title: Global proteomic approach unmasks involvement of keratins 8 and 18 in the delivery of cystic fibrosis transmembrane conductance regulator (CFTR)/deltaF508-CFTR to the plasma membrane.
abstract: Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF gene (cftr). Physiologically, CF is characterized by an abnormal chloride secretion in epithelia due to a dysfunction of a mutated cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a cAMP-dependent chloride channel whose most frequent mutation, deltaF508, leads to an aberrantly folded protein which causes a dysfunction of the channel. However, a growing number of reports suggest that modifier genes and environmental factors are involved in the physiology of CF. To identify proteins whose expression depends on wild-type WT-CFTR or deltaF508-CFTR, we chose a global proteomic approach based on the use of two-dimensional gel electrophoresis (2-DE) and mass spectrometry. The experiments were carried out with HeLa cells stably transfected with WT-CFTR (pTCFWT) or deltaF508-CFTR (pTCFdeltaF508). These experiments unmasked keratin 8 (K8) and 18 (K18) which were differentially expressed in pTCFWT vs. pTCFdeltaF508. An immunoblot of K18 confirmed the 2-DE results. Intracellular localization experiments of WT-CFTR, deltaF508-CFTR, K8, and K18 suggest that the expression of these proteins are linked, and that the concentrations of K8 and K18 and/or their distribution may be involved in the traffic of WT-CFTR/deltaF508-CFTR. A functional assay for CFTR revealed that specifically lowering K18 expression or changing its distribution leads to the delivery of functional deltaF508-CFTR to the plasma membrane. This work suggests a novel function of K18 in CF.
authors.affiliation: Inserm U467, Faculté de Médecine Necker Enfants Malades, Paris, France.
authors.forename: Noélie
authors.initials: N
authors.lastname: Davezac
authors.forename: Danielle
authors.initials: D
authors.lastname: Tondelier
authors.forename: Joanna
authors.initials: J
authors.lastname: Lipecka
authors.forename: Pascale
authors.initials: P
authors.lastname: Fanen
authors.forename: France
authors.initials: F
authors.lastname: Demaugre
authors.forename: Janusz
authors.initials: J
authors.lastname: Debski
authors.forename: Michal
authors.initials: M
authors.lastname: Dadlez
authors.forename: André
authors.initials: A
authors.lastname: Schrattenholz
authors.forename: Michael A
authors.initials: MA
authors.lastname: Cahill
authors.forename: Aleksander
authors.initials: A
authors.lastname: Edelman
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D015151:Immunoblotting; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D007525:Isoelectric Focusing; D053538:Keratin-18; D053533:Keratin-8; D007633:Keratins / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D008856:Microscopy, Fluorescence; D009154:Mutation; D021381:Protein Transport; D040901:Proteomics / Q000379:methods*; D011806:Quinolinium Compounds / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D013997:Time Factors; D014162:Transfection
chemical_list: C505032:CFTR protein, human; C488421:KRT18 protein, human; C426203:KRT8 protein, human; D053538:Keratin-18; D053533:Keratin-8; D011806:Quinolinium Compounds; D034741:RNA, Small Interfering; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C070311:6-methoxy-N-ethylquinolinium; D007633:Keratins