ABCMdb

ABCC7 p.Val510Asp

Predicted by SNAP2:	A: N (82%), C: N (72%), D: N (57%), E: N (72%), F: N (72%), G: N (72%), H: N (66%), I: N (93%), K: N (82%), L: N (87%), M: N (87%), N: N (72%), P: N (66%), Q: N (82%), R: N (78%), S: N (72%), T: N (87%), W: D (66%), Y: N (78%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, W: N, Y: N,

[switch to compact view]
Comments [show]

None has been submitted yet.

Publications
[hide] Correctors promote maturation of cystic fibrosis t... J Biol Chem. 2007 Nov 16;282(46):33247-51. Epub 2007 Oct 2. Wang Y, Loo TW, Bartlett MC, Clarke DM
Correctors promote maturation of cystic fibrosis transmembrane conductance regulator (CFTR)-processing mutants by binding to the protein.
J Biol Chem. 2007 Nov 16;282(46):33247-51. Epub 2007 Oct 2., 2007-11-16 [PMID:17911111]

Abstract [show]
The most common cause of cystic fibrosis (CF) is defective folding of a cystic fibrosis transmembrane conductance regulator (CFTR) mutant lacking Phe(508) (DeltaF508). The DeltaF508 protein appears to be trapped in a prefolded state with incomplete packing of the transmembrane (TM) segments, a defect that can be repaired by expression in the presence of correctors such as corr-4a, VRT-325, and VRT-532. To determine whether the mechanism of correctors involves direct interactions with CFTR, our approach was to test whether correctors blocked disulfide cross-linking between cysteines introduced into the two halves of a Cys-less CFTR. Although replacement of the 18 endogenous cysteines of CFTR with Ser or Ala yields a Cys-less mutant that does not mature at 37 degrees C, we found that maturation could be restored if Val(510) was changed to Ala, Cys, Ser, Thr, Gly, Ala, or Asp. The V510D mutation also promoted maturation of DeltaF508 CFTR. The Cys-less/V510A mutant was used for subsequent cross-linking analysis as it yielded relatively high levels of mature protein that was functional in iodide efflux assays. We tested for cross-linking between cysteines introduced into TM6 and TM7 of Cys-less CFTR/V510A because cross-linking between TM6 and TM7 of P-glycoprotein, the sister protein of CFTR, was inhibited with the corrector VRT-325. Cys-less CFTR/V510A mutant containing cysteines at I340C(TM6) and S877C(TM7) could be cross-linked with a homobifunctional cross-linker. Correctors and the CFTR channel blocker benzbromarone, but not P-glycoprotein substrates, inhibited cross-linking of mutant I340C(TM6)/S877C(TM7). These results suggest that corrector molecules such as corr-4a interact directly with CFTR.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Atomic model of human cystic fibrosis transmembran... Cell Mol Life Sci. 2008 Aug;65(16):2594-612. Mornon JP, Lehn P, Callebaut I
Atomic model of human cystic fibrosis transmembrane conductance regulator: membrane-spanning domains and coupling interfaces.
Cell Mol Life Sci. 2008 Aug;65(16):2594-612., [PMID:18597042]

Abstract [show]
We describe herein an atomic model of the outward-facing three-dimensional structure of the membrane-spanning domains (MSDs) and nucleotide-binding domains (NBDs) of human cystic fibrosis transmembrane conductance regulator (CFTR), based on the experimental structure of the bacterial transporter Sav1866. This model, which is in agreement with previous experimental data, highlights the role of some residues located in the transmembrane passages and directly involved in substrate translocation and of some residues within the intracellular loops (ICL1-ICL4) making MSD/NBD contacts. In particular, our model reveals that D173 ICL1 and N965 ICL3 likely interact with the bound nucleotide and that an intricate H-bond network (involving especially the ICL4 R1070 and the main chain of NBD1 F508) may stabilize the interface between MSD2 and the NBD1F508 region. These observations allow new insights into the ATP-binding sites asymmetry and into the molecular consequences of the F508 deletion, which is the most common cystic fibrosis mutation.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Mechanisms for rescue of correctable folding defec... Mol Biol Cell. 2009 Sep;20(18):4059-69. Epub 2009 Jul 22. Grove DE, Rosser MF, Ren HY, Naren AP, Cyr DM
Mechanisms for rescue of correctable folding defects in CFTRDelta F508.
Mol Biol Cell. 2009 Sep;20(18):4059-69. Epub 2009 Jul 22., [PMID:19625452]

Abstract [show]
Premature degradation of CFTRDeltaF508 causes cystic fibrosis (CF). CFTRDeltaF508 folding defects are conditional and folding correctors are being developed as CF therapeutics. How the cellular environment impacts CFTRDeltaF508 folding efficiency and the identity of CFTRDeltaF508's correctable folding defects is unclear. We report that inactivation of the RMA1 or CHIP ubiquitin ligase permits a pool of CFTRDeltaF508 to escape the endoplasmic reticulum. Combined RMA1 or CHIP inactivation and Corr-4a treatment enhanced CFTRDeltaF508 folding to 3-7-fold greater levels than those elicited by Corr-4a. Some, but not all, folding defects in CFTRDeltaF508 are correctable. CHIP and RMA1 recognize different regions of CFTR and a large pool of nascent CFTRDeltaF508 is ubiquitinated by RMA1 before Corr-4a action. RMA1 recognizes defects in CFTRDeltaF508 related to misassembly of a complex that contains MSD1, NBD1, and the R-domain. Corr-4a acts on CFTRDeltaF508 after MSD2 synthesis and was ineffective at rescue of DeltaF508 dependent folding defects in amino-terminal regions. In contrast, misfolding caused by the rare CF-causing mutation V232D in MSD1 was highly correctable by Corr-4a. Overall, correction of folding defects recognized by RMA1 and/or global modulation of ER quality control has the potential to increase CFTRDeltaF508 folding and provide a therapeutic approach for CF.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Cystic fibrosis transmembrane regulator fragments ... Biochem J. 2010 Jan 27;426(1):19-29. Pagano MA, Marin O, Cozza G, Sarno S, Meggio F, Treharne KJ, Mehta A, Pinna LA
Cystic fibrosis transmembrane regulator fragments with the Phe508 deletion exert a dual allosteric control over the master kinase CK2.
Biochem J. 2010 Jan 27;426(1):19-29., 2010-02-15 [PMID:19925455]

