Received for publication November 26, 2007.
Revised January 28, 2008.
Accepted for publication January 28, 2008.

Proteasome-dependent pharmacological rescue of CFTR revealed by mutation of glycine 622

Abstract

The most common mutation F508del causing Cystic Fibrosis results in misfolding of CFTR leading to its degradation via the proteasome pathway. To study the mechanism of action of several pharmacological chaperones benzo[c]quinolizinium (MPB), we have analysed their effects on two CF mutations; F508del- and G622D-CFTR. The replacement of G622 by an aspartic acid (G622D) alters the trafficking and activity of the protein. G622D, like 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. Co-immunoprecipitation experiments on human airway epithelial F508del/F508del CF15 cells showed that MPB did not prevent the interaction of F508del-CFTR with HSP-70, HSP-90 or calnexin. Functional rescue of F508del-CFTR by MPB and miglustat was abolished by brefeldin A but potentiated by thapsigargin and geldanamycin. The proteasome inhibitor MG132 potentiated the effect of miglustat but only modestly the MPB one. Importantly, MPB inhibited proteasome activity in F508del-CFTR expressing cells but did not affect directly the activity of purified 20S proteasome. With the mutant G622D-CFTR, MPB did not inhibit proteasome activity, like in mock transfected cells. Inhibition of cellular degradation machinery by MPB is not only CFTR-dependent but also follows similar structure-activity relationship as demonstrated for functional correction. We conclude that G622D is a partial trafficking-deficient mutant with dysfunctional chloride channel activity, and that G622 is part of the putative site for interaction of MPB with CFTR, protecting the channel from proteasome-mediated degradation.

Key words: F508del, G622D, benzoquinolizinium compounds, cystic fibrosis, pharmacological chaperone, proteasome