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Article Dans Une Revue Journal of Pharmacology and Experimental Therapeutics Année : 2008

Proteasome-Dependent Pharmacological Rescue of Cystic Fibrosis Transmembrane Conductance Regulator Revealed by Mutation of Glycine 622

Résumé

The most common mutation (F508del) causing cystic fibrosis (CF) results in misfolding of the CF transmembrane conductance regulator (CFTR), leading to its degradation via the proteasome pathway. To study the mechanism of action of several pharmacological chaperones benzo[c]quinolizinium (MPB), we analyzed their effects on two CF mutations; F508del-CFTR and G622D-CFTR. The replacement of Gly622 by an aspartic acid (G622D) alters the trafficking and activity of the protein. G622D, similar to F508del, was functionally rescued by the glucosidase inhibitor miglustat but, unlike F508del, could not be rescued by MPB. A structure-activity relationship for F508del functional correction revealed the following profile: MPB-104-91-07-80 > 05 > 89 >> 9-hydroxyphenanthrene = phenanthrene. Coimmunoprecipitation experiments on human airway epithelial F508del/F508del CF15 cells showed that MPB did not prevent the interaction of F508del-CFTR with heat shock protein (HSP)70, HSP90, or calnexin. Functional rescue of F508del-CFTR by MPB and miglustat was abolished by brefeldin A (BFA) but potentiated by thapsigargin (TG) and geldanamycin. The proteasome inhibitor MG132 potentiated the effect of miglustat but only modestly affected that of MPB. It is noteworthy that MPB inhibited proteasome activity in F508del-CFTR-expressing cells but did not directly affect the activity of purified 20S proteasome. With the mutant G622D-CFTR, MPB did not inhibit proteasome activity, as in mock-transfected cells. Inhibition of cellular degradation machinery by MPB is not only CFTR-dependent, but it also follows similar structure-activity relationship as demonstrated by functional correction. We conclude that G622D is a partial trafficking-deficient mutant with dysfunctional chloride channel activity, and that Gly622 is part of the putative site for interaction of MPB with CFTR, protecting the channel from proteasome-mediated degradation.
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hal-00392917 , version 1 (09-06-2009)

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Caroline Norez, Frédéric Bilan, Alain Kitzis, Yvette Mettey, Frédéric Becq. Proteasome-Dependent Pharmacological Rescue of Cystic Fibrosis Transmembrane Conductance Regulator Revealed by Mutation of Glycine 622. Journal of Pharmacology and Experimental Therapeutics, 2008, 25 (1), pp.89-99. ⟨10.1124/jpet.107.134502⟩. ⟨hal-00392917⟩
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