Abstract [show]
Cystic fibrosis mostly follows a single Phe508 deletion in CFTR (cystic fibrosis transmembrane regulator) (CFTRDeltaF508), thereby causing premature fragmentation of the nascent protein with concomitant alterations of diverse cellular functions. We show that CK2, the most pleiotropic protein kinase, undergoes allosteric control of its different cellular forms in the presence of short CFTR peptides encompassing the Phe508 deletion: these CFTRDeltaF508 peptides drastically inhibit the isolated catalytic subunit (alpha) of the kinase and yet up-regulate the holoenzyme, composed of two catalytic and two non-catalytic (beta) subunits. Remarkable agreement between in silico docking and our biochemical data point to different sites for the CFTRDeltaF508 peptide binding on isolated CK2alpha and on CK2beta assembled into the holoenzyme, suggesting that CK2 targeting may be perturbed in cells expressing CFTRDeltaF508; this could shed light on some pleiotropic aspects of cystic fibrosis disease.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Structure and dynamics of NBD1 from CFTR character... J Mol Biol. 2010 Feb 19;396(2):406-30. Epub 2009 Nov 26. Lewis HA, Wang C, Zhao X, Hamuro Y, Conners K, Kearins MC, Lu F, Sauder JM, Molnar KS, Coales SJ, Maloney PC, Guggino WB, Wetmore DR, Weber PC, Hunt JF
Structure and dynamics of NBD1 from CFTR characterized using crystallography and hydrogen/deuterium exchange mass spectrometry.
J Mol Biol. 2010 Feb 19;396(2):406-30. Epub 2009 Nov 26., 2010-02-19 [PMID:19944699]

Abstract [show]
The DeltaF508 mutation in nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the predominant cause of cystic fibrosis. Previous biophysical studies on human F508 and DeltaF508 domains showed only local structural changes restricted to residues 509-511 and only minor differences in folding rate and stability. These results were remarkable because DeltaF508 was widely assumed to perturb domain folding based on the fact that it prevents trafficking of CFTR out of the endoplasmic reticulum. However, the previously reported crystal structures did not come from matched F508 and DeltaF508 constructs, and the DeltaF508 structure contained additional mutations that were required to obtain sufficient protein solubility. In this article, we present additional biophysical studies of NBD1 designed to address these ambiguities. Mass spectral measurements of backbone amide (1)H/(2)H exchange rates in matched F508 and DeltaF508 constructs reveal that DeltaF508 increases backbone dynamics at residues 509-511 and the adjacent protein segments but not elsewhere in NBD1. These measurements also confirm a high level of flexibility in the protein segments exhibiting variable conformations in the crystal structures. We additionally present crystal structures of a broader set of human NBD1 constructs, including one harboring the native F508 residue and others harboring the DeltaF508 mutation in the presence of fewer and different solubilizing mutations. The only consistent conformational difference is observed at residues 509-511. The side chain of residue V510 in this loop is mostly buried in all non-DeltaF508 structures but completely solvent exposed in all DeltaF508 structures. These results reinforce the importance of the perturbation DeltaF508 causes in the surface topography of NBD1 in a region likely to mediate contact with the transmembrane domains of CFTR. However, they also suggest that increased exposure of the 509-511 loop and increased dynamics in its vicinity could promote aggregation in vitro and aberrant intermolecular interactions that impede trafficking in vivo.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] The V510D suppressor mutation stabilizes DeltaF508... Biochemistry. 2010 Aug 3;49(30):6352-7. Loo TW, Bartlett MC, Clarke DM
The V510D suppressor mutation stabilizes DeltaF508-CFTR at the cell surface.
Biochemistry. 2010 Aug 3;49(30):6352-7., 2010-08-03 [PMID:20590134]

Abstract [show]
Deletion of Phe508 (DeltaF508) in the first nucleotide-binding domain (NBD1) of CFTR causes cystic fibrosis. The mutation severely reduces the stability and folding of the protein by disrupting interactions between NBD1 and the second transmembrane domain (TMD2). We found that replacement of Val510 with acidic residues (but not neutral or positive residues) promoted maturation of DeltaF508-CFTR with V510D more efficiently than V510E. Promotion of DeltaF508-CFTR maturation did not require NBD2 as introduction of V510D into a DeltaNBD2/DeltaF508-CFTR mutant restored maturation to levels similar to that of full-length protein. The V510D mutation increased the half-life of mature DeltaF508-CFTR at the cell surface by about 5-fold to resemble the half-life of wild-type CFTR. It was also observed that introduction of the V510R/R1070D mutations into DeltaF508-CFTR also promoted maturation whereas the V510D/R1070A mutations did not. We propose that the V510D mutation in NBD1 promotes maturation and stabilizes DeltaF508-CFTR at the cell surface through formation of a salt bridge with Arg1070 in TMD2.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Thermal unfolding studies show the disease causing... Protein Sci. 2010 Oct;19(10):1917-31. Protasevich I, Yang Z, Wang C, Atwell S, Zhao X, Emtage S, Wetmore D, Hunt JF, Brouillette CG
Thermal unfolding studies show the disease causing F508del mutation in CFTR thermodynamically destabilizes nucleotide-binding domain 1.
Protein Sci. 2010 Oct;19(10):1917-31., [PMID:20687133]

