ABCC7 p.Val510Cys
| 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, |
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[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.
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No. Sentence Comment
3 Although replacement of the 18 endogenous cysteines of CFTR with Ser or Ala yields a Cys-less mutant that does not mature at 37 °C, we found that maturation could be restored if Val510 was changed to Ala, Cys, Ser, Thr, Gly, Ala, or Asp.
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ABCC7 p.Val510Cys 17911111:3:183
status: NEW65 Fig. 2A shows that only the V510C mutation promoted maturation of Cys-less CFTR at 37 °C.
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ABCC7 p.Val510Cys 17911111:65:28
status: NEW70 Stable BHK cell lines expressing mutants V510A, V510C, V510S, V510G, or V510D were generated for use in iodide efflux assays.
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ABCC7 p.Val510Cys 17911111:70:48
status: NEW73 The results of mutants V510C, V510S, or V510G are not shown for clarity.
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ABCC7 p.Val510Cys 17911111:73:23
status: NEW82 ACCELERATED PUBLICATION: CFTR TM Domain Cross-linking 33248 To test whether the Val510 changes could also promote maturation of ⌬F508 CFTR (in a wild-type background), we introduced the Val510 mutations that promoted maturation of Cys-less CFTR (Val510 to Cys, Gly, Ala, Ser, Asp, or Thr) into ⌬F508 CFTR.
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ABCC7 p.Val510Cys 17911111:82:254
status: NEW[hide] Phenylalanine-508 mediates a cytoplasmic-membrane ... Proc Natl Acad Sci U S A. 2008 Mar 4;105(9):3256-61. Epub 2008 Feb 27. Serohijos AW, Hegedus T, Aleksandrov AA, He L, Cui L, Dokholyan NV, Riordan JR
Phenylalanine-508 mediates a cytoplasmic-membrane domain contact in the CFTR 3D structure crucial to assembly and channel function.
Proc Natl Acad Sci U S A. 2008 Mar 4;105(9):3256-61. Epub 2008 Feb 27., 2008-03-04 [PMID:18305154]
Abstract [show]
Deletion of phenylalanine-508 (Phe-508) from the N-terminal nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ATP-binding cassette (ABC) transporter family, disrupts both its folding and function and causes most cystic fibrosis. Most mutant nascent chains do not pass quality control in the ER, and those that do remain thermally unstable, only partially functional, and are rapidly endocytosed and degraded. Although the lack of the Phe-508 peptide backbone diminishes the NBD1 folding yield, the absence of the aromatic side chain is primarily responsible for defective CFTR assembly and channel gating. However, the site of interdomain contact by the side chain is unknown as is the high-resolution 3D structure of the complete protein. Here we present a 3D structure of CFTR, constructed by molecular modeling and supported biochemically, in which Phe-508 mediates a tertiary interaction between the surface of NBD1 and a cytoplasmic loop (CL4) in the C-terminal membrane-spanning domain (MSD2). This crucial cytoplasmic membrane interface, which is dynamically involved in regulation of channel gating, explains the known sensitivity of CFTR assembly to many disease-associated mutations in CL4 as well as NBD1 and provides a sharply focused target for small molecules to treat CF. In addition to identifying a key intramolecular site to be repaired therapeutically, our findings advance understanding of CFTR structure and function and provide a platform for focused biochemical studies of other features of this unique ABC ion channel.
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No. Sentence Comment
84 Nevertheless, the proximity or relative orientation of the F508C/F1068C, F508C/G1069C, and V510C/G1069C pairs permitted very little disulfide bond formation on oxidation catalyzed by copper phenanthroline, i.e., only a very small proportion of mature band was converted to cross-linked band Fig. 2.
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ABCC7 p.Val510Cys 18305154:84:91
status: NEW[hide] Processing mutations disrupt interactions between ... J Biol Chem. 2008 Oct 17;283(42):28190-7. Epub 2008 Aug 16. Loo TW, Bartlett MC, Clarke DM
Processing mutations disrupt interactions between the nucleotide binding and transmembrane domains of P-glycoprotein and the cystic fibrosis transmembrane conductance regulator (CFTR).
