ABCMdb

ABCC7 p.Val510Asp

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Publications

PMID: 17911111 [PubMed] Wang Y et al: "Correctors promote maturation of cystic fibrosis transmembrane conductance regulator (CFTR)-processing mutants by binding to the protein."

No. Sentence Comment

4 The V510D mutation also promoted maturation of ⌬F508 CFTR. Login to comment
70 Stable BHK cell lines expressing mutants V510A, V510C, V510S, V510G, or V510D were generated for use in iodide efflux assays. Login to comment
72 The results of the most active (V510A) and least active (V510D) mutants are shown in Fig. 2C. Login to comment
98 C, iodide efflux assays were performed on stable BHK cell lines expressing Cys-less/V510A CFTR (open circles) or Cys-less/V510D CFTR (closed squares). Login to comment
124 Position 510 appears to be particularly important for CFTR maturation as introduction of the suppressor mutation V510D into ⌬F508 CFTR also promoted maturation of the protein (Fig. 2D). Login to comment
128 The V510D mutation may counter the effects of ⌬F508 by acting as a suppressor mutation, perhaps by forming a salt bridge with a positive amino acid located in one of the intracellular loops. Login to comment
131 Although V510D was the most efficient suppressor mutation because it promoted maturation of both Cys-less and ⌬F508 CFTRs, it was less useful than the V510A change in Cys-less CFTR because it showed reduced iodide efflux activity. Login to comment
136 The proposed effects of the V510D mutation and correctors on maturation of ⌬F508 CFTR are shown in the models in supplemental Fig. 1. Login to comment
143 The V510D suppressor mutation is predicted to overcome the effects of the ⌬F508 CFTR mutation by promoting interactions between TMD1 and NBD1 (supplemental Fig. 1B). Login to comment

PMID: 18597042 [PubMed] Mornon JP et al: "Atomic model of human cystic fibrosis transmembrane conductance regulator: membrane-spanning domains and coupling interfaces."

No. Sentence Comment

239 Interestingly, it was very recently shown that the V510D mutation in the DF508 protein promotes its maturation [72]. Login to comment
240 This might be explained by the fact that the V510D mutation may restore the ICL4/NBD1 contacts missing in DF508. Login to comment
241 Indeed, as shown in Supplementary Data 6 (http://www.impmc.jussieu.fr/~callebau/CFTR.html), our refined model suggests that two novel, nearly symmetrical contacts [R1070 NH1 - D510 OD1 (3.0 ); R1070 NH1 - D510 OD2 (3.3 )] may appear in V510D DF508. Login to comment
313 This hypothesis is supported by the restoration of DF508 maturation by the V510D mutation [72]. Login to comment

PMID: 19625452 [PubMed] Grove DE et al: "Mechanisms for rescue of correctable folding defects in CFTRDelta F508."

No. Sentence Comment

261 Indeed, introduction of the V510D suppressor point mutation into CFTR⌬F508 partially restored folding of some CFTR⌬F508 V510D (Wang et al., 2007), but the majority of CFTR⌬F508 V510D accumulated in its B-form (Figure 6A), and the level of repair remained below the observed levels of folded wild-type CFTR (compare to CFTR panel in Figure 3B). Login to comment
262 Corr-4a was able to enhance the accumulation of the folded C-form of CFTR⌬F508 V510D (Figure 6A). Login to comment
263 Pulse-chase analysis indicated that Corr-4a stabilizes the B-form and enhances the folding efficiency of CFTR⌬F508 V510D (Figure 6B). Login to comment
265 Overall, the V510D suppressor mutation and Corr-4a appear to act in an additive manner to restore the folding of CFTR⌬F508 toward wild-type levels. Login to comment
268 To test this model, we asked if the V510D suppressor mutation permits Corr-4a to correct the misfolding of CFTR 1172X⌬F508. Login to comment
269 Suppression of CFTR 1172X⌬F508 misfolding upon introduction of the V510D mutation was similar to the folding correction observed with CFTR⌬F508 V510D (Figure 6A). Login to comment
270 Corr-4a was now able to promote the proper folding of a small pool of CFTR 1172X⌬F508 V510D and enhance the accumulation of its maturely glycosylated C-form. Login to comment
271 However, again the level of correction was low and an increase in the folding efficiency of CFTR 1172X⌬F508 V510D was not detected in pulse-chase experiments (Figure 6B). Login to comment
276 (A) HEK293 cells were transfected with 1 ␮g of either CFTR⌬F508, CFTR⌬F508 V510D, CFTR 1172X⌬F508, or CFTR 1172X⌬F508 V510D. Login to comment
312 Yet, Corr-4a is significantly more effective at promoting the folding of CFTR⌬F508 V510D than CFTR⌬F508. Login to comment
313 The V510D mutant is designed to facilitate interactions between NBD1 and MSD2 that are defective when F508 is deleted (Mornon et al., 2008). Login to comment

PMID: 19925455 [PubMed] Pagano MA et al: "Cystic fibrosis transmembrane regulator fragments with the Phe508 deletion exert a dual allosteric control over the master kinase CK2."

No. Sentence Comment

51 Also of note in Figure 1 is the effect of mutating Val510 to aspartate either in the context of a wild-type peptide or its F508 equivalent, the latter combination having been shown to partially restore functionality of CFTR F508 [30]; in our hands this V510D mutation significantly abrogates the inhibitory efficacy of the CFTR F508 peptide, yet has a diametrically opposed effect when present in the wild-type peptide, whose moderate inhibitory efficiency is increased almost equalling that of the CFTR F508 peptide. Login to comment

PMID: 19944699 [PubMed] Lewis HA et al: "Structure and dynamics of NBD1 from CFTR characterized using crystallography and hydrogen/deuterium exchange mass spectrometry."

No. Sentence Comment

294 Notably, a V510D mutation has been reported to suppress the trafficking defect caused by ΔF508, restoring export of a significant fraction of ΔF508-CFTR to the cell surface.73 The conformational change caused by the ΔF508 mutation dramatically alters the surface topography and chemical characteristics in the immediate vicinity of the 509-511 loop (Fig. 9) but not elsewhere in NBD1 (Figs. 3a and 8a). Login to comment
318 The efficacy of V510D in rescuing the ΔF508 trafficking defect suggests that this suppressor mutation changes the conformation of the 509-511 loop, stabilizes the folding of the ABCα subdomain, or blocks chaperone interactions, because it seems unlikely to directly improve NBD1 binding to the TMDs. Login to comment

PMID: 20590134 [PubMed] Loo TW et al: "The V510D suppressor mutation stabilizes DeltaF508-CFTR at the cell surface."