Abstract [show]
Misfolding and degradation of CFTR is the cause of disease in patients with the most prevalent CFTR mutation, an in-frame deletion of phenylalanine (F508del), located in the first nucleotide-binding domain of human CFTR (hNBD1). Studies of (F508del)CFTR cellular folding suggest that both intra- and inter-domain folding is impaired. (F508del)CFTR is a temperature-sensitive mutant, that is, lowering growth temperature, improves both export, and plasma membrane residence times. Yet, paradoxically, F508del does not alter the fold of isolated hNBD1 nor did it seem to perturb its unfolding transition in previous isothermal chemical denaturation studies. We therefore studied the in vitro thermal unfolding of matched hNBD1 constructs +/-F508del to shed light on the defective folding mechanism and the basis for the thermal instability of (F508del)CFTR. Using primarily differential scanning calorimetry (DSC) and circular dichroism, we show for all hNBD1 pairs studied, that F508del lowers the unfolding transition temperature (T(m)) by 6-7 degrees C and that unfolding occurs via a kinetically-controlled, irreversible transition in isolated monomers. A thermal unfolding mechanism is derived from nonlinear least squares fitting of comprehensive DSC data sets. All data are consistent with a simple three-state thermal unfolding mechanism for hNBD1 +/- F508del: N(+/-MgATP) <==> I(T)(+/-MgATP) --> A(T) --> (A(T))(n). The equilibrium unfolding to intermediate, I(T), is followed by the rate-determining, irreversible formation of a partially folded, aggregation-prone, monomeric state, A(T), for which aggregation to (A(T))(n) and further unfolding occur with no detectable heat change. Fitted parameters indicate that F508del thermodynamically destabilizes the native state, N, and accelerates the formation of A(T).

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Integrated biophysical studies implicate partial u... Protein Sci. 2010 Oct;19(10):1932-47. Wang C, Protasevich I, Yang Z, Seehausen D, Skalak T, Zhao X, Atwell S, Spencer Emtage J, Wetmore DR, Brouillette CG, Hunt JF
Integrated biophysical studies implicate partial unfolding of NBD1 of CFTR in the molecular pathogenesis of F508del cystic fibrosis.
Protein Sci. 2010 Oct;19(10):1932-47., [PMID:20687163]

Abstract [show]
The lethal genetic disease cystic fibrosis is caused predominantly by in-frame deletion of phenylalanine 508 in the cystic fibrosis transmembrane conductance regulator (CFTR). F508 is located in the first nucleotide-binding domain (NBD1) of CFTR, which functions as an ATP-gated chloride channel on the cell surface. The F508del mutation blocks CFTR export to the surface due to aberrant retention in the endoplasmic reticulum. While it was assumed that F508del interferes with NBD1 folding, biophysical studies of purified NBD1 have given conflicting results concerning the mutation's influence on domain folding and stability. We have conducted isothermal (this paper) and thermal (accompanying paper) denaturation studies of human NBD1 using a variety of biophysical techniques, including simultaneous circular dichroism, intrinsic fluorescence, and static light-scattering measurements. These studies show that, in the absence of ATP, NBD1 unfolds via two sequential conformational transitions. The first, which is strongly influenced by F508del, involves partial unfolding and leads to aggregation accompanied by an increase in tryptophan fluorescence. The second, which is not significantly influenced by F508del, involves full unfolding of NBD1. Mg-ATP binding delays the first transition, thereby offsetting the effect of F508del on domain stability. Evidence suggests that the initial partial unfolding transition is partially responsible for the poor in vitro solubility of human NBD1. Second-site mutations that increase the solubility of isolated F508del-NBD1 in vitro and suppress the trafficking defect of intact F508del-CFTR in vivo also stabilize the protein against this transition, supporting the hypothesize that it is responsible for the pathological trafficking of F508del-CFTR.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] The endoplasmic reticulum-associated Hsp40 DNAJB12... Mol Biol Cell. 2011 Feb 1;22(3):301-14. Epub 2010 Dec 9. Grove DE, Fan CY, Ren HY, Cyr DM
The endoplasmic reticulum-associated Hsp40 DNAJB12 and Hsc70 cooperate to facilitate RMA1 E3-dependent degradation of nascent CFTRDeltaF508.
Mol Biol Cell. 2011 Feb 1;22(3):301-14. Epub 2010 Dec 9., 2011-02-01 [PMID:21148293]

Abstract [show]
Relative contributions of folding kinetics versus protein quality control (QC) activity in the partitioning of non-native proteins between life and death are not clear. Cystic fibrosis transmembrane conductance regulator (CFTR) biogenesis serves as an excellent model to study this question because folding of nascent CFTR is inefficient and deletion of F508 causes accumulation of CFTRDeltaF508 in a kinetically trapped, but foldable state. Herein, a novel endoplasmic reticulum (ER)-associated Hsp40, DNAJB12 (JB12) is demonstrated to play a role in control of CFTR folding efficiency. JB12 cooperates with cytosolic Hsc70 and the ubiquitin ligase RMA1 to target CFTR and CFTRDeltaF508 for degradation. Modest elevation of JB12 decreased nascent CFTR and CFTRDeltaF508 accumulation while increasing association of Hsc70 with ER forms of CFTR and the RMA1 E3 complex. Depletion of JB12 increased CFTR folding efficiency up to threefold and permitted a pool of CFTRDeltaF508 to fold and escape the ER. Introduction of the V510D misfolding suppressor mutation into CFTRDeltaF508 modestly increased folding efficiency, whereas combined inactivation of JB12 and suppression of intrinsic folding defects permitted CFTRDeltaF508 to fold at 50% of wild-type efficiency. Therapeutic correction of CFTRDeltaF508 misfolding in cystic fibrosis patients may require repair of defective folding kinetics and suppression of ER QC factors, such as JB12.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Benzbromarone stabilizes DeltaF508 CFTR at the cel... Biochemistry. 2011 May 31;50(21):4393-5. Epub 2011 May 3. Loo TW, Bartlett MC, Clarke DM
Benzbromarone stabilizes DeltaF508 CFTR at the cell surface.
Biochemistry. 2011 May 31;50(21):4393-5. Epub 2011 May 3., 2011-05-31 [PMID:21520952]

Abstract [show]
Deletion of Phe508 from the first nucleotide-binding domain of the CFTR chloride channel causes cystic fibrosis because it inhibits protein folding. Indirect approaches such as incubation at low temperatures can partially rescue DeltaF508 CFTR, but the protein is unstable at the cell surface. Here, we show that direct binding of benzbromarone to the transmembrane domains promoted maturation and stabilized DeltaF508 CFTR because its half-life at the cell surface was ~10-fold longer than that for low-temperature rescue. Therefore, a search for small molecules that can rescue and stabilize DeltaF508 CFTR could lead to the development of an effective therapy for cystic fibrosis.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] The W232R suppressor mutation promotes maturation ... Biochemistry. 2011 Feb 8;50(5):672-85. Epub 2011 Jan 11. Loo TW, Bartlett MC, Clarke DM
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.
Biochemistry. 2011 Feb 8;50(5):672-85. Epub 2011 Jan 11., 2011-02-08 [PMID:21182301]

