PMID: 23890012

Farinha CM, King-Underwood J, Sousa M, Correia AR, Henriques BJ, Roxo-Rosa M, Da Paula AC, Williams J, Hirst S, Gomes CM, Amaral MD
Revertants, low temperature, and correctors reveal the mechanism of F508del-CFTR rescue by VX-809 and suggest multiple agents for full correction.
Chem Biol. 2013 Jul 25;20(7):943-55. doi: 10.1016/j.chembiol.2013.06.004., [PubMed]
Sentences
No. Mutations Sentence Comment
16 ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:16:441
status: NEW
view ABCC7 p.Arg1070Trp details
ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:16:257
status: NEW
view ABCC7 p.Gly550Glu details
ABCC7 p.Val510Asp
X
ABCC7 p.Val510Asp 23890012:16:413
status: NEW
view ABCC7 p.Val510Asp details
The second F508del-associated defect impairs CFTR interdomain folding, namely, (1) the NBD1-NBD2 dimerization interface (critical for channel activation and accounting for the F508del-CFTR gating defect; Dalemans et al., 1991), which can be rescued by the G550E revertant, and (2) the interaction of NBD1 with the fourth intracellular loop (ICL4) of TMD2 (Serohijos et al., 2008), shown to be reverted by either V510D (Loo et al., 2010) or R1070W, which both fill the pocket left empty by F508del (Thibodeau et al., 2010). Login to comment
23 ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:23:365
status: NEW
view ABCC7 p.Arg1070Trp details
ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:23:246
status: NEW
view ABCC7 p.Gly550Glu details
ABCC7 p.Arg555Lys
X
ABCC7 p.Arg555Lys 23890012:23:281
status: NEW
view ABCC7 p.Arg555Lys details
ABCC7 p.Val510Asp
X
ABCC7 p.Val510Asp 23890012:23:401
status: NEW
view ABCC7 p.Val510Asp details
Herein, we explored the MoA of VX-809 by analyzing its synergistic/additive effect with those of previously characterized genetic revertants, which rescue F508del-CFTR by causing different effects: 4RK affecting traffic (Roxo-Rosa et al., 2006), G550E (Roxo-Rosa et al., 2006) and R555K increasing channel gating by strengthening the NBD1:NBD2 dimer interface, and R1070W (Serohijos et al., 2008) and V510D (Wang et al., 2007a; Loo et al., 2010) by filling the NBD1:ICL4 interface. Login to comment
36 ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:36:443
status: NEW
view ABCC7 p.Arg1070Trp details
ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:36:432
status: NEW
view ABCC7 p.Gly550Glu details
ABCC7 p.Arg555Lys
X
ABCC7 p.Arg555Lys 23890012:36:469
status: NEW
view ABCC7 p.Arg555Lys details
ABCC7 p.Val510Asp
X
ABCC7 p.Val510Asp 23890012:36:455
status: NEW
view ABCC7 p.Val510Asp details
VX-809 Adds to VRT-325 and Corr-4a to Rescue F508del-CFTR but Exhibits Variable Effects on Genetic Revertants In order to characterize the rescue mechanism of VX-809 on F508del-CFTR, we then tested the effect of incubating it together with VRT-325 and Corr-4a on BHK cells stably expressing this mutant alone or in cis with the following genetic revertants: (1) 4RK (where the four AFTs were simultaneously mutated to lysines), (2) G550E, (3) R1070W, (4) V510D, or (5) R555K (Figures 1A-1E). Login to comment
41 ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:41:159
status: NEW
view ABCC7 p.Gly550Glu details
ABCC7 p.Arg555Lys
X
ABCC7 p.Arg555Lys 23890012:41:168
status: NEW
view ABCC7 p.Arg555Lys details
Interestingly, however, analysis of the effects of the three compounds upon the revertants showed that VX-809 (but neither VRT-325 nor Corr-4a) is additive to G550E or R555K in correcting F508del-CFTR (by 38% and 32%, respectively), strongly suggesting that VX-809 acts differently from these two revertants. Login to comment
42 ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:42:77
status: NEW
view ABCC7 p.Arg1070Trp details
Moreover, whereas both VRT-325 and VX-809 add significantly to the effect of R1070W by 17% (p = 0.011) and 28% (p = 1.6 10 5 ), respectively, Corr-4a does not. Login to comment
44 ABCC7 p.Val510Asp
X
ABCC7 p.Val510Asp 23890012:44:77
status: NEW
view ABCC7 p.Val510Asp details
Curiously, all the three compounds further increase band C levels of F508del-V510D-CFTR, but VRT-325 produces the greatest effect. Login to comment
46 ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:46:147
status: NEW