No. Sentence Comment

0 pubs.acs.org/Biochemistry Published on Web 06/30/2010 r 2010 American Chemical Society 6352 Biochemistry 2010, 49, 6352-6357 DOI: 10.1021/bi100807h The V510D Suppressor Mutation Stabilizes ΔF508-CFTR at the Cell Surface† Tip W. Loo, M. Claire Bartlett, and David M. Clarke* Departments of Medicine and Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada Received March 30, 2010; Revised Manuscript Received June 29, 2010 ABSTRACT: Deletion of Phe508 (ΔF508) in the first nucleotide-binding domain (NBD1) of CFTR causes cystic fibrosis. Login to comment
2 We found that replacement of Val510 with acidic residues (but not neutral or positive residues) promoted maturation of ΔF508-CFTR with V510D more efficiently than V510E. Login to comment
3 Promotion of ΔF508-CFTR maturation did not require NBD2 as introduction of V510D into a ΔNBD2/ΔF508-CFTR mutant restored maturation to levels similar to that of full-length protein. Login to comment
4 The V510D mutation increased the half-life of mature ΔF508-CFTR at the cell surface by about 5-fold to resemble the half-life of wild-type CFTR. Login to comment
5 It was also observed that introduction of the V510R/ R1070D mutations into ΔF508-CFTR also promoted maturation whereas the V510D/R1070A mutations did not. Login to comment
6 We propose that the V510D mutation in NBD1 promotes maturation and stabilizes ΔF508-CFTR at the cell surface through formation of a salt bridge with Arg1070 in TMD2. Login to comment
16 The V510D suppressor mutation is interesting because Val510 is predicted to reside at the domain-domain interface between NBD1 and TMD2 in close proximity to Phe508 (8). Login to comment
17 A negative charge appeared to be important because the V510D mutation promoted maturation of ΔF508-CFTR while mutation of Val510 to neutral amino acids (Cys, Gly, Ala, Ser, Asn, Pro, Thr, Tyr) did not (9). Login to comment
18 In this study, we tested whether the V510D mutation would reduce the turnover of ΔF508-CFTR at the cell surface. Login to comment
25 Generation of stable BHK cell lines expressing wild-type or mutant ΔF508/V510D-CFTRs was performed as described previously (9). Login to comment
27 HEK 293 cells were transfected with W356C/W1145C-, ΔF508/W356C/W1145C-, or ΔF508/V510D/W356C/W1145C-CFTR cDNAs, and the cells were incubated for 4 h at 37 °C. Login to comment
40 clarke@utoronto.ca.1 Abbreviations: TM, transmembrane; NBD, nucleotide-binding domain; HEK, human embryonic kidney; BHK, baby hamster kidney. after oxidation of carbohydrate with sodium periodate (14) were performed using HEK 293 cells transfected with A52-tagged wild-type, ΔF508-, or ΔF508/V510D-CFTR cDNAs as described previously. Login to comment
45 Measurement of cAMP-stimulated iodide efflux was performed on baby hamster kidney (BHK) cells or BHK cells expressing wild-type or mutant ΔF508/V510D-CFTRs as described previously (9, 15). Login to comment
49 In a modeling study, it was predicted that the negative charge of the V510D mutation promoted maturation of ΔF508-CFTR be- causeitrestoresdefective NBD1-TMD2interactionsbyforming a salt bridge with Arg1070 (TMD2) (17). Login to comment
51 HEK 293 cells weretransfected withthe cDNAof wild-type CFTR (Figure 1E) or mutants ΔF508 (Figure 1, panels E-H), V510D/ΔF508 (Figure 1F), V510E/ΔF508 (Figure 1G), and V510R/ΔF508 (Figure 1H), and whole cell SDS extracts were subjected to immunoblot analysis 18 h after transfection. Login to comment
53 The V510E mutation also promoted maturation, but it was less efficient than the V510D mutation (12% and 35%, respectively) (Figure 1, panels F and G). Login to comment
55 It was possible that the high level of expression of V510D/ ΔF508 in the transiently transfected HEK 293 cells was also a factor in enhancing maturation of the mutant. Login to comment
56 To examine the extent of maturation at lower levels of expression, HEK 293 cells were transfected with lower concentrations of plasmid containing V510D/ΔF508 cDNA and whole cell extracts subjected to immunoblot analysis 42 h after transfection. Login to comment
58 It was observed that expression of CFTR was reduced when cells were transfected with lower concentrations of plasmid(Figure1I) butthe relativesteady-state level of mature V510D/ΔF508-CFTR remained at about 50% of total CFTR (Figure 1J). Login to comment
59 To test if the mutant was active, BHK cell lines were generated that stably expressed wild-type or V510D/ΔF508-CFTRs for use in iodide efflux assays. Login to comment
60 The relative expression level of mature to total CFTR for mutant V510D/ΔF508 in BHK cells was also found to be about 50% (data not shown). Login to comment
62 Cells expressing wild-type or V510D/ΔF508-CFTRs exhibited iodide efflux upon addition of forskolin (Figure 2). Login to comment
66 We introduced the V510D mutation into ΔF508-CFTR lacking NBD2 (truncated after residue 1196 and containing the epitope for monoclonal antibody A52 (Δ1197-1480)-A52) to test if it still promoted maturation. Login to comment
67 Immunoblot analysis of whole cell SDS extracts (Figure 3, left panel) showed that ΔF508/V510D/ΔNBD2-A52 (Δ1197-1480) had a maturation efficiency (about 50%) that was similar to that of the full-length ΔF508/V510D mutant (about 50%; Figure 1, panel F). Login to comment
72 To test the maturation efficiency of ΔF508- and ΔF508/V510D-CFTRs at various levels of expression, HEK 293 cells were transfected with various levels of plasmid followed by immunoblot analysis and enhanced chemiluminescence 42 h after transfection (I). Login to comment