Abstract [show]
ATP-binding cassette (ABC) proteins contain two nucleotide-binding domains (NBDs) and two transmembrane (TM) domains (TMDs). Interdomain interactions and packing of the TM segments are critical for function, and disruption by genetic mutations contributes to disease. P-glycoprotein (P-gp) is a useful model to identify mechanisms that repair processing defects because numerous arginine suppressor mutations have been identified in the TM segments. Here, we tested the prediction that a mechanism of arginine rescue was to promote intradomain interactions between TM segments and restore interdomain assembly. We found that suppressor W232R(TM4/TMD1) rescued mutants with processing mutations in any domain and restored defective NBD1-NBD2, NBD1-TMD2, and TMD1-TMD2 interactions. W232R also promoted packing of the TM segments because it rescued a truncation mutant lacking both NBDs. The mechanism of W232R rescue likely involved intradomain hydrogen bond interactions with Asn296(TM5) since only N296A abolished rescue by W232R and rescue was only observed when Trp232 was replaced with hydrogen-bonding residues. In TMD2, suppressor T945R(TM11) also promoted packing of the TM segments because it rescued the truncation mutant lacking the NBDs and suppressed formation of alternative topologies. We propose that T945R rescue was mediated by interactions with Glu875(TM10) since T945E/E875R promoted maturation while T945R/E875A did not.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Fixing cystic fibrosis by correcting CFTR domain a... J Cell Biol. 2012 Oct 15;199(2):199-204. doi: 10.1083/jcb.201208083. Okiyoneda T, Lukacs GL
Fixing cystic fibrosis by correcting CFTR domain assembly.
J Cell Biol. 2012 Oct 15;199(2):199-204. doi: 10.1083/jcb.201208083., [PMID:23071149]

Abstract [show]
For cystic fibrosis (CF) patients most therapies focus on alleviating the disease symptoms. Yet the cellular basis of the disease has been well studied; mutations in the CF gene can impair folding, secretion, cell surface stability, and/or function of the CFTR chloride channel. Correction of these basic defects has been a challenge, but indicates that a deeper understanding of the molecular and cellular mechanism of mutations is a prerequisite for developing more efficient therapies.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Allosteric modulation balances thermodynamic stabi... J Mol Biol. 2012 May 25;419(1-2):41-60. Epub 2012 Mar 8. Aleksandrov AA, Kota P, Cui L, Jensen T, Alekseev AE, Reyes S, He L, Gentzsch M, Aleksandrov LA, Dokholyan NV, Riordan JR
Allosteric modulation balances thermodynamic stability and restores function of DeltaF508 CFTR.
J Mol Biol. 2012 May 25;419(1-2):41-60. Epub 2012 Mar 8., [PMID:22406676]

Abstract [show]
Most cystic fibrosis is caused by a deletion of a single residue (F508) in CFTR (cystic fibrosis transmembrane conductance regulator) that disrupts the folding and biosynthetic maturation of the ion channel protein. Progress towards understanding the underlying mechanisms and overcoming the defect remains incomplete. Here, we show that the thermal instability of human DeltaF508 CFTR channel activity evident in both cell-attached membrane patches and planar phospholipid bilayers is not observed in corresponding mutant CFTRs of several non-mammalian species. These more stable orthologs are distinguished from their mammalian counterparts by the substitution of proline residues at several key dynamic locations in first N-terminal nucleotide-binding domain (NBD1), including the structurally diverse region, the gamma-phosphate switch loop, and the regulatory insertion. Molecular dynamics analyses revealed that addition of the prolines could reduce flexibility at these locations and increase the temperatures of unfolding transitions of DeltaF508 NBD1 to that of the wild type. Introduction of these prolines experimentally into full-length human DeltaF508 CFTR together with the already recognized I539T suppressor mutation, also in the structurally diverse region, restored channel function and thermodynamic stability as well as its trafficking to and lifetime at the cell surface. Thus, while cellular manipulations that circumvent its culling by quality control systems leave DeltaF508 CFTR dysfunctional at physiological temperature, restoration of the delicate balance between the dynamic protein's inherent stability and channel activity returns a near-normal state.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Disruption of cytokeratin-8 interaction with F508d... Hum Mol Genet. 2012 Feb 1;21(3):623-34. Epub 2011 Oct 28. Colas J, Faure G, Saussereau E, Trudel S, Rabeh WM, Bitam S, Guerrera IC, Fritsch J, Sermet-Gaudelus I, Davezac N, Brouillard F, Lukacs GL, Herrmann H, Ollero M, Edelman A
Disruption of cytokeratin-8 interaction with F508del-CFTR corrects its functional defect.
Hum Mol Genet. 2012 Feb 1;21(3):623-34. Epub 2011 Oct 28., [PMID:22038833]

Abstract [show]
We have previously reported an increased expression of cytokeratins 8/18 (K8/K18) in cells expressing the F508del mutation of cystic fibrosis transmembrane conductance regulator (CFTR). This is associated with increased colocalization of CFTR and K18 in the vicinity of the endoplasmic reticulum, although this is reversed by treating cells with curcumin, resulting in the rescue of F508del-CFTR. In the present work, we hypothesized that (i) the K8/K18 network may interact physically with CFTR, and that (ii) this interaction may modify CFTR function. CFTR was immunoprecipitated from HeLa cells transfected with either wild-type (WT) CFTR or F508del-CFTR. Precipitates were subjected to 2D-gel electrophoresis and differential spots identified by mass spectrometry. K8 and K18 were found significantly increased in F508del-CFTR precipitates. Using surface plasmon resonance, we demonstrate that K8, but not K18, binds directly and preferentially to the F508del over the WT human NBD1 (nucleotide-binding domain-1). In vivo K8 interaction with F508del-CFTR was confirmed by proximity ligation assay in HeLa cells and in primary cultures of human respiratory epithelial cells. Ablation of K8 expression by siRNA in F508del-expressing HeLa cells led to the recovery of CFTR-dependent iodide efflux. Moreover, F508del-expressing mice topically treated with K8-siRNA showed restored nasal potential difference, equivalent to that of WT mice. These results show that disruption of F508del-CFTR and K8 interaction leads to the correction of the F508del-CFTR processing defect, suggesting a novel potential therapeutic target in CF.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] CFTR: folding, misfolding and correcting the Delta... Trends Mol Med. 2012 Feb;18(2):81-91. Epub 2011 Dec 3. Lukacs GL, Verkman AS
CFTR: folding, misfolding and correcting the DeltaF508 conformational defect.
Trends Mol Med. 2012 Feb;18(2):81-91. Epub 2011 Dec 3., [PMID:22138491]

