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

• 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
• 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

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.

项目总结(见说明)： CFTR在对肺健康至关重要的离子/液体动态平衡中起着重要的调节作用，对阴离子(直接)和阳离子(间接)运输都有影响。Cftr功能的复杂性需要一个复杂而动态的结构，该结构是近年来进化而来的独特的ABC转运蛋白家族成员。与所有蛋白质一样，CFTR进化必须在结构复杂性和热力学稳定性之间取得平衡，结果是合成的野生型多肽链中只有25%达到稳定状态。 AF508突变加剧了低效的生物发生，将这一比例降至基本上为零。一些操作，包括降低温度、下调质量控制组件和所谓的“校正”小分子，改善了细胞的处理和运输。然而，这些动作并不能恢复热稳定性(TS)，在摄氏30度以下的部分通道功能在摄氏37度时会迅速丧失。 因此，有效的治疗需要恢复TS的试剂。在这里，我们将重点放在决定TS的变构耦合途径上，以了解其机制基础，以便合理地操纵它。SA 1将阐明NBD1中调节性插入(RL)的不稳定影响以及其切除如何恢复稳定和功能。SA 2将使用两种互补的方法来确定恢复TS的其他变化，第一种利用分子动力学，第二种探索AF508 CFRR保留TS的基础 其他一些稳定的物种。这些方法已经揭示，在偶联途径的关键位置引入脯氨酸残基可以稳定AF508CFTR。SA3将利用CFTR的小分子和大分子粘结剂作为热稳定剂。首先，将通过计算(实验验证)筛选小分子文库，以结合到与TS相关的蛋白质区域。其次，我们将确定更大的蛋白质/多肽结合体，包括合成抗体和纳米抗体。与这两个领域的领先实验室合作，已经开发出有效的初始试剂。除了提供了一种全新的范式和综合的计算和实验方法来解决CF问题外，我们还为这个项目小组的其他项目提供了必要的CFTR工具和方法。