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

Abstract

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.

Rattlesnake Phospholipase A2 Increases CFTR-Chloride Channel Current and Corrects ∆ F508CFTR Dysfunction: Impact in Cystic Fibrosis

“…During investigation of the cytokeratin 8 -NBD1 interaction 10 and using CB as a negative control, we discovered a surprisingly high binding affinity of CB for NBD1. Viperidae snake venom PLA 2 (structurally homologous to inflammatory, nonpancreatic human sPLA 2 -IIA) are known to possess a large spectrum of pharmacological functions.…”

“…To gain further insight into the mechanisms involved in the interactions between CB and F508CFTR, we performed SPR competition experiments, taking advantage of the known interaction between NBD1/F508NBD1 and keratin 8 (K8) 10 . Previously, SPR results showed that K8 binds to F508NBD1 with nanomolar affinity (Colas et al, 2012). Now, as indicated in Fig.…”

Finding new drugs to enhance anion secretion in cystic fibrosis: Toward suitable systems for better drug screening. Report on the pre-conference meeting to the 12th ECFS Basic Science Conference, Albufeira, 25–28 March 2015

“…The interruption of this interaction allows targeting of functional F508del-CFTR to the plasma membrane with rescue of epithelial Cl − transport. It has been proposed that the site of interaction between F508del-CFTR and K8 is a target for F508del pharmacotherapy [13,14]. Using a hypothesisdriven approach based on a molecular dynamic model of NBD1/F508del-NBD1 [16], molecular docking, and functional and biochemical assays, two abnormal pockets on F508del-NBD1 have been identified.…”

Structural Dynamics of the Vimentin Coiled-coil Contact Regions Involved in Filament Assembly as Revealed by Hydrogen-Deuterium Exchange

“…Intermediate filament (IF) 3 proteins form highly resilient filaments that are markedly resistant to mechanical stress. Mediated by prominent cytolinker proteins of the plakin family and motor proteins, they integrate actin filaments and microtubules to establish a functional cytoskeleton in metazoan cells and promote optimal tissue function (1).…”

“…Mediated by prominent cytolinker proteins of the plakin family and motor proteins, they integrate actin filaments and microtubules to establish a functional cytoskeleton in metazoan cells and promote optimal tissue function (1). Apart from their basic mechanical function in maintaining cell flexibility, they are also involved in multiple cellular activities that range from cell division and motility to the topological organization of transmembrane channels (2,3). Because of these multifunctional properties of IF proteins, mutations in IF-encoding genes cause almost 100 different inherited diseases in humans (4,5).…”