Abstract [show]
Cystic fibrosis (CF), the most common lethal genetic disease in the Caucasian population, is caused by loss-of-function mutations of the CF transmembrane conductance regulator (CFTR), a cyclic AMP-regulated plasma membrane chloride channel. The most common mutation, deletion of phenylalanine 508 (DeltaF508), impairs CFTR folding and, consequently, its biosynthetic and endocytic processing as well as chloride channel function. Pharmacological treatments may target the DeltaF508 CFTR structural defect directly by binding to the mutant protein and/or indirectly by altering cellular protein homeostasis (proteostasis) to promote DeltaF508 CFTR plasma membrane targeting and stability. This review discusses recent basic research aimed at elucidating the structural and trafficking defects of DeltaF508 CFTR, a prerequisite for the rational design of CF therapy to correct the loss-of-function phenotype.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Correction of both NBD1 energetics and domain inte... Cell. 2012 Jan 20;148(1-2):150-63. Rabeh WM, Bossard F, Xu H, Okiyoneda T, Bagdany M, Mulvihill CM, Du K, di Bernardo S, Liu Y, Konermann L, Roldan A, Lukacs GL
Correction of both NBD1 energetics and domain interface is required to restore DeltaF508 CFTR folding and function.
Cell. 2012 Jan 20;148(1-2):150-63., [PMID:22265408]

Abstract [show]
The folding and misfolding mechanism of multidomain proteins remains poorly understood. Although thermodynamic instability of the first nucleotide-binding domain (NBD1) of DeltaF508 CFTR (cystic fibrosis transmembrane conductance regulator) partly accounts for the mutant channel degradation in the endoplasmic reticulum and is considered as a drug target in cystic fibrosis, the link between NBD1 and CFTR misfolding remains unclear. Here, we show that DeltaF508 destabilizes NBD1 both thermodynamically and kinetically, but correction of either defect alone is insufficient to restore DeltaF508 CFTR biogenesis. Instead, both DeltaF508-NBD1 energetic and the NBD1-MSD2 (membrane-spanning domain 2) interface stabilization are required for wild-type-like folding, processing, and transport function, suggesting a synergistic role of NBD1 energetics and topology in CFTR-coupled domain assembly. Identification of distinct structural deficiencies may explain the limited success of DeltaF508 CFTR corrector molecules and suggests structure-based combination corrector therapies. These results may serve as a framework for understanding the mechanism of interface mutation in multidomain membrane proteins.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Functional Rescue of F508del-CFTR Using Small Mole... Front Pharmacol. 2012 Sep 26;3:160. doi: 10.3389/fphar.2012.00160. eCollection 2012. Molinski S, Eckford PD, Pasyk S, Ahmadi S, Chin S, Bear CE
Functional Rescue of F508del-CFTR Using Small Molecule Correctors.
Front Pharmacol. 2012 Sep 26;3:160. doi: 10.3389/fphar.2012.00160. eCollection 2012., [PMID:23055971]

Abstract [show]
High-throughput screens for small molecules that are effective in "correcting" the functional expression of F508del-CFTR have yielded several promising hits. Two such compounds are currently in clinical trial. Despite this success, it is clear that further advances will be required in order to restore 50% or greater of wild-type CFTR function to the airways of patients harboring the F508del-CFTR protein. Progress will be enhanced by our better understanding of the molecular and cellular defects caused by the F508del mutation, present in 90% of CF patients. The goal of this chapter is to review the current understanding of defects caused by F508del in the CFTR protein and in CFTR-mediated interactions important for its biosynthesis, trafficking, channel function, and stability at the cell surface. Finally, we will discuss the gaps in our knowledge regarding the mechanism of action of existing correctors, the unmet need to discover compounds which restore proper CFTR structure and function in CF affected tissues and new strategies for therapy development.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Correctors of DeltaF508 CFTR restore global confor... FASEB J. 2013 Feb;27(2):536-45. doi: 10.1096/fj.12-216119. Epub 2012 Oct 26. He L, Kota P, Aleksandrov AA, Cui L, Jensen T, Dokholyan NV, Riordan JR
Correctors of DeltaF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.
FASEB J. 2013 Feb;27(2):536-45. doi: 10.1096/fj.12-216119. Epub 2012 Oct 26., [PMID:23104983]

Abstract [show]
Most cystic fibrosis is caused by the deletion of a single amino acid (F508) from CFTR and the resulting misfolding and destabilization of the protein. Compounds identified by high-throughput screening to improve DeltaF508 CFTR maturation have already entered clinical trials, and it is important to understand their mechanisms of action to further improve their efficacy. Here, we showed that several of these compounds, including the investigational drug VX-809, caused a much greater increase (5- to 10-fold) in maturation at 27 than at 37 degrees C (<2-fold), and the mature product remained short-lived (T(1/2) approximately 4.5 h) and thermally unstable, even though its overall conformational state was similar to wild type, as judged by resistance to proteolysis and interdomain cross-linking. Consistent with its inability to restore thermodynamic stability, VX-809 stimulated maturation 2-5-fold beyond that caused by several different stabilizing modifications of NBD1 and the NBD1/CL4 interface. The compound also promoted maturation of several disease-associated processing mutants on the CL4 side of this interface. Although these effects may reflect an interaction of VX-809 with this interface, an interpretation supported by computational docking, it also rescued maturation of mutants in other cytoplasmic loops, either by allosteric effects or via additional sites of action. In addition to revealing the capabilities and some of the limitations of this important investigational drug, these findings clearly demonstrate that DeltaF508 CFTR can be completely assembled and evade cellular quality control systems, while remaining thermodynamically unstable. He, L., Kota, P., Aleksandrov, A. A., Cui, L., Jensen, T., Dokholyan, N. V., Riordan, J. R. Correctors of DeltaF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Cystic fibrosis transmembrane conductance regulato... Cold Spring Harb Perspect Med. 2013 Feb 1;3(2):a009514. doi: 10.1101/cshperspect.a009514. Hunt JF, Wang C, Ford RC
Cystic fibrosis transmembrane conductance regulator (ABCC7) structure.
Cold Spring Harb Perspect Med. 2013 Feb 1;3(2):a009514. doi: 10.1101/cshperspect.a009514., [PMID:23378596]