J Biol Chem. 2008 Oct 17;283(42):28190-7. Epub 2008 Aug 16., 2008-10-17 [PMID:18708637]
Abstract [show]
P-glycoprotein (P-gp, ABCB1) is an ATP-dependent drug pump. Each of its two homologous halves contains a transmembrane domain (TMD) that has six transmembrane (TM) segments and a nucleotide-binding domain (NBD). Determining how the two halves interact may provide insight into the folding of P-gp as the drug-binding pocket and nucleotide-binding sites are predicted to be at the interface between the two halves. Here, we present evidence for NBD1-TMD2 and NBD2-TMD1 interactions. We also show that TMD-NBD interactions in immature and mature P-gp can be affected by the presence of a processing mutation. We found that the NBD-TMD mutants L443C(NBD1)/S909C(TMD2) and A266C(TMD1)/F1086C(NBD2) could be cross-linked at 0 degrees C with oxidant (copper phenanthroline). Cross-linking was inhibited by vanadate-trapping of nucleotide. The presence of a processing mutation (G268V/L443C(NBD1)/S909C(TMD2); L1260A/A266C(TMD1)/F1086C(NBD2)) resulted in the synthesis of the immature (150 kDa) protein as the major product and the mutants could not be cross-linked with copper phenanthroline. Expression of the processing mutants in the presence of a pharmacological chaperone (cyclosporin A), however, resulted in the expression of mature (170 kDa) protein at the cell surface that could be cross-linked. Similarly, CFTR mutants A274C(TMD1)/L1260C(NBD2) and V510C(NBD1)/A1067C(TMD2) could be cross-linked at 0 degrees C with copper phenanthroline. Introduction of DeltaF508 mutation in these mutants, however, resulted in the synthesis of immature CFTR that could not be cross-linked. These results suggest that establishment of NBD interactions with the opposite TMD is a key step in folding of ABC transporters.
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No. Sentence Comment
51 We also constructed the V510C(NBD1)/A1067C(TMD2) CFTR mutant because position V510C or V510A promoted maturation of Cys-less CFTR (22).
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ABCC7 p.Val510Cys 18708637:51:78
status: NEW179 It was previously shown that mutations V510C or V510A promoted maturation of Cys-less CFTR (22).
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ABCC7 p.Val510Cys 18708637:179:39
status: NEW180 It was possible that V510C could interact with TMD2.
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ABCC7 p.Val510Cys 18708637:180:21
status: NEW[hide] A small-molecule modulator interacts directly with... Mol Pharmacol. 2009 Jun;75(6):1430-8. Epub 2009 Apr 1. Wellhauser L, Kim Chiaw P, Pasyk S, Li C, Ramjeesingh M, Bear CE
A small-molecule modulator interacts directly with deltaPhe508-CFTR to modify its ATPase activity and conformational stability.
Mol Pharmacol. 2009 Jun;75(6):1430-8. Epub 2009 Apr 1., [PMID:19339490]
Abstract [show]
The deletion of Phe-508 (DeltaPhe508) constitutes the most prevalent of a number of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) that cause cystic fibrosis (CF). This mutation leads to CFTR misfolding and retention in the endoplasmic reticulum, as well as impaired channel activity. The biosynthetic defect can be partially overcome by small-molecule "correctors"; once at the cell surface, small-molecule "potentiators" enhance the channel activity of DeltaPhe508-CFTR. Certain compounds, such as VRT-532, exhibit both corrector and potentiator functions. In the current studies, we confirmed that the inherent chloride channel activity of DeltaPhe508-CFTR (after biosynthetic rescue) is potentiated in studies of intact cells and membrane vesicles. It is noteworthy that we showed that the ATPase activity of the purified and reconstituted mutant protein is directly modulated by binding of VRT-532 [4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)-phenol] ATP turnover by reconstituted DeltaPhe508-CFTR is decreased by VRT-532 treatment, an effect that may account for the increase in channel open time induced by this compound. To determine whether the modification of DeltaPhe508-CFTR function caused by direct VRT-532 binding is associated with structural changes, we evaluated the effect of VRT-532 binding on the protease susceptibility of the major mutant. We found that binding of VRT-532 to DeltaPhe508-CFTR led to a minor but significant decrease in the trypsin susceptibility of the full-length mutant protein and a fragment encompassing the second half of the protein. These findings suggest that direct binding of this small molecule induces and/or stabilizes a structure that promotes the channel open state and may underlie its efficacy as a corrector of DeltaPhe508-CFTR.
