蛋白脱乙酰化以及 UPR 激活转录因子 ATF6 与 CFTR 最小启动子的结合。有趣的是，UPR 相关的转录抑制仅影响有限数量的基因。关于UPR期间CFTR成熟度降低的机制，我们发现CFTR的促折叠分子伴侣Hsp90的表达在UPR期间降低。相反，在相同条件下促降解因子的表达增加。此外，mRNA 结构在疾病发病机制中的重要性直到最近才被认识到。因此，我们初步研究中的一个非常重要的发现是，流行的蛋白质折叠障碍模型，?F508 CFTR，也表现出 mRNA 结构缺陷。具体来说，3 个碱基的缺失在 NBD1 编码区域中产生了两个扩大的发夹环，从而减慢了翻译速度。这提供了一种有趣的可能性，即 F508 CFTR 折叠至少在某种程度上因 mRNA“错误折叠”引起的翻译率降低而受到损害。 ?F508 CFTR 生物发生的这一方面以前尚未被研究过，并且在其他遗传疾病中具有潜在的深远影响。我们的假设是：1）ER应激激活UPR，UPR通过启动子特异性机制选择性抑制基因转录； 2）UPR通过降低促折叠分子伴侣的表达并提高促降解因子的水平来增强内质网相关降解（ERAD）； 3) ?F508突变导致mRNA结构缺陷，降低翻译速率并可能促进蛋白质错误折叠。具体目标是： 1) 确定 ER 应激期间 CFTR 转录抑制的机制； 2）了解UPR降低CFTR成熟效率的机制； 3) 阐明?F508 CFTR mRNA二级结构改变在蛋白质错误折叠和ERAD中的作用。这些研究将提供有关 UPR 在基因表达调控中的作用以及 mRNA 结构如何促进遗传性疾病发病机制的新信息。 公共健康相关性：拟议研究的目的是检查细胞应激反应如何影响对正常上皮功能至关重要的蛋白质（囊性纤维化跨膜电导调节因子 CFTR）的表达。香烟烟雾等环境应激会引起内质网应激并激活未折叠蛋白反应，这些研究正在研究这种细胞反应对包括 CFTR 在内的许多关键基因的基因表达谱的影响。了解这些过程对于了解包括囊性纤维化和慢性阻塞性肺病 (COPD) 在内的多种肺部病理的细胞基础非常重要。