ABCC7 p.Val232Lys
| ClinVar: |
c.695T>A
,
p.Val232Asp
?
, not provided
|
| CF databases: |
c.695T>A
,
p.Val232Asp
(CFTR1)
D
, This mutation was was detected by DGGE and identified by direct sequencing in the CFTR gene. The defect is a T to A change at nucleotide 827 in exon 6a which would lead to a valine-to-aspartic acid replacement in the protein sequence at residue 232. This nucleotide change has been found in an infertile man with CBAVD having neither manifestation of gastrointestinal nor pulmonary disease but with a sweat teat at mmol/
|
| Predicted by SNAP2: | A: N (53%), C: N (61%), D: D (80%), E: D (71%), F: N (61%), G: D (75%), H: D (75%), I: N (97%), K: D (80%), L: N (93%), M: N (82%), N: D (71%), P: D (75%), Q: D (71%), R: D (80%), S: D (66%), T: D (63%), W: D (85%), Y: D (80%), |
| Predicted by PROVEAN: | A: N, C: D, D: D, E: D, F: N, G: D, H: D, I: N, K: D, L: N, M: N, N: D, P: D, Q: D, R: D, S: D, T: N, W: D, Y: N, |
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[hide] Detergent binding explains anomalous SDS-PAGE migr... Proc Natl Acad Sci U S A. 2009 Feb 10;106(6):1760-5. Epub 2009 Jan 30. Rath A, Glibowicka M, Nadeau VG, Chen G, Deber CM
Detergent binding explains anomalous SDS-PAGE migration of membrane proteins.
Proc Natl Acad Sci U S A. 2009 Feb 10;106(6):1760-5. Epub 2009 Jan 30., 2009-02-10 [PMID:19181854]
Abstract [show]
Migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that does not correlate with formula molecular weights, termed "gel shifting," appears to be common for membrane proteins but has yet to be conclusively explained. In the present work, we investigate the anomalous gel mobility of helical membrane proteins using a library of wild-type and mutant helix-loop-helix ("hairpin") sequences derived from transmembrane segments 3 and 4 of the human cystic fibrosis transmembrane conductance regulator (CFTR), including disease-phenotypic residue substitutions. We find that these hairpins migrate at rates of -10% to +30% vs. their actual formula weights on SDS-PAGE and load detergent at ratios ranging from 3.4-10 g SDS/g protein. We additionally demonstrate that mutant gel shifts strongly correlate with changes in hairpin SDS loading capacity (R(2) = 0.8), and with hairpin helicity (R(2) = 0.9), indicating that gel shift behavior originates in altered detergent binding. In some cases, this differential solvation by SDS may result from replacing protein-detergent contacts with protein-protein contacts, implying that detergent binding and folding are intimately linked. The CF-phenotypic V232D mutant included in our library may thus disrupt CFTR function via altered protein-lipid interactions. The observed interdependence between hairpin migration, SDS aggregation number, and conformation additionally suggests that detergent binding may provide a rapid and economical screen for identifying membrane proteins with robust tertiary and/or quaternary structures.
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No. Sentence Comment
42 We noted that certain hairpins ran as more diffuse bands than others (e.g., P205S and Q220W vs. V232K and E217V, see Fig. 1B).
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ABCC7 p.Val232Lys 19181854:42:96
status: NEW62 V232D, V232A, P205S, and Q220W) migrated as WT within statistical significance; 2 were faster (V232D and V232K); and 4 were slower (G228L, E217V, E217F, and E217S/S222E).
