Journal Article

Disruption of cytokeratin-8 interaction with F508del-CFTR corrects its functional defect

Julien Colas, Grazyna Faure, Emilie Saussereau, Stéphanie Trudel, Wael M. Rabeh, Sara Bitam, Ida Chiara Guerrera, Janine Fritsch, Isabelle Sermet-Gaudelus, Noëlie Davezac, Franck Brouillard, Gergely L. Lukacs, Harald Herrmann, Mario Ollero and Aleksander Edelman

in Human Molecular Genetics

Volume 21, issue 3, pages 623-634
Published in print February 2012 | ISSN: 0964-6906
Published online October 2011 | e-ISSN: 1460-2083 | DOI: http://dx.doi.org/10.1093/hmg/ddr496
Disruption of cytokeratin-8 interaction with F508del-CFTR corrects its functional defect

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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.

Journal Article.  7078 words.  Illustrated.

Subjects: Genetics and Genomics

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