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No. Sentence Comment
208 It is noteworthy that Loo et al. (2008) and Serohijos et al. (2008) showed that deletion of Phe508 impairs the chemical cross-linking that normally occurs between a non-native cysteine residue incorporated into NBD1, in the proximity of Phe508 (V510C), and a non-native cysteine introduced into the fourth intracellular loop (A1067C) Fig. 5.
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ABCC7 p.Val510Cys 19339490:208:245
status: NEW[hide] Correctors enhance maturation of DeltaF508 CFTR by... Biochemistry. 2009 Oct 20;48(41):9882-90. Loo TW, Bartlett MC, Clarke DM
Correctors enhance maturation of DeltaF508 CFTR by promoting interactions between the two halves of the molecule.
Biochemistry. 2009 Oct 20;48(41):9882-90., 2009-10-20 [PMID:19761259]
Abstract [show]
Deletion of Phe508 in cystic fibrosis transmembrane conductance regulator (DeltaF508 CFTR) causes cystic fibrosis. CFTR consists of two homologous halves with each containing a nucleotide-binding domain (NBD) and a transmembrane domain (TMD). DeltaF508 CFTR appears to be trapped in an incompletely folded state. Small molecules (correctors) promote folding of DeltaF508 CFTR with relatively low efficiency. Understanding the mechanism of repair may lead to the development of more effective correctors. Here we tested the effect of correctors and the DeltaF508 mutation on interactions between the halves of CFTR when expressed as separate polypeptides. Glycosylation of C-half CFTR was defective when expressed alone as a mixture of core and unglycosylated proteins was detected. Coexpression of C-half CFTR with either wild-type N-half or DeltaF508/N-half CFTR, however, increased the amount of core-glycosylated protein, but only coexpression with wild-type N-half promoted maturation of C-half CFTR (Endo H resistant). This suggested that the DeltaF508 mutation inhibited some interactions between N-half and C-half CFTRs. Interaction of A52-tagged wild-type N-half or DeltaF508/N-half CFTR with histidine-tagged C-half CFTR was then followed by nickel-chelate chromatography. Coexpression of A52-tagged wild-type N-half or DeltaF508/N-half CFTR with histidine-tagged C-half CFTR resulted in the wild-type N-half CFTR but not DeltaF508/N-half CFTR protein being retained on the column. Coexpression of DeltaF508/N-half and C-half CFTR in the presence correctors VX-325 and corr-4a, however, restored interactions between the two halves. An interaction that was restored was that between NBD1 and TMD2 as the correctors restored cross-linking of mutant DeltaF508/NBD1(V510C)/TMD2(A1067C). Therefore, correctors promote proper interactions between the two halves of CFTR.
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No. Sentence Comment
11 Coexpression of ΔF508/N-half and C-half CFTR in the presence correctors VX-325 and corr-4a, however, restored interactions between the two halves. An interaction that was restored was that between NBD1 and TMD2 as the correctors restored cross-linking of mutant ΔF508/NBD1- (V510C)/TMD2(A1067C).
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ABCC7 p.Val510Cys 19761259:11:287
status: NEW48 The construction of double-cysteine CFTR mutant ΔF508/V510C- (NBD1)/A1067C(TMD2) in a Cys-less background was described previously (17).
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ABCC7 p.Val510Cys 19761259:48:60
status: NEW78 For example, mature CFTR shows cross-linking between cysteines in TM segment 6 (N-half) and TM segment 12 (C-half) or between cysteines in NBD1 [N-half (V510C)] and ICL4 [C-half (A1067C)], but the immature form of ΔF508 does not (17, 25).
