Molecular Chaperone Recognition of CFTR Stability

1个项目摘要 囊性纤维化是一种由囊性纤维化基因突变引起的致死性遗传性肺部疾病。 3跨膜电导调节蛋白(CFTR)，一种上皮性阴离子通道蛋白。最常见的病人 4突变、苯丙氨酸508(ΔF508)的缺失以及1000多个突变中的许多都会破坏CFTR值。不稳定 5ΔF508被分子伴侣识别为未折叠。伴侣最终与突变的cftr结合 6会导致早熟降解，从而导致CF。分子伴侣到底是如何识别ΔF508 cftr的 7具体情况尚不清楚。先前的体外研究表征了CFTR多肽和CFTR中的伴侣结合热点 8个单独的结构域；然而，ΔF508在细胞中展开并暴露的伴侣结合热点仍然存在 9个未知。我们假设结构域和子域级别的ΔF508展开暴露了伴侣结合热点 10由分子伴侣识别。此外，我们假设稳定的ΔF508 cftr已获得食品和药物管理局的批准 11个CF疗法将恢复WT CFTR的域和亚域水平的伴侣识别。在AIM I中，我们 12建议在硅胶中模拟全长WT和ΔF508 CFTR型结构，以确定ΔF508如何偏离 13例WT结构正常。我们可以在罗塞塔建造ΔF508 CFTR模型，将CF药物对接到结构上，以及 通过将模拟结果与已发表的结果进行比较来采样CFTR构象空间的14种基准方法 15个实验数据。在AIM II中，我们提出了特定部位的非正则氨基酸掺入光化学。 16个交联剂，以共价方式捕获与活细胞中CFTR域和亚域结合的分子伴侣。 17我们可以通过亲和纯化质谱学鉴定和定量位点特异性的CFTR相互作用子 18串联质量标记。此外，我们将再次使用CF药物稳定ΔF508 CFTR，以研究如何 19小分子结合影响分子伴侣结合。浅谈药物结合的相互关系 20和伴侣识别是重要的，因为治疗CF的唯一靶向治疗涉及稳定ΔF508 21带有称为药理伴侣的小分子。然而，药理上的伴侣是 22是通过昂贵的表型筛选发现的，其分子机制尚不清楚。我们寻求 23区分药理伴侣是否改变了分子伴侣的识别 24通过变构效应结合结构域或邻近结构域。其他错误折叠的疾病，如长时间心律失常 25 QT综合征，是由相似的膜蛋白突变引起的，但药物治疗是遥不可及的 26到缺乏筛查的分析方法。因此，我们开发了评估药理伴侣及其 27对CFTR结构稳定性的贡献。我们的新方法将阐明不稳定因素之间的相互作用 28个突变体，分子伴侣识别，以及利用和整合的药理伴侣拯救 29来自计算结构生物学和蛋白质组学的数据。这将为基于结构的和 30药理伴侣的计算机辅助药物设计。 31 32

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