Dynamics and Thermal Stability in CFTR Function and Dysfunction

CFTR 功能和功能障碍的动力学和热稳定性

基本信息

批准号：
8249225
负责人：
JOHN R RIORDAN
金额：
$ 36.83万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-05-15 至 2017-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8249225
关键词：
ATP-Binding Cassette Transporters Anions Antibodies Attention Binding Biogenesis Body Temperature Cations Cell membrane Cell physiology Cell surface Chronic Obstructive Airway Disease Collaborations Complex Computing Methodologies Coupling Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Data Defect Disease Docking Down-Regulation Elements Equilibrium Evolution Excision Fab Immunoglobulins Failure Family member Functional disorder Health Homeostasis Human Hydrolysis Indium Instruction Intestines Ion Channel Ions Laboratories Libraries Liquid substance Llama Lung Maintenance Maps Methodology Methods Minor Modification Molecular Molecular Models Mutate Mutation Pathway interactions Peptides Phosphorylation Phosphorylation Site Physiological Play Positioning Attribute Process Proline Protein Region Proteins Quality Control Reagent Rest Role Side Site Structure Surface Sweat Glands Temperature Testing Therapeutic Thermodynamics Tissues Variant base effective therapy improved molecular dynamics molecular modeling mutant nanobodies neglect novel polypeptide reproductive restoration small molecule small molecule libraries thermostability three dimensional structure tool trafficking

项目摘要

PROJECT SUMMARY (See instructions): The CFTR plays a crucial regulatory role in ion/fluid homeostasis essential to lung health, impacting both anion (directly) and cation (indirectly) transport. The complexity of CFTR function requires a complex and dynamic structure that has evolved relatively recently as a unique ABC transporter family member. Like all proteins, CFTR evolution had to achieve a balance between structural complexity and thermodynamic stability with the result that only ~25% of wild-type polypeptide chains synthesized achieve a stable state. The AF508 mutation exacerbates inefficient biogenesis, reducing this proportion to essentially zero. Some manipulations including reduced temperature, down-regulation of quality control components and so-called "corrector" small molecules improve cellular processing and trafficking. However, these maneuvers do not restore Thermal Stability (TS) and the partial channel function at temperatures < 30oC is rapidly lost at 37oC. Thus effective therapies require reagents that restore TS. Here we focus on the allosteric coupling pathways that determine TS to understand its mechanistic basis so that it can be manipulated rationally. SA 1 will elucidate the destabilizing influence of the Regulatory Insertion (Rl) in NBD1 and how its excision restores stability and function. SA 2 will employ two complementary approaches to identify other changes that restoreTS, the first utilizing molecular dynamics and the second exploring the basis of retained TS by AF508 CFTRs of some other species that are stable. These methods have already revealed that the introduction of proline residues at key positions in coupling pathways stabilize AF508 CFTR. SA 3 will utilize both small and larger molecule binders of CFTR as thermal stabilizers. First, small molecule libraries will be screened computationally (validated experimentally) for binding to regions of the protein implicated in TS. Second, we will identify larger protein/peptide binders including synthetic antibodies and nanobodies. Effective initial reagents have already been developed in collaboration with leading laboratories in both fields. In addition to providing an entirely new paradigm with integrated computational and experimental approaches to the CF problem, we provide the other projects of this PPG with essential CFTR tools and methodologies.

项目摘要（请参阅说明）： CFTR在离子/液体稳态中起着至关重要的调节作用，对肺部健康必不可少，影响阴离子（直接）和阳离子（间接）转运。 CFTR函数的复杂性需要一个复杂而动态的结构，该结构最近作为独特的ABC转运蛋白家族成员进化。像所有蛋白质一样，CFTR进化必须在结构复杂性和热力学稳定性之间达到平衡，结果构成了野生型多肽链的约25％达到稳定状态。 AF508突变加剧了效率低下的生物发生，使这一比例基本上为零。一些操作包括降低温度，质量控制成分的下调以及所谓的“校正”小分子改善了细胞处理和运输。但是，这些操作不会恢复热稳定性（TS），并且在温度<30oC时的部分通道功能在37oC时迅速损失。因此，有效的疗法需要恢复TS的试剂。在这里，我们专注于确定TS的变构耦合途径，以理解其机械基础，以便可以合理地操纵它。 SA 1将阐明调节插入（RL）在NBD1中的不稳定影响，以及其切除如何恢复稳定性和功能。 SA 2将采用两种互补方法来识别还原的其他变化，第一种利用分子动力学以及第二个探索AF508 CFTR保留TS的基础的更改在其他一些稳定的物种中。这些方法已经表明，在耦合途径中的关键位置引入脯氨酸残基稳定AF508 CFTR。 SA 3将利用CFTR的小和较大分子粘合剂作为热稳定剂。首先，小分子库将在计算上进行筛选（实验验证），以结合与TS中有关的蛋白质的区域。其次，我们将确定较大的蛋白质/肽结合剂，包括合成抗体和纳米体。有效的初始试剂已经与两个领域的领先实验室合作开发。除了通过针对CF问题的集成计算和实验方法提供全新的范式，我们还提供了本PPG的其他项目，并使用必需的CFTR工具和方法。