74 The positions of mature and immature CFTRs are These results suggest that the V510D suppressor mutation differs from other NBD1 suppressor mutations because the absence of NBD2 did not reduce its effect. Login to comment
77 To test if the position of the truncation affected maturation, mutant ΔF508/V510D/ΔNBD2-A52 (Δ1173-1480) was constructed and expressed in HEK293 cells. Login to comment
78 Immunoblot analysis of whole cell extracts showed that the V510D mutation promoted maturation of the truncation mutant but the relative level of mature was only about 20% of total CFTR protein. Login to comment
79 It was predicted in a modeling study (17) that the V510D mutation may promote NBD1-TMD2 interactions in ΔF508-CFTR by forming a salt bridge with positively charged residue Arg1070 (TMD2). Login to comment
80 We tested the contribution of Arg1070 by introducing the R1070A change intoΔF508/V510D. Login to comment
81 Asshownin Figure 4A, mature protein was not detectable in mutant ΔF508/ V510D/R1070A. Login to comment
89 These results suggest that V510D may form a salt bridge with Arg1070 in ΔF508-CFTR. Login to comment
91 To test if the V510D mutation increased the stability of ΔF508-CFTR, a cross-linking assay was used to measure the half-life of mature CFTR at the cell surface. Login to comment
96 Accordingly, the W356C and W1145C mutations were introduced into wild-type, ΔF508-, and ΔF508/V510D-CFTRs. Login to comment
100 Panels A and B of Figure 5 show that the half-lives of cross-linked wild-type/W356C/W1145C-, ΔF508/ W356C/W1145C-, and ΔF508/V510D/W356C/W1145C-CFTRs were about 12,3,and 14 h, respectively. Login to comment
101 Theseresults indicatethat the stability of mutant V510D/ΔF508-CFTR resembled that of wild-type protein. Login to comment
104 To examine the effect of the V510D mutation on turnover of ΔF508-CFTR without the introduced cysteines, a pulse-chase assay was performed. Login to comment
106 By contrast, the conversion of immature to mature form in mutant ΔF508/V510D was slower since there wasconsiderableamountofimmatureCFTRstillpresentat2-4h (Figure 6A, bottom panel). Login to comment
108 After an 8 h chase, the majority of labeled CFTR in wild-type and mutant ΔF508/V510D was present as the mature protein FIGURE 2: Iodide efflux activity of wild-type and ΔF508/V510D-CFTR. Login to comment
109 Iodide efflux assays were performed on BHK cells stably expressing wild-type CFTR, mutant ΔF508/V510D, or no CFTR (control). Login to comment
112 FIGURE 3: Effect of V510D on maturation of ΔF508/ΔNBD2 mutants. Login to comment
118 Therefore, we used this time point to compare the lifetimes of the mature protein in wild-type and mutant ΔF508/ V510D. Login to comment
120 The results indicate that V510D stabilizes ΔF508-CFTR. Login to comment
121 To test if V510D stabilizes ΔF508-CFTR at the cell surface, cells expressing wild-type, ΔF508-, or ΔF508/V510D-CFTRs werefirst incubated at 30°C for 18 h sothatΔF508-CFTR would be present at the cell surface. Login to comment
125 It was found that mature forms of wild-type or ΔF508/V510D-CFTRs had similar half-lives of about 12 h while ΔF508-CFTR had a short half-life of about 2 h (Figure 7). Login to comment
128 This study showed that the V510D mutation increased the cell surface stability of mature ΔF508-CFTR to levels that were similar to the wild-type protein (Figures 5-7). Login to comment
131 HEK 293 cells expressing wild-type, ΔF508-, or ΔF508/ V510D-CFTRs containing the W356C and W1145C cysteines were treated with the thiol cross-linker BMH. Login to comment
134 HEK 293 cells were transfected with A52-tagged wild-type, ΔF508, or ΔF508/V510D cDNAs. Login to comment
141 HEK 293 cells were transfected with the cDNAs of A52-tagged wild-type, ΔF508-, or ΔF508/V510D-CFTR. Login to comment
142 To compare the half-life of ΔF508-CFTR to wild-type or mutant ΔF508/V510D-CFTR, the cells were first incubated at 30 °C to promote maturation and increase the level of ΔF508-CFTR at the cell surface. Login to comment
148 The position of mature CFTR is had half-lives of about 12 h. Although the effects of these suppressor mutations on full-length CFTR were similar to that of V510D, there were different effects on NBD2 deletion mutants. Login to comment
150 The V510D mutation (present study) appeared to restore stability by promoting NBD1-TMD2 interactions in ΔF508-CFTR since deletion of NBD2 had little effect (Figure 3; Δ1197-1480). Login to comment
156 Other evidence suggesting that ΔF508/ΔNBD2 (Δ1173-1480) was more difficult to rescue was the observation that introduction of V510D into the mutant yielded lower levels of mature protein compared to the ΔF508/ΔNBD2/V510D (Δ1197-1480) construct (Figure 3). Login to comment
158 Mutation of Arg1070 to a neutral amino acid abolished V510D rescue of ΔF508-CFTR while V510R only rescued the mutant when the R1070D change was introduced (Figure 4C). Login to comment
173 In this study we found that the ΔF508/V510D mutant was active while we failed to detect activity when V510D was introduced into Cys-less CFTR (9). Login to comment
174 Although Cys-less/V510D-CFTR matured, its chloride channel activity was below the limits ofdetection inthe iodide efflux assay. Login to comment
178 While introduction of V510D into Cys-less or ΔF508-CFTRs promoted maturation, the Cys-less mutant was different because its maturation could also be promoted by changing Val510 to Thr, Cys, Gly, Ala, or Ser. Login to comment
180 In summary, the V510D mutation increases the stability of ΔF508-CFTR by promoting NBD1-TMD2 interactions likely through formation of a salt bridge with Arg1070 in ICL4. Login to comment