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ABCC7 p.Val510Cys 19761259:78:153
status: NEW101 The black dot indicates the predicted location of an introduced cysteine (A1067C) in TMD2 that will directly cross-link to a cysteine introduced into NBD1 (V510C).
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ABCC7 p.Val510Cys 19761259:101:156
status: NEW[hide] Restoration of domain folding and interdomain asse... FASEB J. 2010 Aug;24(8):3103-12. Epub 2010 Mar 16. He L, Aleksandrov LA, Cui L, Jensen TJ, Nesbitt KL, Riordan JR
Restoration of domain folding and interdomain assembly by second-site suppressors of the DeltaF508 mutation in CFTR.
FASEB J. 2010 Aug;24(8):3103-12. Epub 2010 Mar 16., [PMID:20233947]
Abstract [show]
Deletion of PHE508 (DeltaF508) from the first nucleotide-binding domain (NBD1) of CFTR, which causes most cystic fibrosis, disrupts the folding and assembly of the protein. Although the folding pathways and yield of isolated NBD1 are altered, its global structure is not, and details of the changes in the rest of the protein remain unclear. To gain further insight into how the whole mutant protein is altered, we have determined the influence of known second-site suppressor mutations in NBD1 on the conformation of this domain and key interfaces between domains. We found that the suppressors restored maturation of only those processing mutations located in NBD1, but not in other domains, including those in the C-terminal cytoplasmic loop of the second membrane-spanning domain, which forms an interface with the NBD1 surface. Nevertheless, the suppressors promoted the formation of this interface and others in the absence of F508. The suppressors restored maturation in a DeltaF508 construct from which NBD2 was absent but to a lesser extent than in the full-length, indicating that DeltaF508 disrupts interactions involving NBD2, as well as other domains. Rescue of DeltaF508-CFTR by suppressors required the biosynthesis of the entire full-length protein in continuity, as it did not occur when N- and C-terminal "halves" were coexpressed. Simultaneous with these interdomain perturbations, DeltaF508 resulted in suppressor reversed alterations in accessibility of residues both in the F508-containing NBD1 surface loop and in the Q loop within the domain core. Thus, in the context of the full-length protein, DeltaF508 mutation causes detectable changes in NBD1 conformation, as well as interdomain interactions.
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No. Sentence Comment
123 Cys- pair cross-linking also was not observed with Cys pairs at V510C/G1069C and K564C/G1069C in the NBD1/ CL4 interface of ⌬F508-CFTR (Fig. 3B).
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ABCC7 p.Val510Cys 20233947:123:64
status: NEW126 In addition, Cys pairs V510C/G1069C and K564C/G1069C at the NBD1/CL4 interface also were able to be cross-linked by M3M and M8M (Fig. 3B).
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ABCC7 p.Val510Cys 20233947:126:23
status: NEW181 Using this assay, we compared the accessibility of 491C and V510C in NBD1 of full-length CFTR in the absence or presence of ⌬F508.
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ABCC7 p.Val510Cys 20233947:181:60
status: NEW202 In contrast to the buried 491C, V510C was clearly labeled in both immature and mature forms of WT-CFTR (lane 5).
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ABCC7 p.Val510Cys 20233947:202:32
status: NEW205 This may reflect the involvement of this loop in the NBD1/CL4 interface, as shown with the cross-linking of V510C/ G1069C in the mature but not in the immature form (18).
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ABCC7 p.Val510Cys 20233947:205:108
status: NEW206 In contrast to the more buried 491C, introduction of ⌬F508 did not make V510C more accessible to labeling compared to the immature WT-CFTR (lane 6).
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ABCC7 p.Val510Cys 20233947:206:79
status: NEW207 Introduction of the suppressor mutations did not appear to reduce exposure of V510C in the immature form, as it had the more buried 491C.
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ABCC7 p.Val510Cys 20233947:207:78
status: NEW208 However, the accessibility of V510C in the mature form, which was restored by the suppressors, was similar to that in the WT-CFTR (lane 7).
