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CFTR Biogenesis and Function in Epithelia

CFTR 上皮细胞的生物发生和功能

基本信息

批准号：
8241053
负责人：
ZSUZSA BEBOK
金额：
$ 36.26万
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：
2004
资助国家：
美国
起止时间：
2004-05-01 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8241053
关键词：
5&apos 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

项目摘要

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剂（CFTR）是一种在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在野生型（WT）和最常见的致病突变体的生物发生上。F508雌性生殖道。基于令人信服的初步结果，本更新申请集中在两个主要主题：1)未折叠蛋白反应（UPR）调节基因表达的分子机制；2)以CFTR为模型研究点突变对mRNA二级结构改变的影响。我们发现UPR抑制内源性WT CFTR的转录、翻译、成熟效率和功能。我们已经将upr相关的转录抑制定位在CFTR的最小启动子上。其抑制机制包括DNA超甲基化、组蛋白去乙酰化以及upr激活的转录因子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成熟降低的机制，我们发现CFTR的前折叠伴侣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，在我们的初步研究中，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碱基的缺失会在NBD1编码区产生两个放大的发夹环，从而减慢翻译速度。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，是由于mRNA“错误折叠”导致的翻译速率降低而受损的。这方面？F508 CFTR的生物发生机制以前没有被研究过，并且在其他遗传疾病中具有潜在的深远意义。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的转录；2) UPR通过降低促折叠伴侣的表达和提高促降解因子的水平来促进内质网相关降解（ERAD）；3) ？F508突变导致mRNA结构缺陷，降低翻译率并可能促进蛋白质错误折叠。具体目的是：1)确定内质网应激下CFTR转录抑制的机制；2)了解普遍定期审议降低CFTR成熟效率的机制；3)阐明？F508 CFTR mRNA二级结构改变与蛋白错误折叠和ERAD。这些研究将为UPR在基因表达调控中的作用以及mRNA结构如何参与遗传疾病的发病机制提供新的信息。