PMID: 20687133 [PubMed] Protasevich I et al: "Thermal unfolding studies show the disease causing F508del mutation in CFTR thermodynamically destabilizes nucleotide-binding domain 1."

No. Sentence Comment

44 hNBD1 Nameb Termini / Mutationsc Tm d DTm ¼ Tm D508 - Tm wt ( C) PDB ID 1 hNBD1-D(RI,RE) 2935c46917 387-646[D405-436] 57.7 þ 0.2 2PZE 1 (F508del)hNBD1D (RI,RE) 2935c47217 387-646[D405-436, F508del] 51.5 þ 0.3 À6.2 þ 0.3 2PZF 2 387-646[D405-436, V510D] 60.2 þ 0.4 2 387-646[D405-436, V510D, F508del] 53.0 þ 0.1 À7.2 þ 0.4 3 387-646[D405-436, F494N, Q637R] 59.2 3 387-646[D405-436, F494N, Q637R, F508del] 52.8 À6.4 4 387-646[D405-436, G550E, R553Q, R555K] 61.7 4 387-646[D405-436, G550E, R553Q, R555K,F508del] 55.7 À6.0 5 387-678[D405-436] 58.1 5 387-678[D405-436, F508del] 51.7 À6.2 6 hNBDI-315 2935c38217 389-678[F429S, F494N, Q637R] 49.8 þ 0.3 6 hNBDI-3F508del15 2935c37117 389-678[F429S, F494N, Q637R, F508del] 43.6 þ 0.1 À6.3 þ 0.3 2BBS 7 389-678[F429S, F494N, L636E5, Q637R] 50.5 þ 0.2 7 389-678[F429S, F494N, L636E, Q637R, F508del] 44.9 À6.2 þ 0.2 a DSC conducted at 1 mg/mL protein. Login to comment
61 The Teem suppressor triplet (pair 4)29 increases Tm by 4 , the V510D mutation (pair 2)30 by 2.5 , and F494N/ Q637R (pair 3) by 1.5 . Login to comment

PMID: 20687163 [PubMed] Wang C et al: "Integrated biophysical studies implicate partial unfolding of NBD1 of CFTR in the molecular pathogenesis of F508del cystic fibrosis."

No. Sentence Comment

28 Surprisingly, several of these solubilizing surface mutations in hNBD1, identified in a screen focused exclusively on the in vitro solubility of hNBD1, were shown to suppress the in vivo trafficking defect of F508del-CFTR more strongly than the best existing pharmacological agents.32,38 Notably, the mutated residues (e.g., F429S, F494N, and Q637R) are not in direct contact with F508 and do not appear to be allosterically coupled.18 A similar hydrophobic-to-hydrophilic substitution in the immediate vicinity of F508, the V510D mutation, also strongly suppresses the in vivo trafficking defect of F508del-CFTR.39,40 It was proposed that these substitutions could block adventitious chaperone interactions that prevent proper ER export.18 However, there is as yet no concrete evidence explaining the tight correlation between the effects of mutations on the in vitro solubility properties of hNBD1 and the in vivo trafficking properties of human CFTR. Login to comment
156 They were subsequently introduced into intact F508del-CFTR and demonstrated to suppress its aberrant trafficking in vivo in tissue culture cells.37 Introducing all three mutations simultaneously into full-length hNBD1 led to improved yield and stability of the purified soluble domain after overexpression in E. coli.15 The V510D mutation was made to explore the molecular pathology caused by the F508del mutation after crystallographic studies demonstrated that F508del produces a large change in the conformation of val-510.15 The hydrophobic sidechain of val-510 is mostly buried on the surface of hNBD1 in the absence of the F508del mutation,18 at a site where it is likely to make direct packing interactions to the transmembrane domains of CFTR.35 However, its backbone conformation is altered so that it projects from the surface of hNBD1 and is completely solvent-exposed in the presence of the F508del mutation.18 Surprisingly, the V510D mutation strongly suppresses the trafficking defect caused by the F508del mutation in vivo in tissue culture cells.39,40 Finally, the Figure 4. Login to comment
161 These mutations also show significant efficacy in suppressing the trafficking defect caused by the F508del mutation in vivo in tissue culture cells,32 although they are less effective that the Teem suppressors,37 the V510D mutation,39,40 or deletion of the RI.41 The Teem suppressor triplet (Fig. 4A-C), the V510D mutation (Fig. 4D-F), and the F494N/Q637R mutations (Fig. 4G-I) all stabilize hNBD1 against the initial unfolding transition, shifting its midpoint 0.25-0.50 M higher in urea concentration. Login to comment
162 The magnitude of the SLS increase following the initial unfolding transition is greatly reduced by the Teem suppressor triplet and the V510D mutation and significantly reduced by the F494N/Q637R mutations. Login to comment
165 In the presence of the Teem suppressor triplet or the V510D mutation, which significantly stabilize the domain, the urea-induced increase in trp fluorescence (Supporting Information Fig. S8B,E) precedes the initial unfolding transition as monitored by CD spectroscopy (Supporting Information Fig. S8A,D). Login to comment
200 The Teem suppressor triplet and the F494N mutation are located on the opposite face of the ABCa subdomain from the V510D mutation, which is adjacent to the F508del site, and crystallographic studies indicate that the direct stereochemical effects of these different mutations are likely to be unrelated.18 The influence of all of these mutations on the initial unfolding transition suggests that it involves a global change in ABCa subdomain conformation. Login to comment

PMID: 21148293 [PubMed] Grove DE et al: "The endoplasmic reticulum-associated Hsp40 DNAJB12 and Hsc70 cooperate to facilitate RMA1 E3-dependent degradation of nascent CFTRDeltaF508."

No. Sentence Comment

14 Introduction of the V510D misfolding suppressor mutation into CFTRΔF508 modestly increased folding efficiency, whereas combined inactivation of JB12 and suppression of intrinsic folding defects permitted CFTRΔF508 to fold at 50% of wild-type efficiency. Login to comment
261 Folding of CFTRΔF508 increased dramatically upon introduction of the V510D suppressor mutation because CFTRΔF508 V510D accumulated at a C/B ratio of 0.2. Login to comment
262 Strikingly, depletion of JB12 increased the C/B ratio of CFTRΔF508 V510D more than eightfold to an impressive value near 1.6, which is 50% of the ratio observed for wild-type CFTR. Login to comment
263 In pulse-chase experiments, we also observed depletion of JB12 to permit CFTRΔF508 V510D to fold with ~50% of wild-type efficiency (Figure 8B). Login to comment
264 The combination of JB12 kd and Corr-4a treatment enabled CFTRΔF508 V510D to obtain a C/B ratio of 3.5, which is the same as the 3.4 value observed with wild-type CFTR. Login to comment
265 In pulse-chase experiments, we also observed that these conditions permit the B-form of CFTRΔF508 V510D to convert to the C-form with an efficiency that was greater than CFTR (Figure 8B). Login to comment
266 Corr-4a is able to stabilize transmembrane regions of CFTR (Wang et al., 2007; Grove et al., 2009), which may explain the twofold increase in CFTRΔF508 V510D folding efficiency that occurs when Corr-4a is present in JB12- depleted cells. Login to comment
277 We compared the extent to which the introduction of V510D, a misfolding suppressor mutation, into CFTRΔF508 versus inactivation of JB12 enhanced CFTRΔF508 folding (Figure 8). Login to comment
279 The folding of CFTR, CFTRΔF508, and CFTRΔF508 V510D was compared under control conditions, upon JB12 depletion, when cells were treated with the folding corrector Corr-4a (Pedemonte et al., 2005), or with a combination of the above conditions. Login to comment
318 Transfections with JB12 siRNA oligos and pcDNA3.1(+)- CFTR, pcDNA3.1(+)-CFTRΔF508, or pcDNA3.1(+)-CFTRΔF508 V510D were performed as described in Materials and Methods. Login to comment

PMID: 21520952 [PubMed] Loo TW et al: "Benzbromarone stabilizes DeltaF508 CFTR at the cell surface."

