Molecular Chaperone Recognition of CFTR Stability

CFTR 稳定性的分子伴侣识别

• 批准号: 10734051
• 负责人: Eli Fritz McDonald
• 金额: $ 3.3万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10734051
• 关键词: Affinity Chromatography; Amber; Amino Acids; Anions; Arrhythmia; Attenuated; Benchmarking; Binding; Biological Assay; Cardiac; Cells; Computer Assisted; Computer Models; Computer software; Crosslinker; Cryoelectron Microscopy; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Delta F508 mutation; Disease; Docking; Drug Design; Environment; Epithelium; FDA approved; Future; Genetic; Goals; Immunoprecipitation; In Situ; In Vitro; Individual; Individual Differences; Label; Length; Link; Long QT Syndrome; Lung; Lung diseases; Maintenance; Mass Spectrum Analysis; Membrane; Membrane Proteins; Methodology; Methods; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Mutate; Mutation; Organ; Patients; Peptides; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Phenylalanine; Positioning Attribute; Protein Conformation; Proteins; Proteomics; Publishing; Research; Sampling; Scanning; Site; Structural Models; Structural Protein; Structure; Syndrome; Technology; Thermodynamics; crosslink; data integration; disease-causing mutation; experience; experimental study; improved; in silico; molecular recognition; mutant; novel strategies; pharmacologic; premature; protein misfolding; protein structure; proteostasis; screening; simulation; small molecule; structural biology; targeted treatment; tool

1 PROJECT SUMMARY 2 Cystic fibrosis (CF) is a lethal genetic lung disease caused by mutations in the Cystic Fibrosis 3 Transmembrane Conductance Regulator (CFTR), an epithelial anion channel protein. The most common patient 4 mutation, deletion of phenylalanine 508 (ΔF508) and many of over 1000 mutations destabilize CFTR. Unstable 5 ΔF508 is recognized by molecular chaperones as unfolded. Chaperone binding to mutant CFTR eventually 6 results in pre-mature degradation leading to CF. Precisely how molecular chaperones recognize ΔF508 CFTR 7 as unfolded is unclear. Previous in vitro studies characterized chaperone binding hotspots in CFTR peptides and 8 individual domains; however, the chaperone binding hotspots that ΔF508 unfolds and exposes in the cell remains 9 unknown. We hypothesize domain and sub-domain level ΔF508 unfolding exposes chaperone binding hotspots 10 recognized by molecular chaperones. Additionally, we hypothesize stabilizing ΔF508 CFTR with FDA approved 11 CF therapies will restore domain and sub-domain level chaperone recognition towards WT CFTR. In aim I, we 12 propose simulating full-length WT and ΔF508 CFTR structures in silico to determine how ΔF508 deviates from 13 normal WT structure. We can build ΔF508 CFTR models in Rosetta, dock CF drugs to the structures, and 14 benchmark methods for sampling CFTR conformational space by comparing simulations results to published 15 experimental data. In aim II, we propose site-specific non-canonical amino acid incorporation of photochemical 16 crosslinkers to covalently capture molecular chaperone binding to CFTR domains and sub-domains in live cells. 17 We can identify and quantify site-specific CFTR interactors by affinity-purification mass spectrometry with 18 Tandem Mass Tag labeling. Furthermore, we will again stabilize ΔF508 CFTR with CF drugs to examine how 19 small molecule binding impacts molecular chaperone binding. Studying the relationship between drug binding 20 and chaperone recognition is important because the only targeted treatment for CF involved stabilizing ΔF508 21 CFTR with small molecules called pharmacological chaperones. However, pharmacological chaperones are 22 discovered through expensive phenotypic screens and their molecular mechanisms remain unclear. We seek to 23 distinguish whether pharmacological chaperones change molecular chaperone recognition in the domain of 24 binding or nearby domains through allosteric effects. Other misfolding diseases, such as cardiac arrythmia Long 25 QT Syndrome, are caused by mutations in similar membrane proteins, but drug treatments lay out of reach due 26 to lack of assays for screening. Thus, we developed methods to evaluate pharmacological chaperones and their 27 contributions to CFTR structural stability. Our novel approach will elucidate the interplay between unstable 28 mutants, molecular chaperone recognition, and pharmacological chaperone rescue by leveraging and integrating 29 data from computational structural biology and proteomics. This will pave the way for structure-based and 30 computer aided drug design of pharmacological chaperones. 31 32

1个项目摘要 2囊性纤维化（CF）是由囊性纤维化突变引起的致命遗传肺疾病 3跨膜电导调节剂（CFTR），一种上皮阴离子通道蛋白。最常见的病人 4突变，苯丙氨酸508（ΔF508）的缺失以及1000多个突变中的许多突变破坏CFTR的稳定。不稳定 5ΔF508被分子伴侣识别为展开。伴侣最终与突变体CFTR结合 6导致成熟前降解导致CF。恰好是分子伴侣如何识别ΔF508CFTR 7随着展开不清楚。以前的体外研究表征了CFTR肽中的伴侣结合热点和 8个单个领域；但是，ΔF508在细胞中展开和暴露的伴侣结合热点仍然存在 9未知。我们假设域和子域级别ΔF508展开露出伴侣结合点 10被分子伴侣识别。此外，我们假设使用FDA批准稳定ΔF508CFTR 11 CF疗法将恢复对WT CFTR的域和亚域级分子伴侣识别。在目标一世中，我们 12提出模拟全长WT和ΔF508CFTR结构的提案，以确定ΔF508如何偏离 13正常的WT结构。我们可以在Rosetta中构建ΔF508CFTR模型，将CF CF药物固定到结构，并且 通过将模拟结果与已发布的模拟结果进行比较，用于对CFTR构象空间进行采样的14个基准方法 15个实验数据。在AIM II中，我们提出了由光化学的特定地点特异性的非经典氨基酸 16个交联，以共价捕获活细胞中与CFTR结构域和亚域结合的分子链酮。 17我们可以通过与亲和力纯化质谱法识别和量化位点特异性CFTR相互作用。 18串联质量标签标签。此外，我们将再次使用CF药物稳定ΔF508CFTR，以检查如何 19小分子结合会影响分子伴侣结合。研究药物结合之间的关系 20和伴侣识别很重要，因为CF的唯一靶向治疗涉及稳定ΔF508 21 CFTR具有称为药理伴侣的小分子。但是，药物伴侣是 22通过昂贵的表型筛选发现，其分子机制仍不清楚。我们寻求 23药物链酮是否改变了分子链酮在域中的分子链酮的识别 24通过变构效应结合或附近的域。其他错误折叠疾病，例如心脏雅利亚氏症长 25 QT综合征是由类似膜蛋白突变引起的，但药物治疗范围不足 26缺乏筛查测定法。这，我们开发了评估药物伴侣及其的方法 27对CFTR结构稳定性的贡献。我们的新方法将阐明不稳定之间的相互作用 28个突变体，分子伴侣识别和通过利用和整合的药物伴侣救援 29来自计算结构生物学和蛋白质组学的数据。这将为基于结构和 30次计算机辅助药物设计的药物伴侣。 31 32