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Molecular Chaperone Recognition of CFTR Stability

CFTR 稳定性的分子伴侣识别

基本信息

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
项目类别：
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

项目摘要

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）是一种致命的遗传性肺部疾病，由囊性纤维化基因突变引起。 3跨膜电导调节因子（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）缺失和超过1000个突变中的许多突变使CFTR不稳定。不稳定 5 ΔF508被分子伴侣识别为未折叠的。伴侣蛋白最终与突变CFTR结合 6导致过早降解，导致CF。分子伴侣如何精确识别ΔF508 CFTR 第7章不清楚先前的体外研究表征了CFTR肽中的伴侣结合热点， 8个单独的结构域;然而，ΔF508在细胞中展开和暴露的伴侣蛋白结合热点仍然存在， 9未知我们假设结构域和亚结构域水平的ΔF508去折叠暴露了分子伴侣结合热点 10个被分子伴侣识别。此外，我们假设稳定FDA批准的ΔF508 CFTR 11 CF疗法将恢复结构域和亚结构域水平的分子伴侣对WT CFTR的识别。在aim I中，我们 12建议模拟全长WT和ΔF508 CFTR结构，以确定ΔF508如何偏离 13例正常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，通过将模拟结果与已发表的结果进行比较， 15实验数据在目标II中，我们提出了位点特异性非典型氨基酸掺入的光化学方法。 16交联剂，以共价捕获与活细胞中的CFTR结构域和亚结构域结合的分子伴侣。 17我们可以通过亲和纯化质谱鉴定和定量位点特异性CFTR相互作用物， 18 Tandem Mass Tag标签。此外，我们将再次用CF药物稳定ΔF508 CFTR，以研究 19小分子结合影响分子伴侣结合。研究药物结合与 20和分子伴侣识别是重要的，因为CF的唯一靶向治疗涉及稳定ΔF508 21 CFTR与称为药理学伴侣的小分子。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。我们寻求 23区分药理学伴侣是否改变分子伴侣识别的领域， 24结合或附近的结构域通过变构效应。其他错误折叠疾病，如心律失常 QT综合征是由类似膜蛋白的突变引起的，但由于药物治疗无法实现， 26例为缺乏筛查试验。因此，我们开发了评价药理学伴侣及其活性的方法。对CFTR结构稳定性的27项贡献。我们的新方法将阐明不稳定的 28突变体，分子伴侣识别，和药理学伴侣救援，通过杠杆作用和整合 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药理学伴侣的计算机辅助药物设计。 31 32