view ABCC7 p.Arg1070Trp details
ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:46:109
status: NEW
view ABCC7 p.Gly550Glu details
These analyses were performed for three of the revertants with different F508del-CFTR effects (4RK, traffic; G550E, NBD1:NBD2 dimer interface; and R1070W, NBD1:ICL4 interface). Login to comment
48 ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:48:36
status: NEW
view ABCC7 p.Gly550Glu details
For example, an increase in F508del-G550E-CFTR processing is detected at 0.3 mM VX-809, whereas a 10-fold higher concentration is needed for significant rescue of F508del-CFTR (Figure 2C). Login to comment
57 ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:57:180
status: NEW
view ABCC7 p.Arg1070Trp details
ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:57:169
status: NEW
view ABCC7 p.Gly550Glu details
ABCC7 p.Arg555Lys
X
ABCC7 p.Arg555Lys 23890012:57:207
status: NEW
view ABCC7 p.Arg555Lys details
ABCC7 p.Val510Asp
X
ABCC7 p.Val510Asp 23890012:57:192
status: NEW
view ABCC7 p.Val510Asp details
Effect of Small Molecule Correctors on F508del-CFTR and Genetic Revertants (A-F) BHK cell lines stably expressing CFTR bearing F508del alone (A) or in cis with 4RK (B), G550E (C), R1070W (D), V510D (E), and R555K (F) were incubated for 24 hr with 6.7 mM VRT-325, 10 mM Corr-4a, or 3 mM VX-809 alone or in combination. Login to comment
72 ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:72:52
status: NEW
view ABCC7 p.Arg1070Trp details
ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:72:42
status: NEW
view ABCC7 p.Gly550Glu details
ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:72:113
status: NEW
view ABCC7 p.Gly550Glu details
We then assessed the effect of revertants G550E and R1070W by modeling (Figure 3F; Figures S2B and S2C), whereby G550E allows a salt bridge to form across the ATP binding site with Lys1250 and other residues from NBD2 (Figure 3F). Login to comment
73 ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:73:13
status: NEW
view ABCC7 p.Arg1070Trp details
By contrast, R1070W (Figure S2A) plausibly fills in the space left empty by absence of F508 at the NBD1:ICL4 interface as previously proposed (Thibodeau et al., 2010). Login to comment
87 ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:87:220
status: NEW
view ABCC7 p.Arg1070Trp details
ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:87:213
status: NEW
view ABCC7 p.Gly550Glu details
ABCC7 p.Arg555Lys
X
ABCC7 p.Arg555Lys 23890012:87:244
status: NEW
view ABCC7 p.Arg555Lys details
ABCC7 p.Val510Asp
X
ABCC7 p.Val510Asp 23890012:87:233
status: NEW
view ABCC7 p.Val510Asp details
Rescue of F508del-CFTR by Low Temperature Is Additive to Genetic Revertants To learn more about how low temperature rescues F508del-CFTR, we assessed its combined effect with that of the above genetic revertants: G550E, R1070W, 4RK, V510D, and R555K (Figures 4A and 4B). Login to comment
88 ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:88:140
status: NEW
view ABCC7 p.Arg1070Trp details
ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:88:133
status: NEW
view ABCC7 p.Gly550Glu details
ABCC7 p.Arg555Lys
X
ABCC7 p.Arg555Lys 23890012:88:157
status: NEW
view ABCC7 p.Arg555Lys details
ABCC7 p.Val510Asp
X
ABCC7 p.Val510Asp 23890012:88:126
status: NEW
view ABCC7 p.Val510Asp details
Results show that low temperature further increases processing levels of F508del-CFTR by the five genetic revertants, namely, V510D, G550E, R1070W, 4RK, and R555K, by an additional 35%, 65%, 38%, 27%, and 22%, respectively (compare gray and black bars in Figure 4B). Login to comment
89 ABCC7 p.Arg560Thr
X
ABCC7 p.Arg560Thr 23890012:89:128
status: NEW
view ABCC7 p.Arg560Thr details
As a negative control, we also analyzed the effect of low temperature on CFTR bearing another class II disease-causing mutation R560T (Roxo-Rosa et al., 2006), confirming its previously described insensitivity to low temperature rescue (Figures 4A and 4B). Login to comment
96 ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:96:82
status: NEW
view ABCC7 p.Gly550Glu details
(F) Change in F508del NBD1-NBD2 heterodimer interface by the second-site mutation G550E. Login to comment