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ABCC7 p.Val510Cys 20233947:208:30
status: NEW[hide] Regulatory insertion removal restores maturation, ... J Mol Biol. 2010 Aug 13;401(2):194-210. Epub 2010 Jun 16. Aleksandrov AA, Kota P, Aleksandrov LA, He L, Jensen T, Cui L, Gentzsch M, Dokholyan NV, Riordan JR
Regulatory insertion removal restores maturation, stability and function of DeltaF508 CFTR.
J Mol Biol. 2010 Aug 13;401(2):194-210. Epub 2010 Jun 16., 2010-08-13 [PMID:20561529]
Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) epithelial anion channel is a large multidomain membrane protein that matures inefficiently during biosynthesis. Its assembly is further perturbed by the deletion of F508 from the first nucleotide-binding domain (NBD1) responsible for most cystic fibrosis. The mutant polypeptide is recognized by cellular quality control systems and is proteolyzed. CFTR NBD1 contains a 32-residue segment termed the regulatory insertion (RI) not present in other ATP-binding cassette transporters. We report here that RI deletion enabled F508 CFTR to mature and traffic to the cell surface where it mediated regulated anion efflux and exhibited robust single chloride channel activity. Long-term pulse-chase experiments showed that the mature DeltaRI/DeltaF508 had a T(1/2) of approximately 14 h in cells, similar to the wild type. RI deletion restored ATP occlusion by NBD1 of DeltaF508 CFTR and had a strong thermostabilizing influence on the channel with gating up to at least 40 degrees C. None of these effects of RI removal were achieved by deletion of only portions of RI. Discrete molecular dynamics simulations of NBD1 indicated that RI might indirectly influence the interaction of NBD1 with the rest of the protein by attenuating the coupling of the F508-containing loop with the F1-like ATP-binding core subdomain so that RI removal overcame the perturbations caused by F508 deletion. Restriction of RI to a particular conformational state may ameliorate the impact of the disease-causing mutation.
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No. Sentence Comment
154 HEK 293 cells were transiently transfected with Cys-less ΔF508 CFTR with Cys pairs introduced at the CL2-NBD2 (C276-Q1280C) or CL4-NBD1 (V510C-G1069C) interfaces in the absence or presence of ΔRI.
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ABCC7 p.Val510Cys 20561529:154:143
status: NEW[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.
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No. Sentence 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.
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ABCC7 p.Val510Cys 20590134:178:176
status: NEW[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.
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No. Sentence Comment
30 Figure 1C shows that 50 μM benzbromarone inhibited cross-linking between TMD1 and TMD2 [M348C(TM6)/ T1142C(TM12)] but not between NBD1 and TMD2 [V510C- (NBD1)/A1067C(ICL4)].
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ABCC7 p.Val510Cys 21520952:30:151
status: NEW50 (C) Effect of benzbromarone on cross-linking (X-link) between cysteines in TMD1 and TMD2 (M348C/T1142C) or NBD1 and TMD2 (V510C/A1067C).7 (D) Immunoblot of cells expressing CFTR TMD1þ2 in the absence (À) or presence (þ) of 0.05 mM benzbromarone.
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ABCC7 p.Val510Cys 21520952:50:122
status: NEW[hide] Repair of CFTR folding defects with correctors tha... Methods Mol Biol. 2011;741:23-37. Loo TW, Clarke DM
Repair of CFTR folding defects with correctors that function as pharmacological chaperones.
Methods Mol Biol. 2011;741:23-37., [PMID:21594776]
Abstract [show]
The major cause of cystic fibrosis is the presence of processing mutations in CFTR (such as deletion of Phe-508 (F508del-CFTR)) that disrupt folding of the protein and trafficking to the cell surface. Processing mutations appear to inhibit folding of CFTR so that it accumulates in the endoplasmic reticulum as a partially folded protein. Expressing the proteins in the presence of small molecules called correctors can repair CFTR folding defects. Some correctors appear to function as pharmacological chaperones that specifically bind to the CFTR processing mutants and induce them to complete the folding process. In this chapter, we describe techniques to examine the effects of correctors on folding of CFTR processing mutants.
