TY - JOUR
T1 - Disruption of cytokeratin-8 interaction with F508del-CFTR corrects its functional defect
AU - Colas, Julien
AU - Faure, Grazyna
AU - Saussereau, Emilie
AU - Trudel, Stéphanie
AU - Rabeh, Wael M.
AU - Bitam, Sara
AU - Guerrera, Ida Chiara
AU - Fritsch, Janine
AU - Sermet-gaudelus, Isabelle
AU - Davezac, Noëlie
AU - Brouillard, Franck
AU - Lukacs, Gergely L.
AU - Herrmann, Harald
AU - Ollero, Mario
AU - Edelman, Aleksander
N1 - Funding Information:
This work was supported by European Commission grant ‘NEUPROCF’ (FP6) (to A.E.); by French Agence Nationale de la Recherche grant ‘EICOCF’ (to A.E., M.O.); by the associations Vaincre la Mucoviscidose (to A.E., M.O.) and Mucoviscidose: ABCF2 (to A.E., I.S.-G.); Legs Poix-University of Paris 5 (to A.E., M.O.); the Canadian Institutes of Health Research; and NIH-NIDDK and Canadian Cystic Fibrosis Foundation (to G.L.L.).
PY - 2012/2
Y1 - 2012/2
N2 - We have previously reported an increased expression of cytokeratins 8/18 (K8/K18) in cells expressing the F508del mutation of cystic fibrosis transmembrane conductance regulator (CFTR). This is associated with increased colocalization of CFTR and K18 in the vicinity of the endoplasmic reticulum, although this is reversed by treating cells with curcumin, resulting in the rescue of F508del-CFTR. In the present work, we hypothesized that (i) the K8/K18 network may interact physically with CFTR, and that (ii) this interaction may modify CFTR function. CFTR was immunoprecipitated from HeLa cells transfected with either wild-type (WT) CFTR or F508del-CFTR. Precipitates were subjected to 2D-gel electrophoresis and differential spots identified by mass spectrometry. K8 and K18 were found significantly increased in F508del-CFTR precipitates. Using surface plasmon resonance, we demonstrate that K8, but not K18, binds directly and preferentially to the F508del over the WT human NBD1 (nucleotide-binding domain-1). In vivo K8 interaction with F508del-CFTR was confirmed by proximity ligation assay in HeLa cells and in primary cultures of human respiratory epithelial cells. Ablation of K8 expression by siRNA in F508del-expressing HeLa cells led to the recovery of CFTR-dependent iodide efflux. Moreover, F508del-expressing mice topically treated with K8-siRNA showed restored nasal potential difference, equivalent to that of WT mice. These results show that disruption of F508del-CFTR and K8 interaction leads to the correction of the F508del-CFTR processing defect, suggesting a novel potential therapeutic target in CF.
AB - We have previously reported an increased expression of cytokeratins 8/18 (K8/K18) in cells expressing the F508del mutation of cystic fibrosis transmembrane conductance regulator (CFTR). This is associated with increased colocalization of CFTR and K18 in the vicinity of the endoplasmic reticulum, although this is reversed by treating cells with curcumin, resulting in the rescue of F508del-CFTR. In the present work, we hypothesized that (i) the K8/K18 network may interact physically with CFTR, and that (ii) this interaction may modify CFTR function. CFTR was immunoprecipitated from HeLa cells transfected with either wild-type (WT) CFTR or F508del-CFTR. Precipitates were subjected to 2D-gel electrophoresis and differential spots identified by mass spectrometry. K8 and K18 were found significantly increased in F508del-CFTR precipitates. Using surface plasmon resonance, we demonstrate that K8, but not K18, binds directly and preferentially to the F508del over the WT human NBD1 (nucleotide-binding domain-1). In vivo K8 interaction with F508del-CFTR was confirmed by proximity ligation assay in HeLa cells and in primary cultures of human respiratory epithelial cells. Ablation of K8 expression by siRNA in F508del-expressing HeLa cells led to the recovery of CFTR-dependent iodide efflux. Moreover, F508del-expressing mice topically treated with K8-siRNA showed restored nasal potential difference, equivalent to that of WT mice. These results show that disruption of F508del-CFTR and K8 interaction leads to the correction of the F508del-CFTR processing defect, suggesting a novel potential therapeutic target in CF.
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U2 - 10.1093/hmg/ddr496
DO - 10.1093/hmg/ddr496
M3 - Article
C2 - 22038833
AN - SCOPUS:84856096159
SN - 0964-6906
VL - 21
SP - 623
EP - 634
JO - Human Molecular Genetics
JF - Human Molecular Genetics
IS - 3
M1 - ddr496
ER -