101 ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:101:132
status: NEW
view ABCC7 p.Arg1070Trp details
ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:101:114
status: NEW
view ABCC7 p.Gly550Glu details
ABCC7 p.Val510Asp
X
ABCC7 p.Val510Asp 23890012:101:121
status: NEW
view ABCC7 p.Val510Asp details
Interestingly, these additive effects were observed not only for revertants promoting protein-autonomous folding (G550E, V510D, and R1070W) but also for the 4RK revertant, which bypasses the AFT-mediated ER retention. Login to comment
102 ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:102:266
status: NEW
view ABCC7 p.Arg1070Trp details
ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:102:233
status: NEW
view ABCC7 p.Gly550Glu details
ABCC7 p.Val510Asp
X
ABCC7 p.Val510Asp 23890012:102:256
status: NEW
view ABCC7 p.Val510Asp details
Combination of Different Genetic Revertants Is Also Additive Next, to assess the full potential for F508del-CFTR rescue, we combined the effects of folding and traffic revertants by producing stable BHK cell lines expressing F508del-G550E-CFTR, where 4RK, V510D, or R1070W were also added in cis, and analyzed processing (Figures 4C and 4D). Login to comment
103 ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:103:47
status: NEW
view ABCC7 p.Arg1070Trp details
ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:103:86
status: NEW
view ABCC7 p.Gly550Glu details
ABCC7 p.Val510Asp
X
ABCC7 p.Val510Asp 23890012:103:36
status: NEW
view ABCC7 p.Val510Asp details
Results in Figure 4C show that 4RK, V510D, and R1070W further increased processing of G550E-F508del-CFTR by another 12%, 59%, and 70%, respectively. Login to comment
104 ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:104:60
status: NEW
view ABCC7 p.Arg1070Trp details
ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:104:33
status: NEW
view ABCC7 p.Gly550Glu details
ABCC7 p.Val510Asp
X
ABCC7 p.Val510Asp 23890012:104:51
status: NEW
view ABCC7 p.Val510Asp details
In fact, the combined effects of G550E with either V510D or R1070W bring F508del-CFTR processing to z80%, i.e., close to levels of WT-CFTR, which can be further increased at 26 C reaching 88%. Login to comment
105 ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:105:19
status: NEW
view ABCC7 p.Gly550Glu details
The combination of G550E with 4RK, although additive, has a more modest effect (32% in total) and is still additive to low temperature (Figure 4D, far right bar). Login to comment
106 ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:106:59
status: NEW
view ABCC7 p.Gly550Glu details
Low Temperature Kinetics of F508del-CFTR Alone or with 4RK/G550E The increased effect of combining correctors, revertants, and low temperature is strongly suggestive of different MoAs. Login to comment
107 ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:107:158
status: NEW
view ABCC7 p.Gly550Glu details
However, to further characterize the MoA of low temperature rescue, we studied the turnover rate and processing efficiency of F508del-CFTR or in cis with 4RK/G550E at 26 C in comparison to these variants at 37 C. Login to comment
123 ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:123:59
status: NEW
view ABCC7 p.Gly550Glu details
The dotted line corresponds to levels of band C in F508del-G550E- CFTR. Login to comment
124 ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:124:142
status: NEW
view ABCC7 p.Gly550Glu details
We then similarly assessed how low temperature affects the turnover and processing of F508del-CFTR bearing the two genetic revertants 4RK and G550E (Figure 6). Login to comment
125 ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:125:48
status: NEW
view ABCC7 p.Gly550Glu details
Results from pulse-chase experiments of F508del-G550E-CFTR show a dramatic reduction in the turnover of the immature form (Figure 6A, left panel), which also corresponds to an increase in its processing efficiency (band C levels) when the chase was performed at 26 C (Figure 6A). Login to comment
128 ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:128:47
status: NEW
view ABCC7 p.Gly550Glu details
