Disruption of cytokeratin-8 interaction with F508del-CFTR corrects its functional defect.
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.
Faculté de Médecine Paris-Descartes, INSERM, U845, Paris, France.
Julien
J
Colas
Grazyna
G
Faure
Emilie
E
Saussereau
Stéphanie
S
Trudel
Wael M
WM
Rabeh
Sara
S
Bitam
Ida Chiara
IC
Guerrera
Janine
J
Fritsch
Isabelle
I
Sermet-Gaudelus
Noëlie
N
Davezac
Franck
F
Brouillard
Gergely L
GL
Lukacs
Harald
H
Herrmann
Mario
M
Ollero
Aleksander
A
Edelman
D000818:Animals; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D005260:Female; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D053538:Keratin-18 / Q000378:metabolism; D053533:Keratin-8 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008297:Male; D051379:Mice; D009154:Mutation; D009666:Nose / Q000166:cytology; D054730:Protein Interaction Domains and Motifs
D053538:Keratin-18; D053533:Keratin-8; D019005:Cystic Fibrosis Transmembrane Conductance Regulator