ABCMdb

PMID: 18230692

Norez C, Bilan F, Kitzis A, Mettey Y, Becq F
Proteasome-dependent pharmacological rescue of cystic fibrosis transmembrane conductance regulator revealed by mutation of glycine 622.
J Pharmacol Exp Ther. 2008 Apr;325(1):89-99. Epub 2008 Jan 29., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Gly622Asp 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. Login to comment 2 ABCC7 p.Gly622Asp ABCC7 p.Gly622Asp The replacement of Gly622 by an aspartic acid (G622D) alters the trafficking and activity of the protein. Login to comment 3 ABCC7 p.Gly622Asp G622D, similar to F508del, was functionally rescued by the glucosidase inhibitor miglustat but, unlike F508del, could not be rescued by MPB. Login to comment 9 ABCC7 p.Gly622Asp With the mutant G622D-CFTR, MPB did not inhibit proteasome activity, as in mock-transfected cells. Login to comment 11 ABCC7 p.Gly622Asp 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. Login to comment 25 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Several other less frequent CF mutations such as G551D or G1349D are classified as class 3 because the corresponding proteins, although correctly located at the plasma membrane, have a dysfunctional regulation (see for a recent review, MacDonald et al., 2007). Login to comment 26 ABCC7 p.Gly622Asp The G622D-CFTR mutant, in which the pathophysiology is unclear, has been provisionally classified as a class 3 missense mutation after its identification in CF patients (http://www.genet.sickkids.on.ca/cftr) (Vankeerberghen et al., 1998). Login to comment 28 ABCC7 p.Gly622Asp The G622D mutant forms a cAMP-regulated chloride channel with significantly lower Po than the wild-type channels (Vankeerberghen et al., 1998). Login to comment 34 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp In previous studies, we identified benzo[c]quinolizinium (MPB) derivatives (several chemical structures are shown in Fig. 1B together with two phenanthrene derivatives) as activators of wild-type CFTR, and G551D, G1349D, and F508del mutants (Becq et al., 1999; Dormer et al., 2001a; Marivingt-Mounir et al., 2004; Melin et al., 2004). Login to comment 36 ABCC7 p.Gly622Asp In the present work, we performed a comparative analysis of the effect of several chemically diverse MPB and phenanthrene derivatives on two CFTR mutants, F508del and G622D. Login to comment 38 ABCC7 p.Gly622Asp A N C Wt 620H-E-G-S-S- G622D 620H-E-D-S-S- NBD1 NBD2 R-domain N C Wt 505N-I-I-F-G- F508del 505N-I-I-G NBD1 NBD2 R-domain N+ OH Cl Cl5-butyl-10-chloro-6-hydroxybenzo[c]quinolizinium chloride MPB-91 phenanthrene OH 9-hydroxyphenanthrene MPB-104 NCl OH Cl MPB-89 NCl B N+ OH Cl6-hydroxybenzo[c]quinolizinium chloride MPB-05 N+ Cl OH Cl6-hydroxy-10-chlorobenzo[c]quinolizinium chloride MPB-07 MPB-07 N+ OH F Cl- 10-fluoro-6-hydroxybenzo[c]quinolizinium MPB80MPB-80MPB-05 N+ OH F Cl- 10-fluoro-6-hydroxybenzo[c]quinolizinium MPB80 MPB-80 MPB-91 Fig. 1. Login to comment 39 ABCC7 p.Gly622Asp A, scheme illustrating the position of F508del and G622D mutations on a CFTR protein cartoon. Login to comment 42 ABCC7 p.Gly622Asp For this study, we used the human nasal airway epithelial cell line JME/CF15, derived from a CF patient homozygous for the F508del mutation (Jefferson et al., 1990), and COS-7 cells stably transfected with green fluorescent protein (GFP)-tagged CFTR vectors containing either wild-type (wt)-, F508del-, or G622D-CFTR. Login to comment 59 ABCC7 p.Gly622Asp The wt-, F508del-, and G622D-CFTR Cl-channel activities were assayed by measuring the rate of iodide (125 I) efflux from CF15 cells and COS-7 cells as described previously (Melin et al., 2004; Norez et al., 2006a). Login to comment 77 ABCC7 p.Gly622Asp Results MPB Compounds Rescue F508del-CFTR but Not G622D-CFTR. Login