The differential behavior observed for F508del-G550E- and F508del-4RK-CFTR at 26 C becomes most evident when these data are plotted as the total amount of band B + band C against time (Figures 6B and 6C). Login to comment
129 ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:129:44
status: NEW
view ABCC7 p.Gly550Glu details
Indeed, whereas the total amount of F508del-G550E-CFTR at 26 C (Figure 6B, gray/dotted bars) onlybarelydecreasesincomparisontoitstotalat37 C(Figure6B, Figure 5. Login to comment
139 ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:139:103
status: NEW
view ABCC7 p.Gly550Glu details
Altogether they indicate that 4RK is not as additive to low temperature in stabilizing F508del-CFTR as G550E. Login to comment
158 ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:158:48
status: NEW
view ABCC7 p.Gly550Glu details
ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:158:117
status: NEW
view ABCC7 p.Gly550Glu details
ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:158:256
status: NEW
view ABCC7 p.Gly550Glu details
Low Temperature Stabilizes the Immature Form of G550E and 4RK Variants of F508del-CFTR and Acts Synergistically with G550E, but not 4RK, to Rescue F508del-CFTR (A-C) Pulse-chase experiments followed by IP were performed in BHK cells expressing (A) F508del-G550E (left panel) or F508del-4RK-CFTR (right panel) incubated at 26 C for 48 hr. After labeling with [35 S] methionine for 3 hr, chase was performed at 26 C (last five lanes in each panel). Login to comment
160 ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:160:75
status: NEW
view ABCC7 p.Gly550Glu details
ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:160:297
status: NEW
view ABCC7 p.Gly550Glu details
Percentage of bands B and C present at each time point of chase in F508del-G550E- (B) or F508del4RK-CFTR (C) incubated for the duration of the experiment (pulse + chase) at either 26 C or 37 C is shown in graph bars to illustrate the additive effect of low temperature and the genetic revertant G550E, but not 4RK, on F508del-CFTR rescue. Login to comment
164 ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:164:80
status: NEW
view ABCC7 p.Gly550Glu details
ABCC7 p.Arg555Lys
X
ABCC7 p.Arg555Lys 23890012:164:90
status: NEW
view ABCC7 p.Arg555Lys details
Indeed, VX-809 (in contrast to either VRT-325 or Corr-4a) adds to the rescue by G550E and R555K (both acting at the NBD1:NBD2 interface), indicating that the compound and these two revertants probably correct distinct conformational cues of F508del-CFTR. Login to comment
165 ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:165:46
status: NEW
view ABCC7 p.Arg1070Trp details
ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:165:140
status: NEW
view ABCC7 p.Arg1070Trp details
ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:165:161
status: NEW
view ABCC7 p.Gly550Glu details
ABCC7 p.Arg555Lys
X
ABCC7 p.Arg555Lys 23890012:165:167
status: NEW
view ABCC7 p.Arg555Lys details
ABCC7 p.Val510Asp
X
ABCC7 p.Val510Asp 23890012:165:57
status: NEW
view ABCC7 p.Val510Asp details
ABCC7 p.Val510Asp
X
ABCC7 p.Val510Asp 23890012:165:147
status: NEW
view ABCC7 p.Val510Asp details
In contrast, the additive effect of VX-809 to R1070W and V510D is rather modest, thus suggesting that this corrector acts more similarly to R1070W/V510D than to G550E/R555K. Login to comment
166 ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:166:143
status: NEW
view ABCC7 p.Arg1070Trp details
ABCC7 p.Val510Asp
X
ABCC7 p.Val510Asp 23890012:166:159
status: NEW
view ABCC7 p.Val510Asp details
VRT-325 in turn (and comparatively to its modest effect alone) significantly increases (by z29% versus VRT-325 alone) the rescue efficiency of R1070W and also V510D, suggesting effects at distinct sites. Login to comment
168 ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:168:21
status: NEW
view ABCC7 p.Gly550Glu details
For example, F508del-G550E-CFTR required a 10-fold lower dose of VX-809 than F508del-CFTR for significant rescue. Login to comment
171 ABCC7 p.Asp567Ala
X
ABCC7 p.Asp567Ala 23890012:171:155
status: NEW
view ABCC7 p.Asp567Ala details
