Article Figures & Data
Figures
- Fig. 1.
Class I corrector VX-809 stabilizes the interaction between MSD1 and MSD2. (A, panel i) HEK293 cells were transiently transfected with MSD1 or MSD2-NBD2, or cotransfected with MSD1 and MSD2-NBD2 domains. (B, panel i) HEK293 cells were transiently transfected with MSD1 or MSD2, or cotransfected with MSD1 and MSD2 domains. Cells were treated for 24 hours at 37°C with DMSO, VX-809 (3 μM). The MSD1 fragment was detected with an antibody against the N-terminus of CFTR (i.e., mAb MM13-4), and the MSD2-NBD2 fragment was detected with an antibody against the C-terminus of CFTR (mAb 596) and MSD2 fragment with antibody A52. (A, panel ii) Bar graphs show the mean (±S.E.M.) of the percentage of abundance of MSD1 normalized to calnexin loading. Data are representative of four biologic studies (**P < 0.01; ***P < 0.001). (A, panel iii) Bar graphs show the mean (±S.E.M.) of the percentage of abundance of MSD2-NBD2 normalized to calnexin loading. Data are representative of four biologic studies (****P < 0.0001). (B, panel ii) Bar graphs show the mean (±S.E.M.) of the percentage of abundance of MSD1 normalized to calnexin loading. Data are representative of four experiments (*P < 0.05; ***P < 0.001; ****P < 0.0001). (B, panel iii) Bar graphs show the mean (±S.E.M.) of the percentage of abundance of MSD2 normalized to calnexin loading. Data are representative of four biologic studies (***P < 0.001). n.s., not significant.
- Fig. 2.
The class II corrector C4 stabilizes only MSD2-NBD2. (A, panel i) HEK293 cells were transiently transfected with MSD1 or MSD2-NBD2, or cotransfected with MSD1 and MSD2-NBD2 domains and treated for 24 hours at 37°C with DMSO or C4 (10 μM). The MSD1 fragment was detected with an antibody against the N-terminus of CFTR (i.e., mAb MM13-4), and the MSD2-NBD2 fragment was detected with an antibody against the C-terminus of CFTR (mAb 596). (A, panel ii) Bar graphs show the mean (±S.E.M.) of the percentage of abundance of MSD1 normalized to calnexin loading. Data are representative of four biologic studies. (A, panel iii) Bar graphs show the mean (±S.E.M.) of the percentage of abundance of MSD2-NBD2 normalized to calnexin loading. Data are representative of three biologic studies (****P < 0.0001). n.s., not significant.
- Fig. 3.
C4 directly stabilizes MSD2 of CFTR. (A) Endoglycosidase sensitivity of the MSD2-CFTR fragment. Whole cell extracts of HEK293 cells transfected with A52-tagged MSD2-CFTR cDNA were treated with endoglycosidase H (H), peptide-N-glycosidase F (F), or untreated (–). (B) Effect of C4 (10 µM), TMA (500 nM), or VX-809 (3 µM) on the abundance of A52-tagged MSD2-CFTR in HEK293 cells. (C and D) MSD1 or MSD2 domains of CFTR were expressed in HEK293 cells in the presence or absence of C4 (10 μM) at 27°C. After 24 hours, protein synthesis was inhibited by addition of cycloheximide (0.5 mg/ml) with or without C4 (10 μM), and cells were collected after the indicated times for western blot analysis of whole cell extracts. (C, panel i; and D, panel i) The amount of CFTR protein at each time point was quantitated and expressed relative to that at time 0 and calnexin loading. Data are representative of three biologic studies (**P < 0.01).
- Fig. 4.
Additive effect of VX-809 and C4 on correction of F508del-CFTR. (A–C) HEK293 cells were transiently transfected with F508del-CFTR, R170G/F508del, or F508del/R1070W-CFTR, and treated for 24 hours at 37°C with DMSO, VX-809 (3 μM) and/or C4 (10 μM). F508del-CFTR, R170G/F508del-CFTR, and F508del/R1070W-CFTR were detected with an antibody against the N-terminus of CFTR (MM13-4). (C) Mature, complex-glycosylated CFTR; (B) immature, core-glycosylated CFTR. (A, panel i; B, panel i; and C, panel i) Bars represent the mean (±S.E.M.) of the ratio C/(C+B) and are representative of three biologic studies (*P < 0.05; **P < 0.01; ***P < 0.001). (A, panel ii; B, panel ii; and C, panel ii) Bars represent the B+C and band B forms, normalized to DMSO and are representative of three biologic studies (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Stippled horizontal lines in (A and C) show prediction for additive effect of VX-809 plus C4 on C/C+B (left bar graph) or band C (right bar graph). Dotted horizontal line in (B) shows prediction for additive effect on band B (right bar graph). n.s., not significant.
- Fig. 5.
Graphic hypothesis depicting putative interaction between VX-809 and C4 and key role of ICL1 (CL1) in corrector activity. (Upper panel) The illustration shows the MSD1 and F508del-NBD1 as peach colored and MSD2 and NBD2 as cyan colored. (Lower panel) The illustration on the left-hand side highlights the putative stabilizing effect of VX-809 (red) on MSD1 with its allosteric effect on MSD2. The illustration in the middle shows the putative stabilizing effect of C4 (green) on MSD2. The illustration on the right-hand side shows the putative interaction of VX-809 plus C4 on MSD2 (orange) and the primary role of CL1 on mediating correction by the combination.
Additional Files
Data Supplement
- Supplemental Figures -
Supplemental Figure 1 - C4 stabilizes MSD2-NBD2 of CFTR
Supplemental Figure 2 - Additive effect of VX-809 and C4 on processing of R170G-CFTR
Supplemental Figure 3 - VX-809, C4 and glycerol correction of F508del-CFTR is additive with the R1070W second-site mutation
- Supplemental Figures -