Abstract [show]
Structural studies of the cystic fibrosis transmembrane conductance regulator (CFTR) are reviewed. Like many membrane proteins, full-length CFTR has proven to be difficult to express and purify, hence much of the structural data available is for the more tractable, independently expressed soluble domains. Therefore, this chapter covers structural data for individual CFTR domains in addition to the sparser data available for the full-length protein. To set the context for these studies, we will start by reviewing structural information on model proteins from the ATP-binding cassette (ABC) transporter superfamily, to which CFTR belongs.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Dynamics intrinsic to cystic fibrosis transmembran... Cold Spring Harb Perspect Med. 2013 Mar 1;3(3):a009522. doi: 10.1101/cshperspect.a009522. Chong PA, Kota P, Dokholyan NV, Forman-Kay JD
Dynamics intrinsic to cystic fibrosis transmembrane conductance regulator function and stability.
Cold Spring Harb Perspect Med. 2013 Mar 1;3(3):a009522. doi: 10.1101/cshperspect.a009522., [PMID:23457292]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) requires dynamic fluctuations between states in its gating cycle for proper channel function, including changes in the interactions between the nucleotide-binding domains (NBDs) and between the intracellular domain (ICD) coupling helices and NBDs. Such motions are also linked with fluctuating phosphorylation-dependent binding of CFTR's disordered regulatory (R) region to the NBDs and partners. Folding of CFTR is highly inefficient, with the marginally stable NBD1 sampling excited states or folding intermediates that are aggregation-prone. The severe CF-causing F508del mutation exacerbates the folding inefficiency of CFTR and leads to impaired channel regulation and function, partly as a result of perturbed NBD1-ICD interactions and enhanced sampling of these NBD1 excited states. Increased knowledge of the dynamics within CFTR will expand our understanding of the regulated channel gating of the protein as well as of the F508del defects in folding and function.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Corrector VX-809 stabilizes the first transmembran... Biochem Pharmacol. 2013 Sep 1;86(5):612-9. doi: 10.1016/j.bcp.2013.06.028. Epub 2013 Jul 5. Loo TW, Bartlett MC, Clarke DM
Corrector VX-809 stabilizes the first transmembrane domain of CFTR.
Biochem Pharmacol. 2013 Sep 1;86(5):612-9. doi: 10.1016/j.bcp.2013.06.028. Epub 2013 Jul 5., [PMID:23835419]

Abstract [show]
Processing mutations that inhibit folding and trafficking of CFTR are the main cause of cystic fibrosis (CF). A potential CF therapy would be to repair CFTR processing mutants. It has been demonstrated that processing mutants of P-glycoprotein (P-gp), CFTR's sister protein, can be efficiently repaired by a drug-rescue mechanism. Many arginine suppressors that mimic drug-rescue have been identified in the P-gp transmembrane (TM) domains (TMDs) that rescue by forming hydrogen bonds with residues in adjacent helices to promote packing of the TM segments. To test if CFTR mutants could be repaired by a drug-rescue mechanism, we used truncation mutants to test if corrector VX-809 interacted with the TMDs. VX-809 was selected for study because it is specific for CFTR, it is the most effective corrector identified to date, but it has limited clinical benefit. Identification of the VX-809 target domain will help to develop correctors with improved clinical benefits. It was found that VX-809 rescued truncation mutants lacking the NBD2 and R domains. When the remaining domains (TMD1, NBD1, TMD2) were expressed as separate polypeptides, VX-809 only increased the stability of TMD1. We then performed arginine mutagenesis on TM6 in TMD1. Although the results showed that TM6 had distinct lipid and aqueous faces, CFTR was different from P-gp as no arginine promoted maturation of CFTR processing mutants. The results suggest that TMD1 contains a VX-809 binding site, but its mechanism differed from P-gp drug-rescue. We also report that V510D acts as a universal suppressor to rescue CFTR processing mutants.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Revertants, low temperature, and correctors reveal... Chem Biol. 2013 Jul 25;20(7):943-55. doi: 10.1016/j.chembiol.2013.06.004. 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., [PMID:23890012]

Abstract [show]
Cystic fibrosis is mostly caused by the F508del mutation, which impairs CFTR protein from exiting the endoplasmic reticulum due to misfolding. VX-809 is a small molecule that rescues F508del-CFTR localization, which recently went into clinical trial but with unknown mechanism of action (MoA). Herein, we assessed if VX-809 is additive or synergistic with genetic revertants of F508del-CFTR, other correctors, and low temperature to determine its MoA. We explored and integrated those various agents in combined treatments, showing how they add to each other to identify their complementary MoA upon correction of F508del-CFTR. Our experimental and modeling data, while compatible with putative binding of VX-809 to NBD1:ICL4 interface, also indicate scope for further synergistic F508del-CFTR correction by other compounds at distinct conformational sites/cellular checkpoints, thus suggesting requirement of combined therapies to fully rescue F508del-CFTR.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] VX-809 corrects folding defects in cystic fibrosis... Mol Biol Cell. 2013 Oct;24(19):3016-24. doi: 10.1091/mbc.E13-05-0240. Epub 2013 Aug 7. Ren HY, Grove DE, De La Rosa O, Houck SA, Sopha P, Van Goor F, Hoffman BJ, Cyr DM
VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1.
Mol Biol Cell. 2013 Oct;24(19):3016-24. doi: 10.1091/mbc.E13-05-0240. Epub 2013 Aug 7., [PMID:23924900]