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ABCC7 p.Val232Lys 19181854:62:105
status: NEW85 Gel shifts, SDS binding, helicity, and column MW of TM3/4 hairpins Hairpin* Gel shift (dMW, %) Bound SDS (g/g) Helicity (MRE X 103)† Column MW (mut-wt, %)‡ V232Dcf -11 Ϯ 2.6 3.4 Ϯ 0.9 -17 Ϯ 1.2 ϩ19 Ϯ 1.5 V232K -10 Ϯ 3.0 3.8 Ϯ 0.6 -16 Ϯ 1.1 ϩ5.6 Ϯ 1.6 A204L -2.2 Ϯ 2.3 6.0 Ϯ 0.7 -18 Ϯ 1.3 ϩ3.4 Ϯ 1.6 P205A/V232Dcf ϩ0.12 Ϯ 5.2 4.7 Ϯ 0.4 -19 Ϯ 2.6 ϩ21 Ϯ 0.6 WT ϩ0.42 Ϯ 4.5 5.4 Ϯ 1.4 -18 Ϯ 2.2 0.0 Ϯ 0.79 V232A ϩ3.6 Ϯ 3.7 5.2 Ϯ 0.4 -18 Ϯ 0.9 ϩ6.1 Ϯ 1.9 P205Scf ϩ4.7 Ϯ 6.0 4.7 Ϯ 1.0 -18 Ϯ 0.7 ϩ4.4 Ϯ 1.6 Q220W ϩ6.3 Ϯ 2.4 5.0 Ϯ 0.7 -21 Ϯ 2.7 -4.9 Ϯ 1.3 G228L ϩ14 Ϯ 5.1 6.9 Ϯ 1.4 -23 Ϯ 1.7 ϩ14 Ϯ 2.6 E217V ϩ28 Ϯ 1.3 6.7 Ϯ 1.0 -25 Ϯ 1.7 ϩ32 Ϯ 4.0 E217F ϩ29 Ϯ 2.8 9.4 Ϯ 1.9 -28 Ϯ 2.6 ϩ17 Ϯ 1.1 E217S/S222E ϩ29 Ϯ 7.6 10 Ϯ 2.3 -25 Ϯ 2.8 ϩ35 Ϯ 0.9 Glycophorin§ - 3.4 Ϯ 0.6 -9.5 Ϯ 1.2 ϩ83 Ϯ 5.1 *Mutant hairpins are listed in order of increasing gel shift.
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ABCC7 p.Val232Lys 19181854:85:248
status: NEW[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.
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No. Sentence Comment
134 To test if other charged amino acids inhibited maturation, we constructed mutants V232R and V232K.
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ABCC7 p.Val232Lys 24412276:134:92
status: NEW160 We tested if mutations to Val232 that severely inhibited maturation (V232E, V232K, or V232R) could be rescued with other correctors predicted to act as pharmacological chaperones such as 4a [33], VX-809 [19], 2b [35], 5a [12], or VX-325 [36].
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ABCC7 p.Val232Lys 24412276:160:76
status: NEW164 Mutant V232K was rescued with correctors 4a, VX-809 and VX-325 (25-60% mature product) (Fig. 4B and D) while mutant V232R could only be rescued with VX-809 (about 25% mature product) (Fig. 4C and D).
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ABCC7 p.Val232Lys 24412276:164:7
status: NEW171 Accordingly, we introduced the V510D suppressor mutation into mutants V232E, V232K, or V232R.
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ABCC7 p.Val232Lys 24412276:171:77
status: NEW175 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).
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ABCC7 p.Val232Lys 24412276:175:62
status: NEW176 The V510D/V232K mutant however, could still be efficiently rescued with corrector VX-809 to yield mature CFTR as the major product (about 70%).
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ABCC7 p.Val232Lys 24412276:176:10
status: NEW177 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.
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ABCC7 p.Val232Lys 24412276:177:153
status: NEW188 Fig. 4. Rescue of V232E, V232K, and V232R mutants with correctors.
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ABCC7 p.Val232Lys 24412276:188:25
status: NEW189 Whole cell extracts of cells expressing mutants V232E (A), V232K (B), or V232R (C) in the absence (none) or presence of various correctors were subjected to immunoblot analysis.
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ABCC7 p.Val232Lys 24412276:189:59
status: NEW195 The L305R(TM5) P-gp mutant resembles CFTR mutants V232D, V232E, and V232K The characteristics of the V232D/Q207X (Fig. 1) and V232X (Fig. 3) mutants suggest that the mechanism of the V232D mutation involves disruption of a hydrophobic pocket between TM segments.
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ABCC7 p.Val232Lys 24412276:195:68
status: NEW204 It appears that the CFTR V232D, V232E, and V232K processing mutationsresemble P-gp mutantsthatcontain acharged residueata hydrophobic interface between adjacent TM segments because they can be efficiently rescued (with VX-809).
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ABCC7 p.Val232Lys 24412276:204:43
status: NEW222 (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.
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ABCC7 p.Val232Lys 24412276:222:70
status: NEW