Molecular Mechanisms of CFTR Regulation

CFTR调控的分子机制

• 批准号: 6630262
• 负责人: JOHN A.A. LADIAS
• 金额: $ 26.18万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6630262
• 关键词: X ray crystallography; chloride channels; membrane activity; molecular biology; phosphorylation; protein protein interaction; protein purification; protein structure function; recombinant proteins; sodium hydrogen exchanger; syntaxin

DESCRIPTION (provided by applicant): The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-regulated chloride channel that determines the rate of electrolyte and fluid transport in the apical membrane of epithelial cells. Abnormal CFTR function is associated with the pathogenesis of cystic fibrosis and secretory diarrhea. Our long-term objective is to understand the molecular mechanisms underlying the regulation of CFTR at the atomic level and develop novel strategies for modulating the activity of this channel and treating the CFTR-associated diseases. The CFTR topology consists of two membrane-spanning domains and five cytoplasmic domains: an N-terminal domain (NTD), two nucleotide-binding domains, a regulatory domain (R) and a C-terminal domain (CTD). The CFTR activity is modulated through phosphorylation of the R domain, ATP hydrolysis by the NBDs, and interactions of its NTD and CTD domains with syntaxin 1A and NHERF proteins, respectively. However the regulatory mechanisms remain unknown primarily because the three-dimensional structure of the CFTR domains and the structural basis of their interaction with intracellular regulatory proteins remain elusive. This proposal addresses these questions and focuses on the structural analysis of cytoplasmic CFTR domains and their complexes with regulatory proteins, using molecular biology techniques and X-ray crystallography. The specific aims are: 1. To dissect the structural basis of CFTR channel gating mediated through the interaction of the CFTR CTD with the NHERF PDZ1 and PDZ2 domains. 2. To elucidate the molecular mechanisms underlying the regulation of CFTR channel activity through the interaction of the CFTR NTD with syntaxin 1A. 3. To determine the three-dimensional atomic structures of the CFTR NBD1 and NBD2 domains. These studies will provide the first high-resolution three-dimensional structures of four cytoplasmic CFTR domains and the structural basis of CFTR regulation by proteins syntaxin 1A and NHERF. This information is an essential step towards elucidating the basic molecular mechanisms that control the CFTR channel gating. Importantly, the atomic coordinates of these complexes could be used for structure-based rational design of drugs that would modify selectively the CFTR activity with clinical applications in the treatment of cystic fibrosis and secretory diarrhea.

描述(申请人提供)：囊性纤维化跨膜电导调节器(CFTR)是一种受ATP调节的氯离子通道，它决定了上皮细胞顶膜中电解质和液体的转运速度。CFTR功能异常与囊性纤维化和分泌性腹泻的发病机制有关。我们的长期目标是在原子水平上了解cftr调控的分子机制，并开发新的策略来调节这一通道的活性和治疗cftr相关疾病。CFTR拓扑结构由两个跨膜结构域和五个细胞质结构域组成：一个N-末端结构域(NTD)、两个核苷酸结合结构域、一个调节结构域(R)和一个C-末端结构域(CTD)。CFTR的活性分别通过R结构域的磷酸化、NBD对ATP的水解以及其NTD和CTD结构域与Synaxin 1A和NHERF蛋白的相互作用来调节。然而，调控机制仍不清楚，主要是因为CFTR结构域的三维结构及其与细胞内调控蛋白相互作用的结构基础仍然难以捉摸。这项建议解决了这些问题，并侧重于利用分子生物学技术和X射线结晶学对细胞质CFTR结构域及其与调节蛋白的复合体的结构分析。具体目标是： 1.剖析CFTRCTD与NHERF PDZ1和PDZ2结构域相互作用所介导的CFTR通道门控的结构基础。 2.阐明CFTRNTD与Synaxin 1A相互作用调节CFTR通道活性的分子机制。 3.确定CFTRNbd1和Nbd2结构域的三维原子结构。 这些研究将首次提供四个细胞质CFTR结构域的高分辨率三维结构，并提供蛋白质Synaxin 1A和NHERF调控CFTR的结构基础。这些信息是阐明控制CFTR通道门控的基本分子机制的重要一步。重要的是，这些化合物的原子坐标可以用于基于结构的合理药物设计，选择性地修饰CFTR活性，用于治疗囊性纤维化和分泌性腹泻。