Molecular Chaperone Recognition of CFTR Stability

CFTR 稳定性的分子伴侣识别

• 批准号: 10538012
• 负责人: Eli Fritz McDonald
• 金额: $ 3.2万
• 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/10538012
• 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; Epithelial; FDA approved; Future; Genetic; Goals; In Situ; In Vitro; Individual; Individual Differences; Label; Length; Link; Long QT Syndrome; Lung; Lung diseases; Maintenance; Mass Spectrum Analysis; Membrane Proteins; Methodology; Methods; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Mutate; Mutation; Patients; Peptides; Pharmaceutical Preparations; Pharmacology; Pharmacotherapy; Phenotype; Phenylalanine; Positioning Attribute; Protein Conformation; Proteins; Proteomics; Publishing; Research; Sampling; Scanning; Site; Structural Models; Structural Protein; Structure; Syndrome; Technology; Thermodynamics; base; crosslink; disease-causing mutation; experience; experimental study; improved; in silico; molecular recognition; mutant; novel strategies; 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 项目概要 2 囊性纤维化（CF）是一种由囊性纤维化突变引起的致命性遗传性肺部疾病 3 跨膜电导调节器 (CFTR)，一种上皮阴离子通道蛋白 最常见的患者。 4 突变、苯丙氨酸 508 (ΔF508) 的缺失以及超过 1000 个突变中的许多突变都会破坏 CFTR 的稳定性。 5 ΔF508 最终被分子伴侣识别为与突变体 CFTR 结合。 6 导致过早降解，从而导致 CF。 分子伴侣准确地识别 ΔF508 CFTR。 7 的展开情况尚不清楚，之前的体外研究对 CFTR 肽和分子伴侣结合热点的特征尚不清楚。 8 个单独的结构域；然而，ΔF508 在细胞中展开和暴露的分子伴侣结合热点仍然存在 9 未知。我们参与了结构域和子结构域水平 ΔF508 的解折叠，暴露了分子伴侣结合热点。 10 得到分子伴侣的认可 此外，我们还勇敢地稳定了 ΔF508 CFTR 并获得 FDA 批准。 11 CF 疗法将恢复对 WT CFTR 的域和子域水平伴侣识别。 12 建议在计算机中模拟全长 WT 和 ΔF508 CFTR 结构，以确定 ΔF508 如何偏离 13 正常的WT结构 我们可以在Rosetta中构建ΔF508 CFTR模型，将CF药物对接到结构上，并 通过将模拟结果与已发布的结果进行比较，对 CFTR 构象空间进行采样的 14 种基准方法 15个实验数据在目标II中，我们提出了光化学的位点特异性非规范氨基酸掺入。 16 个交联剂可共价捕获与活细胞中 CFTR 结构域和子结构域结合的分子伴侣。 17 我们可以通过亲和纯化质谱法识别和量化位点特异性 CFTR 相互作用因子 18 Tandem Mass Tag 标记 此外，我们将再次用 CF 药物稳定 ΔF508 CFTR，以研究如何稳定。 19 小分子结合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 具有小分子，称为药理学伴侣然而，药理学伴侣是。 22 通过昂贵的表型筛选发现，其分子机制仍不清楚。 23 区分药理伴侣是否改变分子伴侣识别领域 24 通过变构效应结合或附近的结构域 其他错误折叠疾病，例如心律失常。 25 QT 综合征是由类似膜蛋白突变引起的，但由于药物治疗无法实现 26 缺乏筛选分析因此，我们开发了评估药理学伴侣及其的方法。 27 对 CFTR 结构稳定性的贡献我们的新颖方法将阐明不稳定之间的相互作用。 28个突变体、分子伴侣识别和药理伴侣拯救 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蛋白质组学铺平道路。 30 种药理学伴侣的计算机辅助药物设计。 31 32