to comment 78 ABCC7 p.Gly622Asp The plasmid construction (see Materials and Methods) allowed stable expression in COS-7 cells of GFP-tagged F508del-CFTR and GFP-tagged G622D-CFTR. Login to comment 82 ABCC7 p.Gly622Asp Figure 2A shows an example of CFTR-dependent iodide efflux in F508del-, G622D-, and wt-CFTR-expressing COS-7 cells. Login to comment 84 ABCC7 p.Gly622Asp For G622D-CFTR-expressing cells (Fig. 2A, black triangles), Fsk ϩ Gst stimulated the iodide efflux (kpeak - kbasal ϭ 0.046 Ϯ 0.020 min-1 ) to a level Ϸ3-fold lower than that of wt-CFTR cells (Fig. 2A, black squares; kpeak - kbasal ϭ 0.155 Ϯ 0.009 min-1 ). Login to comment 86 ABCC7 p.Gly622Asp These results suggest that, although functional (in agreement with Vankeerberghen et al., 1998), G622D-expressing COS-7 cells have a reduced transport activity compared with wt-CFTR cells. Login to comment 88 ABCC7 p.Gly622Asp An alternative explanation would be that the trafficking of G622D is abnormal. Login to comment 92 ABCC7 p.Gly622Asp Figure 2B presents typical iodide efflux responses, showing that in G622D-CFTR cells incubated with miglustat (black triangles), Fsk ϩ Gst increased (Ϸ2-fold) iodide efflux compared to untreated cells (Fig. 2B, open squares; p Ͻ 0.001). Login to comment 93 ABCC7 p.Gly622Asp However, with G622D-CFTR cells incubated with MPB-104 (Fig. 2B, black circles), the Fsk ϩ Gst-stimulated iodide efflux was not different from control. Login to comment 95 ABCC7 p.Gly622Asp Therefore, these results show a partial trafficking defect of G622D-CFTR that can be reversed by miglustat as with the F508del mutant. Login to comment 98 ABCC7 p.Gly622Asp It is surprising to note that the incubation of cells with any of the MPB correctors did not rescue G622D-CFTR (Fig. 2C, gray bars). Login to comment 99 ABCC7 p.Gly622Asp If G622D is a partial trafficking-deficient mutant, as our functional data suggest, then we should observe less band C forms of the mutant compared to wt-CFTR, indicating a reduced pool of mature proteins. Login to comment 100 ABCC7 p.Gly622Asp Thus, we performed biochemical assays to compare the G622D-CFTR protein expression in the presence or absence of these compounds. Login to comment 103 ABCC7 p.Gly622Asp ABCC7 p.Gly622Asp ABCC7 p.Gly622Asp Anti-GFP immunoblotting in COS-7 cells expressing G622D-CFTR (Fig. 2D, lane 2) shows a reduced amount of band C form but a much higher quantity of band B form for G622D versus wt proteins, confirming the partial trafficking defect of G622D. Login to comment 104 ABCC7 p.Gly622Asp In COS-7 cells expressing G622D-CFTR incubated for 2 h with 100 ␮M miglustat, anti-GFP immunoblotting revealed an increased band C intensity (Fig. 2D, lane 4). Login to comment 107 ABCC7 p.Gly622Asp However, and contrary to F508del-CFTR rescued by MPB derivatives (Dormer et al., 2001a), the abnormal processing of G622D-CFTR cannot be rescued by MPB. Login to comment 124 ABCC7 p.Gly622Asp ABCC7 p.Gly622Asp ABCC7 p.Gly622Asp ABCC7 p.Gly622Asp ABCC7 p.Gly622Asp The efflux Band C Band B wt G622D MPB-104 Miglustat - - - - + - - + Band C Band B wt G622D MPB-104 Miglustat - - - - + - - + Band C Band B wt G622D MPB-104 Miglustat - - - - - - - - + - + - - + - + C A 0.00 0.05 0.10 0.15 0.20 F508del-CFTR G622D-CFTR ns ** ns*** Ctrl MPB-07 MPB-80 MPB-91 - - - - + - - - - + - - - - + Miglustat - - - - + - - - - - - - - - - MPB-104 - - - -- + - - - - + - - - - + - - - - + - - - - + - - - - - - - - - - - - - -- + 12 8 8 8 812 8 8 8 81624 kpeak-kbasal(min-1 ) B 0 2 4 6 8 0.00 0.05 0.10 0.15 0.20 0.25 wt-CFTR G622D-CFTR F508del-CFTR Fsk + Gst Time (min-1 ) k(min-1 ) D 0 2 4 6 8 0.00 0.05 0.10 0.15 0.20 untreated MPB-104 Miglustat Fsk + Gst Time (min) k(min-1 ) 1 2 3 4 Fig. 2. Login to comment 125 ABCC7 p.Gly622Asp Rescue of F508del-CFTR but not of