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No. Sentence Comment
252 The following protocols describe disulfide cross-linking between cysteines introduced into NBD1 (V510C) and the fourth intracellular loop in TMD2 (A1067C) of a F508del-CFTR processing mutant expressed in the presence or the absence of correctors.
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ABCC7 p.Val510Cys 21594776:252:97
status: NEW263 The next day, add 0.17 mg of (V510C)/(A1067C)/ F508del-CFTR cDNA to sterile water to give a total volume of 7.65 ml.
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ABCC7 p.Val510Cys 21594776:263:30
status: NEW282 Effect of correctors on cross-linking of CFTR mutant F508del/V510C (NBD1)/A1067C(TMD2).
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ABCC7 p.Val510Cys 21594776:282:61
status: NEW[hide] Mistargeted MRPdeltaF728 mutant is rescued by intr... FEBS Lett. 2004 Dec 3;578(1-2):145-51. Buyse F, Vandenbranden M, Ruysschaert JM
Mistargeted MRPdeltaF728 mutant is rescued by intracellular GSH.
FEBS Lett. 2004 Dec 3;578(1-2):145-51., [PMID:15581632]
Abstract [show]
The most common cystic fibrosis-causing mutation is the deletion of the widely conserved phenylalanine 508 (DeltaF508) of CFTR. The mutant is unable to fold correctly and to transit to the plasma membrane. MRP1 belongs to the same subfamily of ABC proteins as CFTR and confers resistance to a wide range of chemotherapeutic drugs. By analogy, phenylalanine 728 was deleted in MRP1. Our results shown that MRPDeltaF728 is correctly targeted to the plasma membrane, actively transports doxorubicin (DOX) and vincristine (VCR) and shares a structure identical to MRP1. Intracellular GSH depletion however results in a mistargeted mutant that is retained into the cytoplasm, while in the same conditions wild-type MRP1 is correctly routed to the plasma membrane. The GSH-protein complex could adopt a stable conformation protected against proteolytic degradation and correctly targeted to the plasma membrane.
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No. Sentence Comment
154 HEK 293 cells were transiently transfected with Cys-less ΔF508 CFTR with Cys pairs introduced at the CL2-NBD2 (C276-Q1280C) or CL4- NBD1 (V510C-G1069C) interfaces in the absence or presence of ΔRI.
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ABCC7 p.Val510Cys 15581632:154:144
status: NEW[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.
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No. Sentence Comment
155 Using an otherwise Cys-less construct, we had previously observed methanethiosulfonate-mediated cross-linking between V510C and G1069C in WT CFTR.29,33 Figure 7 shows similar cross-linking of this pair of residues in ΔF/4PT CFTR, providing evidence that the stabilization of ΔF508 NBD1 can Fig. 6.
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ABCC7 p.Val510Cys 22406676:155:118
status: NEW178 HEK293 cells were transiently transfected with Cys-less CFTR or Cys-less ΔF508-CFTR in the presence or absence of the 4PT mutations (S422P/S434P/S492P/A534P/I539T), with the Cys pair V510C/G1069C introduced at the CL4/NBD1 interface.
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ABCC7 p.Val510Cys 22406676:178:188
status: NEW279 Briefly, HEK cells transiently expressing Cys-less CFTR constructs with V510C and G1069C substitutions on the WT or ΔF/4PT CFTR basis grown on 35-mm tissue culture dishes were harvested, washed twice in PBS, and resuspended in 60 μl of PBS.
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ABCC7 p.Val510Cys 22406676:279:72
status: NEW[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.
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No. Sentence Comment
126 HEK293 cells were transiently transfected with Cys-less CFTR or Cys-less èc;F508 CFTR with Cys pairs 276C/Q1280C or V510C/G1069C, introduced at NBD2/CL2 and NBD1/CL4 interfaces, respectively (13).
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ABCC7 p.Val510Cys 23104983:126:120
status: NEW