CFTR with Mutated Diacidic Exit Code Is Rescued by Low Temperature, but Not by Correctors (A) BHK cell lines stably expressing WT-CFTR or bearing the DAA (D567A) mutation alone or in cis with 4RK were cultured at 37 C, incubated at 26 C for 48 hr or for 24hr with VRT-325, Corr-4a, or VX-809, as indicated. CFTR protein was analyzed by WB. Data are representative of n = 5 experiments. Login to comment
174 ABCC7 p.Asp567Ala
X
ABCC7 p.Asp567Ala 23890012:174:84
status: NEW
view ABCC7 p.Asp567Ala details
ABCC7 p.Asp565Ala
X
ABCC7 p.Asp565Ala 23890012:174:77
status: NEW
view ABCC7 p.Asp565Ala details
(C) BHK cell lines stably expressing WT-CFTR or bearing the DD/AA mutations (D565A, D567A) alone or in cis with 4RK were cultured at 37 C, incubated at 26 C for 48 hr or for 24 hr with VRT-325, Corr-4a, or VX-809, as indicated. CFTR protein was analyzed by WB. Data are representative of n = 6 experiments. Login to comment
193 ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:193:62
status: NEW
view ABCC7 p.Arg1070Trp details
ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:193:206
status: NEW
view ABCC7 p.Arg1070Trp details
ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:193:52
status: NEW
view ABCC7 p.Gly550Glu details
ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:193:142
status: NEW
view ABCC7 p.Gly550Glu details
Furthermore, modeling also predicts that revertants G550E and R1070W act at different CFTR interdomain contacts disrupted by F508del: whereas G550E seems to restore the strength of the NBD1:NBD2 interface, R1070W rather promotes the NBD1:ICL4 interaction, as suggested previously (He et al., 2010; Serohijos et al., 2008) and in Figure S2A. Login to comment
195 ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:195:134
status: NEW
view ABCC7 p.Arg1070Trp details
ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:195:8
status: NEW
view ABCC7 p.Gly550Glu details
ABCC7 p.Val510Asp
X
ABCC7 p.Val510Asp 23890012:195:145
status: NEW
view ABCC7 p.Val510Asp details
Indeed, G550E, besides being able to promote rescue of F508del-CFTR (DeCarvalho et al., 2002), shows the largest combined effect with R1070W (or V510D). Login to comment
197 ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:197:210
status: NEW
view ABCC7 p.Arg1070Trp details
ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:197:34
status: NEW
view ABCC7 p.Gly550Glu details
ABCC7 p.Arg555Lys
X
ABCC7 p.Arg555Lys 23890012:197:43
status: NEW
view ABCC7 p.Arg555Lys details
Moreover, the observed synergy of G550E or R555K with VX-809, but not VRT-325, is consistent with this model in which VRT-325 acts on the NBD1:NBD2 dimerization interface (also supported by the synergy between R1070W and VRT-325). Login to comment
198 ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:198:71
status: NEW
view ABCC7 p.Arg1070Trp details
Further modeling is, however, required to examine the observation that R1070W is still additive with VX-809 (albeit modestly). Login to comment
210 ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:210:229
status: NEW
view ABCC7 p.Arg1070Trp details
ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:210:216
status: NEW
view ABCC7 p.Gly550Glu details
ABCC7 p.Arg555Lys
X
ABCC7 p.Arg555Lys 23890012:210:258
status: NEW
view ABCC7 p.Arg555Lys details
ABCC7 p.Val510Asp
X
ABCC7 p.Val510Asp 23890012:210:186
status: NEW
view ABCC7 p.Val510Asp details
Our data also show that low temperature, similar to chemical correctors, further increases processing levels of F508del-CFTR by the five genetic revertants, although to variable levels: V510D (by an additional 35%), G550E (65%), R1070W (38%), 4RK (27%), and R555K (22%). Login to comment
211 ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:211:53
status: NEW
view ABCC7 p.Gly550Glu details
ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:211:335
status: NEW
view ABCC7 p.Gly550Glu details
The strong synergistic effect of low temperature and G550E on the rescue of F508del-CFTR-efficient processing, but only modestly for 4RK, supported by pulse-chase data, suggest that low temperature and 4RK effects are mechanistically closer to each other (and thus related to bypassing the ER quality control) than low temperature and G550E. Login to comment
212 ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:212:54
status: NEW
view ABCC7 p.Gly550Glu details