No. Sentence Comment

14 It was recently reported that the stability of ΔF508 CFTR at the cell surface approached that of wild-type CFTR when NBDÀTMD2 interactions were restored.21 It was shown that introduction of a V510D mutation into NBD1 promoted the maturation and stability of ΔF508 CFTR by forming a a salt bridge with Arg1070 of TMD2.21 Similarly, maturation of ΔF508 CFTR was promoted by a R1070W suppressor mutation in TMD2.5 These suppressor mutation results suggested that direct binding of a compound to the TMDs may promote the maturation and stability of ΔF508 CFTR. Login to comment
38 The slow maturation was similar to what was previously observed with the V510D/ΔF508 CFTR suppressor mutant.8 Maturation of mutant V510D/ΔF508 CFTR required ~4À8 h (Figure 2A) compared to 1À2 h for the wild-type enzyme.21 The half-life of the mature ΔF508 CFTR in the pulseÀchase assays was ~16 h after rescue with benzbromarone (data not shown). Login to comment

PMID: 21182301 [PubMed] Loo TW et al: "The W232R suppressor mutation promotes maturation of a truncation mutant lacking both nucleotide-binding domains and restores interdomain assembly and activity of P-glycoprotein processing mutants."

No. Sentence Comment

332 Similarly, it was suggested that the V510D suppressor mutation in NBD1 promoted folding of ΔF508-CFTR by forming a salt bridge with Arg1070 (64). Login to comment

PMID: 23071149 [PubMed] Okiyoneda T et al: "Fixing cystic fibrosis by correcting CFTR domain assembly."

No. Sentence Comment

166 The V510D suppressor mutation stabilizes DeltaF508-CFTR at the cell surface. Login to comment
164 The V510D suppressor mutation stabilizes DeltaF508-CFTR at the cell surface. Login to comment

PMID: 22406676 [PubMed] Aleksandrov AA et al: "Allosteric modulation balances thermodynamic stability and restores function of DeltaF508 CFTR."

No. Sentence Comment

151 Thus, both the constant and varying temperature experiments demonstrated a requirement for a balance between thermal stability and channel activity in CFTR, consistent with considerations of such a relationship between stability and catalytic function of proteins in general.28 NBD1 stabilization restores an NBD1-CL4 interface In addition to destabilizing NBD1, deletion of F508 also disrupts interdomain contacts including the interface between the NBD1 surface and the cytoplasmic loop (CL)4 in MSD2 in which the residue normally participates.29 Specific second-site mutations on either side of this interface (e.g., R1070W or V510D) have been shown to promote maturation of ΔF508 CFTR.27,30,31 The current observations that the ΔF508 protein with NBD1 strongly stabilized by the proline and I539T substitutions had channel activity similar to the WT at physiological temperature suggested either that the NBD1-CL4 interface is not important for function or that it is adequately restored by NBD1 stabilization. Login to comment
433 The V510D suppressor mutation stabilizes DeltaF508-CFTR at the cell surface. Biochemistry, 49, 6352-6357. 32. Login to comment
436 The V510D suppressor mutation stabilizes DeltaF508-CFTR at the cell surface. Biochemistry, 49, 6352-6357. 32. Login to comment

PMID: 22038833 [PubMed] Colas J et al: "Disruption of cytokeratin-8 interaction with F508del-CFTR corrects its functional defect."

No. Sentence Comment

29 The first one shows experimentally that NBD1 destabilization occurs as a consequence of three solubilizing mutations, namely V510D, F494N and Q637R (21). Login to comment

PMID: 22138491 [PubMed] Lukacs GL et al: "CFTR: folding, misfolding and correcting the DeltaF508 conformational defect."

No. Sentence Comment

95 The cytosolic Nedd4-2 and Fbs1 E3 ligases have also been implicated intheERADofDF508CFTR[58,59].Althoughablationofan E3 ligase or overexpression of a deubiquitinating enzyme were unable to rescue DF508 CFTR processing, perhaps due to the redundancy of the ER quality control machinery, the combination of a second site suppressor mutation (Val510Asp) with inhibition of ubiquitination and exposure to a corrector molecule (Corr-4a) led to the robust maturation of the mutant protein in cell culture models [24,60]. Login to comment

PMID: 22265408 [PubMed] Rabeh WM et al: "Correction of both NBD1 energetics and domain interface is required to restore DeltaF508 CFTR folding and function."