Abstract [show]
Cystic fibrosis (CF) is a fatal genetic disorder associated with defective hydration of lung airways due to the loss of chloride transport through the CF transmembrane conductance regulator protein (CFTR). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain, and its channel assembly requires multiple interdomain contacts. The most common CF-causing mutation, F508del, occurs in NBD1 and results in misfolding and premature degradation of F508del-CFTR. VX-809 is an investigational CFTR corrector that partially restores CFTR function in people who are homozygous for F508del-CFTR. To identify the folding defect(s) in F508del-CFTR that must be repaired to treat CF, we explored the mechanism of VX-809 action. VX-809 stabilized an N-terminal domain in CFTR that contains only MSD1 and efficaciously restored function to CFTR forms that have missense mutations in MSD1. The action of VX-809 on MSD1 appears to suppress folding defects in F508del-CFTR by enhancing interactions among the NBD1, MSD1, and MSD2 domains. The ability of VX-809 to correct F508del-CFTR is enhanced when combined with mutations that improve F508del-NBD1 interaction with MSD2. These data suggest that the use of VX-809 in combination with an additional CFTR corrector that suppresses folding defects downstream of MSD1 may further enhance CFTR function in people with F508del-CFTR.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Allosteric coupling between the intracellular coup... PLoS One. 2013 Sep 18;8(9):e74347. doi: 10.1371/journal.pone.0074347. eCollection 2013. Dawson JE, Farber PJ, Forman-Kay JD
Allosteric coupling between the intracellular coupling helix 4 and regulatory sites of the first nucleotide-binding domain of CFTR.
PLoS One. 2013 Sep 18;8(9):e74347. doi: 10.1371/journal.pone.0074347. eCollection 2013., [PMID:24058550]

Abstract [show]
Cystic fibrosis is caused by mutations in CFTR (cystic fibrosis transmembrane conductance regulator), leading to folding and processing defects and to chloride channel gating misfunction. CFTR is regulated by ATP binding to its cytoplasmic nucleotide-binding domains, NBD1 and NBD2, and by phosphorylation of the NBD1 regulatory insert (RI) and the regulatory extension (RE)/R region. These regulatory effects are transmitted to the rest of the channel via NBD interactions with intracellular domain coupling helices (CL), particularly CL4. Using a sensitive method for detecting inter-residue correlations between chemical shift changes in NMR spectra, an allosteric network was revealed within NBD1, with a construct lacking RI. The CL4-binding site couples to the RI-deletion site and the C-terminal residues of NBD1 that precede the R region in full-length CFTR. 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. Reciprocally, the C-terminal mutation, Q637R, perturbs dynamics in these three sites. This allosteric network suggests a mechanism synthesizing diverse regulatory NBD1 interactions and provides biophysical evidence for the allosteric coupling required for CFTR function.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] The cystic fibrosis V232D mutation inhibits CFTR m... Biochem Pharmacol. 2014 Mar 1;88(1):46-57. doi: 10.1016/j.bcp.2013.12.027. Epub 2014 Jan 9. Loo TW, Clarke DM
The cystic fibrosis V232D mutation inhibits CFTR maturation by disrupting a hydrophobic pocket rather than formation of aberrant interhelical hydrogen bonds.
Biochem Pharmacol. 2014 Mar 1;88(1):46-57. doi: 10.1016/j.bcp.2013.12.027. Epub 2014 Jan 9., [PMID:24412276]

Abstract [show]
Processing mutations that inhibit folding and trafficking of CFTR are the main cause of cystic fibrosis. Repair of CFTR mutants requires an understanding of the mechanisms of misfolding caused by processing mutations. Previous studies on helix-loop-helix fragments of the V232D processing mutation suggested that its mechanism was to lock transmembrane (TM) segments 3 and 4 together by a non-native hydrogen bond (Asp232(TM4)/Gln207(TM3)). Here, we performed mutational analysis to test for Asp232/Gln207 interactions in full-length CFTR. The rationale was that a V232N mutation should mimic V232D and a V232D/Q207A mutant should mature if the processing defect was caused by hydrogen bonds. We report that only Val232 mutations to charged amino acids severely blocked CFTR maturation. The V232N mutation did not mimic V232D as V232N showed 40% maturation compared to 2% for V232D. Mutation of Val232 to large nonpolar residues (Leu, Phe) had little effect. The Q207L mutation did not rescue V232D because Q207L showed about 50% maturation in the presence of corrector VX-809 while V232D/Q207A could no longer be rescued. These results suggest that V232D inhibits maturation by disrupting a hydrophobic pocket between TM segments rather than forming a non-native hydrogen bond. Disulfide cross-linking analysis of cysteines W356C(TM6) and W1145C(TM12) suggest that the V232D mutation inhibits maturation by trapping CFTR as a partially folded intermediate. Since correctors can efficiently rescue V232D CFTR, the results suggest that hydrophilic processing mutations facing a hydrophobic pocket are good candidates for rescue with pharmacological chaperones.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] The role of the cytosolic HSP70 chaperone system i... Dis Model Mech. 2014 Mar;7(3):319-29. doi: 10.1242/dmm.014001. Young JC
The role of the cytosolic HSP70 chaperone system in diseases caused by misfolding and aberrant trafficking of ion channels.
Dis Model Mech. 2014 Mar;7(3):319-29. doi: 10.1242/dmm.014001., [PMID:24609033]

