Trzcinska-Daneluti Agata M, Ly Diane, Huynh Lise, Jiang Chong, Fladd Christopher, Rotin Daniela
Program in Cell Biology, The Hospital for Sick Children, and Biochemistry Department, University of Toronto, Toronto, Ontario M5G 1L7, Canada.
Mol Cell Proteomics. 2009 Apr;8(4):780-90. doi: 10.1074/mcp.M800268-MCP200. Epub 2008 Dec 15.
Cystic Fibrosis is caused by mutations in CFTR, with a deletion of a phenylalanine at position 508 (F508del-CFTR) representing the most common mutation. The F508del-CFTR protein exhibits a trafficking defect and is retained in the endoplasmic reticulum. Here we describe the development of a high-content screen based on a functional assay to identify proteins that correct the F508del-CFTR defect. Using a HEK293 MSR GripTite cell line that stably expresses F508del-CFTR, we individually co-expressed approximately 450 unique proteins fused to the Cl(-)-sensitive YFP(H148Q/I152L) mutant. We then tested correction of F508del-CFTR function by the CI(-)/l(-) exchange method following stimulation with forskolin/IBMX/genistein, using quantitative recordings in multiple individual cells with a high-content (high-throughput) Cellomics KSR imaging system. Using this approach, we identified several known and novel proteins that corrected F508del-CFTR function, including STAT1, Endothelin 1, HspA4, SAPK substrate protein 1, AP2M1, LGALS3/galectin-3, Trk-fused gene, Caveolin 2, PAP/REG3alpha, and others. The ability of these correctors to rescue F508del-CFTR trafficking was then validated by demonstrating their enhancement of maturation (appearance of band C) and by cell surface expression of F508del-CFTR bearing HA tag at the ectodomain using confocal microscopy and flow cytometry. These data demonstrate the utility of high-content analyses for identifying proteins that correct mutant CFTR and discover new proteins that stimulate this correction. This assay can also be utilized for RNAi screens to identify inhibitory proteins that block correction of F508del-CFTR, small molecule, and peptide screens.
囊性纤维化由CFTR基因突变引起，其中第508位苯丙氨酸缺失（F508del-CFTR）是最常见的突变。F508del-CFTR蛋白存在转运缺陷，并滞留在内质网中。在此，我们描述了一种基于功能测定的高内涵筛选方法的开发，以鉴定能够纠正F508del-CFTR缺陷的蛋白质。我们使用稳定表达F508del-CFTR的HEK293 MSR GripTite细胞系，分别共表达了约450种与Cl(-)敏感的YFP(H148Q/I152L)突变体融合的独特蛋白质。然后，在用福斯可林/异丁基甲基黄嘌呤/染料木黄酮刺激后，我们通过Cl(-)/I(-)交换法，使用高内涵（高通量）Cellomics KSR成像系统对多个单个细胞进行定量记录，来测试F508del-CFTR功能的纠正情况。通过这种方法，我们鉴定出了几种已知和新型的能够纠正F508del-CFTR功能的蛋白质，包括信号转导和转录激活因子1（STAT1）、内皮素1、热休克蛋白A4（HspA4）、应激激活蛋白激酶底物蛋白1、衔接蛋白α2亚基（AP2M1）、半乳糖凝集素3（LGALS3/galectin-3）、Trk融合基因、小窝蛋白2、胰腺抗菌肽/再生胰岛衍生蛋白3α（PAP/REG3alpha）等。然后，通过共聚焦显微镜和流式细胞术证明这些校正剂增强了成熟（C条带的出现）以及胞外域带有HA标签的F508del-CFTR的细胞表面表达，从而验证了这些校正剂拯救F508del-CFTR转运的能力。这些数据证明了高内涵分析在鉴定能够纠正突变型CFTR的蛋白质以及发现刺激这种纠正作用的新蛋白质方面的实用性。该测定法还可用于RNA干扰筛选，以鉴定阻断F508del-CFTR纠正的抑制性蛋白质、小分子和肽筛选。