No. Sentence Comment

134 Conversely, stabilization of the NBD1-CL4 interface by the V510D substitution (see bellow) increased the WT CFTR folding efficiency by $2-fold, supporting the critical role of the NBD1-CL4 interface in the coupled domain folding of CFTR (Figure 6B; see below). Login to comment
136 R1070W or V510D substitutions at the interfaces restored the proximity of the DF508 NBD1 and CL4 as shown by Cys crosslinking. Login to comment
137 The NBD1-CL4 interface stabilization can be accomplished by filling the cavity created by the DF508 with the bulky hydrophobic side chain of R1070W or by salt bridge formation between V510D in NBD1 and R1070 in CL4 (Figure S6B) (He et al., 2010; Loo et al., 2008, 2010; Thibodeau et al., 2010). Login to comment
138 R1070W, similar to V510D, alone modestly increased the DF508 CFTR folding efficiency and cellular and PM expression (Figures 6A-6E), in part confirming previous reports (He et al., 2010; Thibodeau et al., 2010; Loo et al., 2010). Login to comment
141 Similarly, the DF508 CFTR folding and expression were synergistically rescued by combining the DF508-NBD1 stabilizing mutation DRI with R1070W or V510D (Figures 6F-6H and S7B), ruling out nonspecific effects of second-site mutations. Login to comment
142 Direct energetic stabilization of the DF508-NBD1-0S and -3S by the V510D mutation was marginal (Figure S5). Login to comment
144 Accordingly, substantially increased folding efficiency of DF508-CFTR-1218X was only achieved by synergistic stabilization of the NBD1-CL4 interface (R1070W or V510D) and NBD1 (3S, -3R, or -R1S) (Figures 7A- 7C, and S7C). Login to comment
174 WT-like domain assembly and stabilization of DF508 CFTR were achieved by a combination of NBD1 and NBD1-CL4 interface-stabilizing mutations (e.g., 3S and R1070W or V510D). Login to comment
175 M2* and red line depict the R1070W mutation. Login to comment
204 Stabilization of the NBD1-CL4 interface by salt bridge formation between V510D and R1070 also improved WT CFTR biogenesis (Figure 6B). Login to comment
135 Conversely, stabilization of the NBD1-CL4 interface by the V510D substitution (see bellow) increased the WT CFTR folding efficiency by 2-fold, supporting the critical role of the NBD1-CL4 interface in the coupled domain folding of CFTR (Figure 6B; see below). Login to comment
139 R1070W, similar to V510D, alone modestly increased the DF508 CFTR folding efficiency and cellular and PM expression (Figures 6A-6E), in part confirming previous reports (He et al., 2010; Thibodeau et al., 2010; Loo et al., 2010). Login to comment
143 Direct energetic stabilization of the DF508-NBD1-0S and -3S by the V510D mutation was marginal (Figure S5). Login to comment
145 Accordingly, substantially increased folding efficiency of DF508-CFTR-1218X was only achieved by synergistic stabilization of the NBD1-CL4 interface (R1070W or V510D) and NBD1 (3S, -3R, or -R1S) (Figures 7A- 7C, and S7C). Login to comment
205 Stabilization of the NBD1-CL4 interface by salt bridge formation between V510D and R1070 also improved WT CFTR biogenesis (Figure 6B). Login to comment

PMID: 23055971 [PubMed] Molinski S et al: "Functional Rescue of F508del-CFTR Using Small Molecule Correctors."

No. Sentence Comment

58 Introduction of R1070W or V510D in the F508del-CFTR protein partially corrects folding of the full-length protein, highlighting the idea that even in the absence of F508, assembly of the CFTR can be partially restored through structural changes at key loci in the protein (Thibodeau et al., 2010; Mendoza et al., 2012). Login to comment

PMID: 23104983 [PubMed] He L et al: "Correctors of DeltaF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein."

No. Sentence Comment

138 The R1070W substitution in CL4 may do so by contributing to interactions among a cluster of aromatic residues at the interface that is weakened by the absence of F508 from the NBD1 surface (11), whereas the V510D mutation was proposed to provide a salt bridge with R1070 (31). Login to comment
139 The V510D mutant on the NBD1 side of the interface is very sensitive to further enhancement of maturation by the compound, whereas that on the CL4 side (R1070W) responds only rather weakly (Fig. 5B). Login to comment
140 This difference may reflect the fact that the V510D substitution stabilizes isolated NBD1 (32) in the absence of the rest of CFTR, as well as influencing the interface, whereas R1070W has only the latter effect. Login to comment
142 Interestingly, as has already been observed by others (9), the influence of the combined V510D and R1070W substitutions is similar to that of V510D alone, which would not be expected if V510D were forming a salt bridge with R1070 but might be if V510D acted primarily to stabilize the NBD1 domain, as has been observed (32). Login to comment
149 B) èc;F508 with NBD1/CL4 interface substitutions R1070W and/or V510D. Login to comment
157 Interestingly, the patterns of enhancement by the compounds were remarkably similar for each of the three classes of NBD1 stabilizing mutations (èc;F/èc;RI, èc;F4S, and èc;F/4PT) with or without one of the interface substitutions (R1070W or V510D). Login to comment

PMID: 23378596 [PubMed] Hunt JF et al: "Cystic fibrosis transmembrane conductance regulator (ABCC7) structure."

No. Sentence Comment

256 Moreover, restoration of the trafficking of F508del-NBD1 by the V510D suppressor mutation, which introduces a negative charge into a generally apolar region J.F. Hunt et al. 16 Cite this article as Cold Spring Harb Perspect Med 2012;3:a009514 www.perspectivesinmedicine.org by Cold Spring Harbor Laboratory Press at SEMMELWEIS UNIV OF MEDICINE on December 5, of the interdomain interface (Fig. 4C,D), is strongly attenuated by introducing the R1070A or R1070D mutations that remove a complementary positive charge from the proximal surface of the TMD (Loo et al. 2010). Login to comment
275 A series of second-site mutations in NBD1 have parallel effects in rescuing the trafficking defect in CFTR in vivo (DeCarvalho et al. 2002; Pissarra et al. 2008; Aleksandrov et al. 2010) and inhibiting molten globule formation by isolated NBD1 in vitro (G550E/R553Q/R555K, F494N/Q637R, or V510D) (Protasevich et al. 2010; Wang et al. 2010). Login to comment

PMID: 23457292 [PubMed] Chong PA et al: "Dynamics intrinsic to cystic fibrosis transmembrane conductance regulator function and stability."

No. Sentence Comment

96 The V510D mutation likely introduces a salt bridge with R1070, strengthening this interface. Login to comment
99 These additional mutations support the idea that the F508del phenotype can be attributed at least partially to a disruption of this interface, although as we discuss below, the V510D mutation also thermodynamically stabilizes NBD1 itself. Login to comment
100 The data on the V510D mutation indicate that interdomain interactions between NBD1 and ICL4 are required for proper folding and maturation of CFTR. Login to comment
201 V510D, which may rescue F508del by facilitating the NBD1-ICL4 interaction, also stabilizes NBD1 (Protasevich et al. 2010), suggesting a dual mechanism of action. Login to comment

PMID: 23835419 [PubMed] Loo TW et al: "Corrector VX-809 stabilizes the first transmembrane domain of CFTR."