This is further supported by the additive effects of G550E and 4RK effects (Figure 4). Login to comment
214 ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:214:198
status: NEW
view ABCC7 p.Gly550Glu details
ABCC7 p.Arg555Lys
X
ABCC7 p.Arg555Lys 23890012:214:127
status: NEW
view ABCC7 p.Arg555Lys details
ABCC7 p.Arg516Lys
X
ABCC7 p.Arg516Lys 23890012:214:106
status: NEW
view ABCC7 p.Arg516Lys details
Although 4RK can also be claimed to impact on F508del-CFTR folding (namely, through its two NBD1 changes: R516K and especially R555K), this is somewhat disproven by the additive effects of 4RK with G550E (Figure 4), the latter truly correcting F508del-NBD1 folding, as assessed by channel gating (Farinha and Amaral, 2005; Rosser et al., 2008; Roxo-Rosa et al., 2006). Login to comment
225 ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:225:606
status: NEW
view ABCC7 p.Arg1070Trp details
ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:225:595
status: NEW
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Assessing the synergistic/additive effects of investigational drug VX-809, one of the most promising to rescue the F508del-CFTR-trafficking defect, with those of genetic revertants as well as other correctors (VRT-325 and Corr-4a) or low temperature pointed to major insights into its MoA: (1) VX-809 is additive to both VRT-325 and Corr-4a, suggesting that each compound operates by a different MoA; (2) VX-809 is additive to low temperature rescue of the mutant almost to WT-CFTR levels; (3) VX-809, VRT-325, and Corr-4a show variable additive effects with the genetic revertants tested (4RK, G550E, and R1070W), thus providing clues for their possible action being exerted at specific protein binding pockets: VX-809 at the NBD1:TMD2 interface (and VRT-325 at NBD1:NBD2) or acting unspecifically (Corr-4a); and (4) VX-809 does not rescue the diacidic code traffic mutant in contrast to low temperature, which seems to act at trafficking surveillance checkpoints. Login to comment
231 ABCC7 p.Arg560Thr
X
ABCC7 p.Arg560Thr 23890012:231:292
status: NEW
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ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:231:142
status: NEW
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ABCC7 p.Arg1070Trp
X
ABCC7 p.Arg1070Trp 23890012:231:219
status: NEW
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ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:231:126
status: NEW
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ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:231:197
status: NEW
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ABCC7 p.Gly550Glu
X
ABCC7 p.Gly550Glu 23890012:231:213
status: NEW
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ABCC7 p.Arg555Lys
X
ABCC7 p.Arg555Lys 23890012:231:103
status: NEW
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ABCC7 p.Arg555Lys
X
ABCC7 p.Arg555Lys 23890012:231:175
status: NEW
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ABCC7 p.Arg516Lys
X
ABCC7 p.Arg516Lys 23890012:231:97
status: NEW
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ABCC7 p.Arg29Lys
X
ABCC7 p.Arg29Lys 23890012:231:92
status: NEW
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ABCC7 p.Val510Asp
X
ABCC7 p.Val510Asp 23890012:231:159
status: NEW
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ABCC7 p.Val510Asp
X
ABCC7 p.Val510Asp 23890012:231:191
status: NEW
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ABCC7 p.Asp567Ala
X
ABCC7 p.Asp567Ala 23890012:231:233
status: NEW
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ABCC7 p.Asp567Ala
X
ABCC7 p.Asp567Ala 23890012:231:267
status: NEW
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ABCC7 p.Asp565Ala
X
ABCC7 p.Asp565Ala 23890012:231:260
status: NEW
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EXPERIMENTAL PROCEDURES Cells and Culture Conditions BHK cell lines expressing F508del-4RK (R29K/R516K/R555K/R716K)-, F508del-G550E-, F508del-R1070W-, F508del-V510D-, F508del-R555K-, F508del-V510D/G550E-, F508del-G550E/R1070W-, DAA (D567A)-, 4RK- DAA-, DD/AA (D565A, D567A)-, 4RK-DD/AA-, and R560T-CFTR were produced and cultured as previously described (Roxo-Rosa et al., 2006). Login to comment