Molecular Pathophysiology of Cystic Fibrosis

囊性纤维化的分子病理生理学

基本信息

批准号：
10417034
负责人：
Tzyh-Chang Hwang
金额：
$ 41.49万
依托单位：
UNIVERSITY OF MISSOURI-COLUMBIA
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-09-30 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10417034
关键词：
ATP Hydrolysis ATP-Binding Cassette Transporters Address Affect Anatomy Anions Aphorisms Binding Binding Sites Biochemical Biological Biophysics Catalysis Chemosensitization Chloride Channels Computing Methodologies Coupled Coupling Cryoelectron Microscopy Cysteine Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Data Development Dimerization Disease Disputes Drug Design Drug Interactions Drug Targeting Electrophysiology (science)FDA approved Family Foundations Functional disorder Genes Genetic Diseases Goals Head Hydrolysis Hydroxyl Radical Investigation Ion Channel Knowledge Learning Life Medical Medicine Methods Mission Modeling Molecular Molecular Conformation Motion Mutation Nonsense Mutation Nucleotides Pathogenicity Pathway interactions Patients Pharmaceutical Preparations Pharmacology Plant Roots Play Positioning Attribute Power stroke Reagent Reporting Research Resolution Role Scanning Side Site Structure Structure-Activity Relationship Tail Technology Testing Therapeutic Time Transmembrane Domain Ursidae Family VX-770 VX-809 Walkers Work base cystic fibrosis patients design dimer disease-causing mutation drug candidate experimental study implementation facilitation improved insight loss of function mutation member next generation novel therapeutics prevent programs small molecule symptom treatment temporal measurement

项目摘要

Loss-of-function mutations in the cftr gene are the root cause of cystic fibrosis (CF), the second most common life-shortening genetic disease in the US. As a member of the ABC (ATP Binding Cassette) transporter superfamily, the CFTR protein comprises two transmembrane domains (TMD1 and TMD2), each followed by a nucleotide binding domain (NBD1 and NBD2 respectively) characterized by the canonical Walker A and B motifs for ATP binding/hydrolysis, and a signature sequence (i.e., LSGGQ) that plays a critical role for the formation of a head-to-tail NBD dimer upon ATP binding. CFTR is unique in that, instead of being an active transporter, CFTR is a bona fide ion channel. Our previous studies have led to a gating mechanism of CFTR that features a probabilistic relationship between ATP-induced NBD dimerization and gate opening in the TMDs. Recent solutions of three cryo-EM structures of CFTR, on one hand, dispute our idea that channel closure does not require a complete separation of the two NBDs, but on the other hand, support our proposition that NBD dimerization does not guarantee gate opening. These cryo-EM data lack high temporal resolution, but their exquisite spatial resolution does offer us an unprecedented opportunity to address several unsettled questions regarding the functional anatomy of CFTR (Aim 1) such as: What is the functional significance of completely separated NBDs shown in the cryo-EM structures? How do the two ATP-binding sites affect each other’s function? The existence of a closed channel with dimerized NBDs not only demands more thorough studies of the gating mechanism but also compels us to challenge the long-held view that only the open channel hydrolyzes ATP. As many of the disease-associated mutations reside in the NBD dimer interface, and thus are excellent subjects facilitating our investigation into NBD/TMD coupling, the proposed studies could also reveal the mechanism by which these mutations cause CF at a molecular level. One important application of our fundamental studies of CFTR gating is to use the knowledge to explore how drugs or drug candidates affect different aspects of CFTR gating (Aim 2), and to determine if and to what extent pathogenic mutations respond to therapeutic reagents (Aim 3). Together with the atomic structures of CFTR in different states, it is timely to probe how reagents, by binding to different regions of CFTR, synergistically activate CFTR. Answering this latter question could serve as a stepping-stone to materialize structure-based drug design.

CFTR基因的功能丧失突变是囊性纤维化（CF）的根本原因，即美国第二大最常见的遗传疾病。作为ABC的成员（ATP结合盒）转运蛋白超家族，CFTR蛋白包括两个跨膜结构域（TMD1和TMD2），随后是核苷酸结合域（分别为nbd1和nbd2）以规范助行器A和B为特征 ATP结合/水解的基序，以及扮演A的签名序列（即LSGGQ）在ATP结合时形成头到尾NBD二聚体的关键作用。 CFTR是唯一的CFTR不是主动转运蛋白，而是一个真正的离子通道。我们以前的研究导致了CFTR的门控机制，该机制具有 ATP诱导的NBD二聚体与门开口之间的概率关系 TMD。一方面，CFTR的三种冷冻EM结构的最新解决方案是争议我们认为渠道关闭不需要两个NBD的观念，而是另一方面，支持我们的提议NBD二聚体不能保证门开场。这些低温EM数据缺乏高临时分辨率，但它们的独家空间决议确实为我们提供了一个前所未有的机会，可以解决一些未解决的问题有关CFTR功能解剖结构的问题（AIM 1），例如：什么是什么冷冻EM结构中显示的完全分离的NBD的功能意义？两个ATP结合站点如何影响彼此的功能？封闭的存在具有二聚NBD的渠道不仅需要对门控进行更彻底的研究机制，但也迫使我们挑战只有公开的长期观点通道水解ATP。由于许多与疾病相关的突变都存在于NBD中二聚体界面，因此是出色的主题，促进了我们对 NBD/TMD耦合，拟议的研究还可以揭示这些突变导致CF在分子水平上。我们的一个重要应用 CFTR幻象的基本研究是利用知识来探索药物或候选药物影响CFTR门控的不同方面（AIM 2），并确定是否以及是否范围致病突变对治疗试剂有反应（AIM 3）。与原子一起 CFTR在不同状态的结构，及时探测试剂如何通过与不同的结合 CFTR区域协同激活CFTR。回答以后的问题可以用作垫脚石以实现基于结构的药物设计。