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High-content functional screen to identify proteins that correct F508del-CFTR function.
Trzcinska-Daneluti, Agata M; Ly, Diane; Huynh, Lise; Jiang, Chong; Fladd, Christopher; Rotin, Daniela.
Afiliação
  • Trzcinska-Daneluti AM; Program in Cell Biology, The Hospital for Sick Children, and Biochemistry Department, University of Toronto, Toronto, Ontario M5G 1L7, Canada.
Mol Cell Proteomics ; 8(4): 780-90, 2009 Apr.
Article em En | MEDLINE | ID: mdl-19088066
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.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Regulador de Condutância Transmembrana em Fibrose Cística / Proteoma / Proteômica Tipo de estudo: Prognostic_studies Limite: Animals / Humans Idioma: En Revista: Mol Cell Proteomics Assunto da revista: BIOLOGIA MOLECULAR / BIOQUIMICA Ano de publicação: 2009 Tipo de documento: Article País de afiliação: Canadá País de publicação: Estados Unidos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Regulador de Condutância Transmembrana em Fibrose Cística / Proteoma / Proteômica Tipo de estudo: Prognostic_studies Limite: Animals / Humans Idioma: En Revista: Mol Cell Proteomics Assunto da revista: BIOLOGIA MOLECULAR / BIOQUIMICA Ano de publicação: 2009 Tipo de documento: Article País de afiliação: Canadá País de publicação: Estados Unidos