G622D-CFTR trafficking by MPB derivatives. Login to comment 126 ABCC7 p.Gly622Asp A, examples of iodide efflux curves obtained in COS-7 cells stably transfected with wt-, F508del-, or G622D-CFTR and stimulated by Fsk/Gst as indicated by the horizontal bar above the traces. Login to comment 127 ABCC7 p.Gly622Asp B, examples of iodide efflux curves obtained for G622D-CFTR-expressing COS-7 cells treated or not with miglustat (100 ␮M, 2 h) or MPB-104 (100 ␮M, 2 h) and stimulated by Fsk/Gst. Login to comment 128 ABCC7 p.Gly622Asp ABCC7 p.Gly622Asp C, bar graph showing the Fsk ϩ Gst-dependent iodide efflux in COS-7 cells stably transfected with F508del-or G622D-CFTR and treated by MPB compounds or miglustat (100 ␮M, 2 h). Login to comment 132 ABCC7 p.Gly622Asp ‫,ءءء‬ p Ͻ 0.001, ns, nonsignificant difference. D, expression of wt-CFTR or G622D-CFTR in COS-7 cells treated as indicated above each lane. Login to comment 209 ABCC7 p.Gly622Asp Because this effect could be attributed to a nonspecific action of MPB, we also measured the proteasome activity in mock COS-7 cells (Fig. 7A) or COS-7 cells stably expressing F508del-CFTR (Fig. 7B) or G622D-CFTR (Fig. 7C). Login to comment 215 ABCC7 p.Gly622Asp Finally, because we showed that MPB correctors are not effective on G622D-CFTR, we asked whether this mutation could also affect the MPB-mediated inhibition of the degradation machinery. Login to comment 216 ABCC7 p.Gly622Asp To study this theory, the proteasome activity was also measured in COS-7 cells expressing G622D proteins. Login to comment 219 ABCC7 p.Gly622Asp Altogether, these results demonstrate that MPB correctors inhibit the cell proteasome activity in a CFTR-dependent manner and suggest that the mutation G622D prevents this inhibition. Login to comment 220 ABCC7 p.Gly622Asp ABCC7 p.Gly622Asp Discussion The present experiments demonstrate the following: 1) MPB derivatives are pharmacological chaperones of F508del-CFTR but not of G622D-CFTR mutants; 2) MPB corrects the F508del-CFTR-trafficking defect via a specific structure-activity relationship; 3) MPB does not prevent the interaction of F508del-CFTR with the ER resident calnexin or cytosolic HSP70 and HSP90 molecular chaperones; 4) MPB correctors inhibit the proteasome activity only in CFTR-expressing cells and have no effect on the activity of purified 20S proteasome; and 5) the mutant G622D-CFTR prevents rescue of CFTR by MPB but also abolishes the inhibition of the proteasome by MPB derivatives. Login to comment 227 ABCC7 p.Gly622Asp ‫,ءءء‬ p Ͻ 0.001 and ‫,ء‬ p Ͻ 0.05, ns, nonsignificant difference. A B C 0 25 50 75 100 125 MG132 Lactacystine MPB-104 MPB-91 MPB-80 MPB-07 MPB-89 phenanthrene DMSO Ctrl + ns ns *** ** ns Proteasome activity in Cos-7 F508del-CFTR cells (%) 0 25 50 75 100 125 MG132 Lactacystine MPB-104 MPB-91 MPB-80 MPB-07 MPB-89 phenanthrene DMSO Ctrl + ns *** ns Proteasome activity in Cos-7 G622D-CFTR cells (%) 0 25 50 75 100 125 MG132 Lactacystine MPB-104 MPB-91 MPB-80 MPB-07 MPB-89 phenanthrene DMSO Ctrl + ns *** ns Proteasome activity in Cos-7 mock cells (%) Fig. 7. Login to comment 229 ABCC7 p.Gly622Asp Histograms showing the effect of MPB correctors on proteasome activity in mock COS-7 cells (A) or COS-7 cells stably transfected with F508del-CFTR (B) or G622D-CFTR (C). Login to comment 234 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp In a previous study analyzing the effect of CFTR channel activators, we reported a structure-activity relationship of MPB compounds (Marivingt-Mounir et al., 2004), and we demonstrated that some of these derivatives were able to stimulate the channel activity of wt-, G551D-, G1349D-, G551D/ G1349D-, and F508del-CFTR (Becq et al., 1999; Dormer et al., 2001a; Marivingt-Mounir et al., 2004; Melin et al., 2004). Login to comment