Abstract [show]
Protein-folding diseases are an ongoing medical challenge. Many diseases within this group are genetically determined, and have no known cure. Among the examples in which the underlying cellular and molecular mechanisms are well understood are diseases driven by misfolding of transmembrane proteins that normally function as cell-surface ion channels. Wild-type forms are synthesized and integrated into the endoplasmic reticulum (ER) membrane system and, upon correct folding, are trafficked by the secretory pathway to the cell surface. Misfolded mutant forms traffic poorly, if at all, and are instead degraded by the ER-associated proteasomal degradation (ERAD) system. Molecular chaperones can assist the folding of the cytosolic domains of these transmembrane proteins; however, these chaperones are also involved in selecting misfolded forms for ERAD. Given this dual role of chaperones, diseases caused by the misfolding and aberrant trafficking of ion channels (referred to here as ion-channel-misfolding diseases) can be regarded as a consequence of insufficiency of the pro-folding chaperone activity and/or overefficiency of the chaperone ERAD role. An attractive idea is that manipulation of the chaperones might allow increased folding and trafficking of the mutant proteins, and thereby partial restoration of function. This Review outlines the roles of the cytosolic HSP70 chaperone system in the best-studied paradigms of ion-channel-misfolding disease--the CFTR chloride channel in cystic fibrosis and the hERG potassium channel in cardiac long QT syndrome type 2. In addition, other ion channels implicated in ion-channel-misfolding diseases are discussed.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Biosynthesis of cystic fibrosis transmembrane cond... Int J Biochem Cell Biol. 2014 Jul;52:26-38. doi: 10.1016/j.biocel.2014.03.020. Epub 2014 Mar 28. Pranke IM, Sermet-Gaudelus I
Biosynthesis of cystic fibrosis transmembrane conductance regulator.
Int J Biochem Cell Biol. 2014 Jul;52:26-38. doi: 10.1016/j.biocel.2014.03.020. Epub 2014 Mar 28., [PMID:24685677]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride (Cl(-)) channel. Mutations of its gene lead to the disease of cystis fibrosis (CF) among which the most common is the deletion of phenylalanine at position 508 (Phe508del). CFTR is a multi-domain glycoprotein whose biosynthesis, maturation and functioning as an anion channel involve multi-level post-translational modifications of CFTR molecules and complex folding processes to reach its native, tertiary conformation. Only 20-40% of the nascent chains achieve folded conformation, while the remaining molecules are targeted for degradation by endoplasmic reticulum, lysosomes, or autophagy. A large number of mutations causing CF impair processing of CFTR. Growing knowledge of CFTR biosynthesis has enabled understanding the cellular basis of CF and has brought to light various potential targets for novel, promising therapies.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Restoration of NBD1 thermal stability is necessary... J Mol Biol. 2015 Jan 16;427(1):106-20. doi: 10.1016/j.jmb.2014.07.026. Epub 2014 Jul 30. He L, Aleksandrov AA, An J, Cui L, Yang Z, Brouillette CG, Riordan JR
Restoration of NBD1 thermal stability is necessary and sufficient to correct F508 CFTR folding and assembly.
J Mol Biol. 2015 Jan 16;427(1):106-20. doi: 10.1016/j.jmb.2014.07.026. Epub 2014 Jul 30., [PMID:25083918]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) (ABCC7), unique among ABC exporters as an ion channel, regulates ion and fluid transport in epithelial tissues. Loss of function due to mutations in the cftr gene causes cystic fibrosis. The most common cystic-fibrosis-causing mutation, the deletion of F508 (DeltaF508) from the first nucleotide binding domain (NBD1) of CFTR, results in misfolding of the protein and clearance by cellular quality control systems. The DeltaF508 mutation has two major impacts on CFTR: reduced thermal stability of NBD1 and disruption of its interface with membrane-spanning domains (MSDs). It is unknown if these two defects are independent and need to be targeted separately. To address this question, we varied the extent of stabilization of NBD1 using different second-site mutations and NBD1 binding small molecules with or without NBD1/MSD interface mutation. Combinations of different NBD1 changes had additive corrective effects on F508 maturation that correlated with their ability to increase NBD1 thermostability. These effects were much larger than those caused by interface modification alone and accounted for most of the correction achieved by modifying both the domain and the interface. Thus, NBD1 stabilization plays a dominant role in overcoming the DeltaF508 defect. Furthermore, the dual target approach resulted in a locked-open ion channel that was constitutively active in the absence of the normally obligatory dependence on phosphorylation by protein kinase A. Thus, simultaneous targeting of both the domain and the interface, as well as being non-essential for correction of biogenesis, may disrupt normal regulation of channel function.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Activation of 3-phosphoinositide-dependent kinase ... J Biol Chem. 2014 Dec 26;289(52):35953-68. doi: 10.1074/jbc.M114.598649. Epub 2014 Nov 10. Caohuy H, Yang Q, Eudy Y, Ha TA, Xu AE, Glover M, Frizzell RA, Jozwik C, Pollard HB
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).
J Biol Chem. 2014 Dec 26;289(52):35953-68. doi: 10.1074/jbc.M114.598649. Epub 2014 Nov 10., [PMID:25384981]

Abstract [show]
Cystic fibrosis (CF) is due to a folding defect in the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, DeltaF508, prevents CFTR from trafficking to the apical plasma membrane. Here we show that activation of the PDK1/SGK1 signaling pathway with C4-ceramide (C4-CER), a non-toxic small molecule, functionally corrects the trafficking defect in both cultured CF cells and primary epithelial cell explants from CF patients. The mechanism of C4-CER action involves a series of mutual autophosphorylation and phosphorylation events between PDK1 and SGK1. Detailed mechanistic studies indicate that C4-CER initially induces autophosphorylation of SGK1 at Ser(422). SGK1[Ser(P)(422)] and C4-CER coincidently bind PDK1 and permit PDK1 to autophosphorylate at Ser(241). Then PDK1[Ser(P)(241)] phosphorylates SGK1[Ser(P)(422)] at Thr(256) to generate fully activated SGK1[Ser(422), Thr(P)(256)]. SGK1[Ser(P)(422),Thr(P)(256)] phosphorylates and inactivates the E3 ubiquitin ligase Nedd4-2. DeltaF508-CFTR is thus free to traffic to the plasma membrane. Importantly, C4-CER-mediated activation of both PDK1 and SGK1 is independent of the PI3K/Akt/mammalian target of rapamycin signaling pathway. Physiologically, C4-CER significantly increases maturation and stability of DeltaF508-CFTR (t(1/2) approximately 10 h), enhances cAMP-activated chloride secretion, and suppresses hypersecretion of interleukin-8 (IL-8). We suggest that candidate drugs for CF directed against the PDK1/SGK1 signaling pathway, such as C4-CER, provide a novel therapeutic strategy for a life-limiting disorder that affects one child, on average, each day.

Comments [show]

None has been submitted yet.

Sentences [show]
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