No. Sentence Comment

29 We also report that V510D acts as a universal suppressor to rescue CFTR processing mutants. Login to comment
180 To test if the V232D or H1085R mutants could be rescued by suppressor mutations in other domains, suppressor mutations in NBD1 (I539T), the NBD1-TMD2 interface (V510D), or TMD2 (R1070W) (only V232D) locations were introduced into the mutants. Login to comment
182 All the mutants could be rescued by the V510D mutation (Fig. 5C). Login to comment
183 This result shows that the V510D and H1085R mutants could indeed be directly rescued by a suppressor mutation. Login to comment
185 We previously reported that the R1070W mutation also reduces the maturation efficiency of wild-type CFTR [32] while V510D does not [33]. Login to comment
186 The results suggest that the V510D is a 'universal suppressor` that is capable of rescuing CFTR mutants with processing mutations in multiple domains. Login to comment
232 Only the V510D suppressor mutation promotes maturation of mutants with processing mutations in multiple domains. Login to comment
237 (C) Extracts of cells expressing processing mutants DF508, V232D, or H1085R with or without the V510D, I539T, or R1070W suppressor mutations were subjected to immunoblot analysis. Login to comment
249 VX-809 shows characteristics similar to the V510D suppressor that is located at the NBD1-ICL2 interface (Fig. 6A) as both can promote maturation of mutants with processing mutations in different domains. Login to comment
259 Finally, the studies suggest that V510D differs from many other suppressor mutations because it acts as a universal suppressor to rescue mutants with mutations in many domains. Login to comment

PMID: 23890012 [PubMed] Farinha CM et al: "Revertants, low temperature, and correctors reveal the mechanism of F508del-CFTR rescue by VX-809 and suggest multiple agents for full correction."

No. Sentence Comment

16 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 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 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
44 Curiously, all the three compounds further increase band C levels of F508del-V510D-CFTR, but VRT-325 produces the greatest effect. Login to comment
57 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
87 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 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
101 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 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 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 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
165 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 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
195 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
210 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
231 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

PMID: 23924900 [PubMed] Ren HY et al: "VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1."

No. Sentence Comment

131 Defective contacts between F508del-NBD1 and ICL4 that limit F508del-CFTR assembly have been partially restored by introduction of the V510D mutation into NBD1 by permitting the formation of a salt bridge between D510 and R1070 of ICL4 (Wang et al., 2007; Figure 6B). Login to comment
132 The V510D mutation partially corrects misfolding of F508del-CFTR, as shown by the C-band for V510D/F508del-CFTR (Figure 6B, lane 4), to levels similar to that for F508del-CFTR in the presence of VX-809. Login to comment
133 Remarkably, VX-809 stimulated the accumulation C-band of V510D/ F508del-CFTR to the levels observed for normal CFTR. Login to comment
134 To determine whether formation of a salt bridge between D510 and R1070 was important for this effect, we introduced the R1070A mutation into V510D/F508del-CFTR. Login to comment
135 In the presence of VX-809, the accumulation of the C-band of R1070A/V510D/ F508del-CFTR was reduced by 75% relative to V510D/F508 -CFTR (Figure 6B, lane 7). Login to comment
136 Yet VX-809 was still able to increase folded R1070A/V510D/F508-CFTR to levels that were significantly higher than those for VX-809-treated F508del-CFTR. Login to comment
137 Because the V510D mutation can modestly improve the thermodynamic stability of purified NBD1 (Lewis et al., 2010; Wang et al., 2010), the residual VX-809 corrector function on R1070A/V501D/F508del-CFTR could result from thermodynamic stabilization of NBD1 that would occur in the absence of salt-bridge formation between D510 and R1070. Login to comment
229 (B) Introduction of the V510D suppressor mutation into NBD1 permits VX-809 to drive high-level folding of F508del-CFTR. Login to comment

PMID: 24058550 [PubMed] Dawson JE et al: "Allosteric coupling between the intracellular coupling helix 4 and regulatory sites of the first nucleotide-binding domain of CFTR."

No. Sentence Comment

5 Titration of CL4 peptide into NBD1 perturbs the conformational ensemble in these sites with similar titration patterns observed in F508del, the major CF-causing mutant, and in suppressor mutants F494N, V510D and Q637R NBD1, as well as in a CL4-NBD1 fusion construct. Login to comment
25 The maturation defects can be partially suppressed by mutations in NBD1 or the CL4 coupling helix, such as V510D and F494N/Q637R [11,16-21], and by the drug VX-809 [22,23], now in clinical trials. Login to comment
50 Similar titration patterns were observed in NBD1 constructs containing Q637R and mutations near the CL4-binding site (F508del, F494N, and V510D), as well as in a CL4-NBD1 fusion. Login to comment
56 Mutant NBD1 constructs with F494N, F508del, V510D, Q637R or F494N/ Q637R were constructed with a Stratagene QuikChange site-directed mutagenesis kit. Login to comment
175 CL4 also binds to NBD1 proteins containing single F508del-suppressor mutations, V510D and F494N, located near the predicted CL4-binding site, and Q637R, which lies between H8 and H9 (Figures 4 and S4). Login to comment
178 In contrast, V510D NBD1 has greater chemical shift changes in the CL4-binding site upon CL4 titration than WT NBD1, relative to changes in other residues (Figures 4E and 4F). Login to comment
201 Correlation Analysis of Titration Data Improves the Detection of Weak but Significant Changes due to CL4 Binding CL4 binding causes relatively small Dvobs, the greatest of which is less than 0.14 ppm (Figure 4E; binding to V510D NBD1). Login to comment
299 Chemical shift changes upon CL4 binding provide evidence for this allosteric network observed with minor variations in five variants of isolated NBD1-WT, F508del, F494N, V510D, and Q637R, in a CL4-NBD1 fusion protein with slightly different CL4 boundaries and in different buffer conditions. Login to comment
309 The mutations that can partially suppress the folding defects of F508del NBD1 are scattered across NBD1- V510D near CL4 in the channel, I539T directly opposite the NBD1:NBD2 interface, and Q637R near the start of the R region [12], hinting at the complex allosteric nature of the NBD1 folding landscape. Login to comment
357 Overlay of apo (black) and 12.5:1 CL4:NBD1 (red) spectra for A. F508del NBD1, B. F494N NBD1, C. V510D NBD1, and D. Q637R NBD1 mutants. Login to comment

PMID: 24412276 [PubMed] Loo TW et al: "The cystic fibrosis V232D mutation inhibits CFTR maturation by disrupting a hydrophobic pocket rather than formation of aberrant interhelical hydrogen bonds."

