CFTR Biogenesis and Function in Epithelia

CFTR 上皮细胞的生物发生和功能

• 批准号: 7791423
• 负责人: ZSUZSA BEBOK
• 金额: $ 36.63万
• 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/7791423
• 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（由申请人提供）：囊性纤维化跨膜传导调节因子（CFTR）是一种在上皮细胞顶面表达的氯离子通道，在囊性纤维化（CF）中存在缺陷。我们以前的项目集中在野生型（WT）和最常见的致病突变体，？F508 CFTR。基于令人信服的初步结果，该更新申请集中在两个主要主题上：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。我们已经将UPR相关的转录抑制定位于CFTR的最小启动子。阻遏的机制涉及DNA超甲基化、组蛋白去乙酰化以及UPR激活的转录因子ATF 6与CFTR最小启动子的结合。有趣的是，UPR相关的转录抑制只影响有限数量的基因。关于机制，减少CFTR成熟在UPR期间，我们表明，表达热休克蛋白90，前折叠伴侣CFTR，减少在UPR期间。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最近才被认识到。因此，在我们的初步研究中，一个非常重要的发现是，蛋白质折叠障碍的流行模型，？F508 CFTR也表现出mRNA结构缺陷。具体地说，3个碱基的缺失在NBD 1编码区产生了两个扩大的发夹环，这减缓了翻译。这提供了一种有趣的可能性，即F508 CFTR折叠至少在一定程度上受到损害，因为mRNA“错误折叠”导致翻译速率降低。这方面的？F508 CFTR的生物发生以前没有被研究过，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）内质网应激激活UPR，UPR通过启动子特异性机制选择性抑制基因转录; 2）UPR通过降低前折叠分子伴侣的表达和提高前降解因子的水平，增强内质网相关降解（ERAD）; 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应激过程中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UPR在CFTR成熟过程中的作用。F508 CFTR mRNA二级结构在蛋白质错误折叠和ERAD中的改变。这些研究将提供有关UPR对基因表达调控的作用以及mRNA结构如何有助于遗传性疾病发病机制的新信息。 公共卫生关系：拟议研究的目标是研究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，囊性纤维化跨膜传导调节因子CFTR的表达。环境应激如香烟烟雾引起内质网应激并激活未折叠蛋白反应，这些研究正在检查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许多肺部病理学的细胞基础非常重要，包括囊性纤维化和慢性阻塞性肺病（COPD）。