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Implication of calnexin in the F508del-CFTR correction by miglustat
Medicine Sciences and Healthcare Journal (MSHJ), Volume 2, Jan 2017

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Abstract
The most common mutation in cystic fibrosis (CF), F508del, results in CFTR (CF transmembrane conductance regulator) protein that is retained in the endoplasmic reticulum (ER). Previously, we have shown that miglustat corrects the defective trafficking of F508del-CFTR and hypothesized that by inhibiting the interaction of F508del-CFTR with calnexin, a lectin implicated in the ERQC (ER quality control), miglustat prevents the retention and the degradation of F508del-CFTR (Norez et al., 2006). However, others contest the role of calnexin in the F508del-CFTR retention (Okiyoneda et al., 2008). The purpose of the study was i) to determine the effect of small interfering RNA (siRNA) calnexin treatment on endogenous F508del-CFTR trafficking, ii) to compare these results with a miglustat induced-correction, iii) to understand whether calnexin is implicated in the F508del-CFTR trafficking and in its correction induced by miglustat. The human CF tracheal cell line CF-KM4 was transfected with a siRNA calnexin (0.5?g/mL, 72h), a siRNA control (0.5?g/mL) or was treated by miglustat (100?M, 2h). Then, the level of calnexin expression was tested by biochemical technique and consequences on CFTR and ENaC activities were assessed using single-cell fluorescence imaging. The results were compared with those obtained on untreated- and reverted- (CF-KM4 stably transfected with the CFTR wild type) CF-KM4 cells. We showed that decreasing calnexin expression (~ 75%) restores F508del-CFTR activity at the plasma membrane in correlation with a decrease (66%) of ENaC activity. Moreover, we found a 1.5 fold higher level of correction induced by miglustat than with siRNA calnexin : this level corresponds to the level of CFTR and ENaC activities measured in reverted CF-KM4. In conclusion, this work is in favor of a role of calnexin in the F508del-CFTR retention and confirms calnexin as a valuable CF therapeutic target. Nevertheless, our results also suggest that inhibition of calnexin/F508del-CFTR interaction is probably not solely sufficient to fully explain the effects of miglustat raising the hypothesis that another molecular target for this drug exists.

Author(s): Dorothee Raveau, Anne Cantereau, Frederic Becq, Caroline Norez
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