No. Sentence Comment

167 3.5. Rescue of Val232 and Gln207 processing mutants with the V510D suppressor mutation Another approach to rescue CFTR processing mutants is to introduce the V510D suppressor mutation [37]. Login to comment
168 The V510D appears to act as a universal suppressor because it can promote maturation of mutants with processing mutations throughout the molecule. Login to comment
169 For example, V510D promotes maturation of mutants with processing mutations in TMD1 (V232D), TMD2 (H1085R) and NBD1 (DF508) whereas other suppressors such as I539T and R1070W promote maturation of DF508 CFTR but not mutants V232D or H1085R [19]. Login to comment
171 Accordingly, we introduced the V510D suppressor mutation into mutants V232E, V232K, or V232R. Login to comment
173 The V510D was particularly effective in promoting maturation of V232E in the absence of VX-809 as the level of mature protein increased from less than 5% (Fig. 4A) to about 35% in mutant V510D/V232E (Fig. 5A and B). Login to comment
174 Expression of V510D/V232E in the presence of VX-809 yielded about 70% mature protein. Login to comment
175 The V510D suppressor caused a small increase in the amount of V232K mature protein (10%) but no detectable increase was observed in mutant V232R when expressed in the absence of VX-809 (Fig. 5A and B). Login to comment
176 The V510D/V232K mutant however, could still be efficiently rescued with corrector VX-809 to yield mature CFTR as the major product (about 70%). Login to comment
177 The relative maturation levels achieved using correctors or the V510D suppressor show that the V232E mutation could be more efficiently rescued than the V232K or V232R mutations. Login to comment
178 The properties of the V232E mutant are likely to be very similar to the V232D mutant as the V510D suppressor mutation [19] could also rescue the V232D mutant. Login to comment
179 If V510D is a universal suppressor, then we predict that it would also promote maturation of the Q207X mutants. Login to comment
180 Accordingly, the V510D mutation was introduced into mutants Q207X (X = A, L, C, E, F, W, N, and S) that were defective in maturation (Fig. 2). Login to comment
182 It was observed that in the absence of VX-809, the V510D mutation significantly improved the maturation of Q207L, Q207C, Q207E, Q207N and Q207S (Fig. 6A and B). Login to comment
183 Mature CFTR was the major product in Q207N/V510D (90% mature product) while mutants Q207L/V510D, Q207C/V510D, Q207E/V510D, and Q207S/V510D showed modest levels of mature CFTR (about 20-40% mature). Login to comment
184 In the presence of corrector VX-809 however, the amount of mature CFTR in mutants V510D/Q207A V510D/Q207L, V510D/Q207C, V510D/Q207E, V510D/Q207F and V510D/Q207S were significantly increased (25-85% mature product). Login to comment
185 Corrector VX-809 did not significantly increase the amount of mature CFTR in mutant V510D/Q207W (Fig. 6A and B). Login to comment
215 To test if the rescued L305R mutant was active, histidine-tagged versions of the mutant and wild-type P-gp were expressed in HEK Fig. 6. Rescue of Q207X mutants with the V510D suppressor mutation. Login to comment
220 An asterisk indicates significant (P < 0.05) difference when compared to the Q207X mutant (without the V510D mutation) that was expressed in the absence of corrector. Login to comment
221 Fig. 5. Rescue of Val232 mutants with the V510D suppressor mutation. Login to comment
222 (A) Whole cell extracts of cells expressing CFTR mutants V232E/V510D, V232K/V510D or V232R/ V510D in the absence () or presence (+) of VX-809 or were subjected to immunoblot analysis. Login to comment
226 An asterisk indicates significant (P < 0.05) difference when compared to the mutant lacking V510D and expressed without corrector. Login to comment
274 In summary, the results suggest that: (1) the mechanism of how V232D causes protein misfolding is unlikely to involve non-native hydrogen bond interactions with Gln207 because V232N yielded about 20-fold more mature CFTR than V232D; (2) it appears that the mechanism of protein misfolding by V232D mutation involves disruption of a hydrophobic pocket since the hydrophobicity of the substituted amino acid at position 232 correlated with the amount of mature product and the ability to correct the defects with correctors or the V510D suppressor mutation; (3) the V232D mutation traps CFTR as a partially folded intermediate that can be rescued by corrector VX-809 to yield a native structure. Login to comment

PMID: 24609033 [PubMed] Young JC et al: "The role of the cytosolic HSP70 chaperone system in diseases caused by misfolding and aberrant trafficking of ion channels."

No. Sentence Comment

141 However, ƊF508 CFTR trafficking was improved by DNAJB12 knockdown combined with another stabilizing V510D mutation within CFTR and a small-molecule stabilizer, Corrector-4 (Grove et al., 2011). Login to comment

PMID: 24685677 [PubMed] Pranke IM et al: "Biosynthesis of cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

1443 The V510D suppressor mutation increased the half-life of mature Phe508del-CFTR at the cell surface (Loo et al., 2010). Login to comment

PMID: 25083918 [PubMed] He L et al: "Restoration of NBD1 thermal stability is necessary and sufficient to correct F508 CFTR folding and assembly."

No. Sentence Comment

35 Rabeh et al. found that, while several of the solubilizing and suppressor mutations caused only a modest promotion of ƊF508 CFTR maturation, their effects were greater when they were combined with the NBD1/CL4 interface substitution R1070W or V510D [33], the latter also having been shown to have a direct effect on NBD1 thermostability [10]. Login to comment
55 In contrast, each of the interface substitutions, V510D (lane 8) and R1070W (lane 9), had much smaller effects. Login to comment
61 The rates of appearance of the mature products and the levels reached were increased further when the R1070W mutation was added to the combined NBD1 changes but not when V510D was added. Login to comment
84 The surface level of ƊF508 CFTR with the combined NBD1 stabilizing mutations (ƊF/combo) reached ~90% that of WT CFTR (Fig. 1c), and the level was somewhat further increased when V510D or R1070W was added to the combination (ƊF/combo/V510D and ƊF/combo/R1070W). Login to comment
88 The addition of V510D to the ƊF508/combo did not further increase its peak of iodide efflux. Login to comment
125 Turning to the interface modified variants (Fig. 3f), BEIA also caused a large further increase in the maturation of the ƊF508/V510D protein. Login to comment
201 We observed that the mutation alone promoted only a low level of maturation as did the other known interface change, V510D [43]. Login to comment

PMID: 25384981 [PubMed] Caohuy H et al: "Activation of 3-phosphoinositide-dependent kinase 1 (PDK1) and serum- and glucocorticoid-induced protein kinase 1 (SGK1) by short-chain sphingolipid C4-ceramide rescues the trafficking defect of DeltaF508-cystic fibrosis transmembrane conductance regulator (DeltaF508-CFTR)."

No. Sentence Comment

427 For instance, the introduction of the suppressor mutation V510D to èc;F508-CFTR mediates rescue of èc;F508-CFTR and increases the half-life of the membrane-bound èc;F508-CFTR (39). Login to comment