MOLECULAR AND CHEMICAL DESCRIPTION OF CFTR FUNCTION

CFTR 功能的分子和化学描述

• 批准号: 6476179
• 负责人: PETER L PEDERSEN
• 金额: $ 22.65万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 2003-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6476179
• 关键词: X ray crystallography; active sites; adenosine triphosphate; adenosinetriphosphatase; affinity chromatography; chemical binding; chloride channels; cystic fibrosis; enzyme activity; enzyme linked immunosorbent assay; hydrolysis; molecular pathology; myosins; nuclear magnetic resonance spectroscopy; polymerase chain reaction; protein folding; protein sequence; protein structure function; transfection

This proposal focuses on the protein called CFTR, mutations in which cause Cystic Fibrosis (CF), a major disease frequently characterized by chronic lung infections. CFTR is an ATP hydrolysis-dependent Cl- channel consisting of 2 membrane domains, 2 nucleotide binding folds (NBF1 plus NBF2), and an R (regulatory) domain. Most cases of CF are caused by a single mutation (deltaF508) in NBF1 which prevents CFTR from trafficking from the E.R. to the plasma membrane where, in the lung, it aids in combating infections. Early work (Science, 1991; JBC, 1992, 93, 94), supported by this grant, demonstrated that NBF1 and NBF2 bind ATP and that the deltaF508 mutation results in a folding problem. Recent work has shown that NBF1 hydrolyzes ATP (JBC, 1995), interacts with the membrane (Biochem., 1997), participates in the formation of an NBF1 plus R/NBF2 complex (In Review), and maintains the critical F508 region as an alpha-helix (In Review). Despite these important advances, essential information is lacking about the catalytic mechanism of NBF1 and NBF2, their "cross-talk" in the NBF1 plus R/NBF2 complex, and the location and role of the F508 region. For these reasons, the following 4 hypotheses will be tested: 1. ATP hydrolysis catalyzed by NBF1 or NBF2 of CFTR proceeds through a reaction pathway similar to that catalyzed by both the F1 moiety of ATP synthase/ATPase complexes and myosin. 2. The rate of ATP hydrolysis is enhanced when the 3 "soluble" domains of CFTR form a NBF1 plus R/NBF2 complex in which both NBF1 NBF2 are catalytic but function in an alternating, cooperative manner. 3. The F508 region of NBF1, which contains a potential catalytic base (E504), is flexible, and contributes to the active "ATPase" site in the NBF1 plus R/NBF2 complex but lies outside this site in NBF1 alone. 4. The deltaF508 mutation responsible for most cases of CF prevents NBF1 from undergoing an ATP-dependent conformational change while reducing the catalytic efficiency of this domain. The proposed studies are fundamental to understanding structure/function relationships within CFTR, to understanding the underlying basis of most cases of CF, and to developing new strategies to treat the disease.

该提案的重点是称为CFTR的蛋白质，其中引起囊性纤维化（CF），这是一种经常以慢性肺部感染为特征的主要疾病。 CFTR是一种ATP水解依赖性CL-通道，该通道由2个膜结构域，2个核苷酸结合褶皱（NBF1 Plus NBF2）和一个R（调节）结构域组成。 大多数CF病例是由NBF1中的单个突变（Deltaf508）引起的，该突变阻止了CFTR从E.R.到质膜的运输，在肺中，它有助于打击感染。 早期工作（Science，1991; JBC，1992，93，94）得到了这项赠款的支持，证明了NBF1和NBF2结合了ATP，并且Deltaf508突变导致折叠问题。 最近的工作表明，NBF1水解ATP（JBC，1995年）与膜相互作用（Biochem。，1997），参与了NBF1 Plus R/NBF2复合物（综述）的形成，并将关键F508区域保持为Alpha-Helix（综述）。尽管有这些重要的进步，但缺乏关于NBF1和NBF2的催化机制，它们在NBF1加R/NBF2复合物中的“串扰”以及F508区域的位置和作用。 由于这些原因，将测试以下4个假设：1。由CFTR的NBF1或NBF2催化的ATP水解通过与ATP合酶/ATPase复合物和肌球蛋白的F1部分相似的反应途径进行。 2。当CFTR的3个“可溶性”结构域形成NBF1加R/NBF2复合物时，ATP水解的速率得到了提高，其中NBF1 NBF2都是催化的，但以交替的合作方式发挥作用。 3。含有潜在催化碱（E504）的NBF1的F508区域是灵活的，并有助于NBF1加上R/NBF2复合物中的主动“ ATPase”位点，但仅在NBF1中仅位于NBF1。 4。导致大多数CF病例的Deltaf508突变可防止NBF1经历ATP依赖性构象变化，同时降低该域的催化效率。拟议的研究对于理解CFTR内的结构/功能关系至关重要，以了解大多数CF病例的基础以及制定治疗疾病的新策略。