ABCC7 p.Thr1471Asp
| Predicted by SNAP2: | A: N (87%), C: N (66%), D: N (53%), E: N (61%), F: N (53%), G: N (82%), H: N (61%), I: N (78%), K: N (72%), L: N (66%), M: N (78%), N: N (72%), P: N (72%), Q: N (72%), R: N (57%), S: N (87%), V: N (78%), W: D (53%), Y: N (53%), |
| 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, V: N, W: N, Y: N, |
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[hide] CFTR mutations altering CFTR fragmentation. Biochem J. 2013 Jan 1;449(1):295-305. doi: 10.1042/BJ20121240. Tosoni K, Stobbart M, Cassidy DM, Venerando A, Pagano MA, Luz S, Amaral MD, Kunzelmann K, Pinna LA, Farinha CM, Mehta A
CFTR mutations altering CFTR fragmentation.
Biochem J. 2013 Jan 1;449(1):295-305. doi: 10.1042/BJ20121240., [PMID:23067305]
Abstract [show]
Most CF (cystic fibrosis) results from deletion of a phenylalanine (F508) in the CFTR {CF transmembrane-conductance regulator; ABCC7 [ABC (ATP-binding cassette) sub-family C member 7]} which causes ER (endoplasmic reticulum) degradation of the mutant. Using stably CFTR-expressing BHK (baby-hamster kidney) cell lines we demonstrated that wild-type CTFR and the F508delCFTR mutant are cleaved into differently sized N- and C-terminal-bearing fragments, with each hemi-CFTR carrying its nearest NBD (nucleotide-binding domain), reflecting differential cleavage through the central CFTR R-domain. Similar NBD1-bearing fragments are present in the natively expressing HBE (human bronchial epithelial) cell line. We also observe multiple smaller fragments of different sizes in BHK cells, particularly after F508del mutation (ladder pattern). Trapping wild-type CFTR in the ER did not generate a F508del fragmentation fingerprint. Fragments change their size/pattern again post-mutation at sites involved in CFTR's in vitro interaction with the pleiotropic protein kinase CK2 (S511A in NBD1). The F508del and S511A mutations generate different fragmentation fingerprints that are each unlike the wild-type; yet, both mutants generate new N-terminal-bearing CFTR fragments that are not observed with other CK2-related mutations (S511D, S422A/D and T1471A/D). We conclude that the F508delCFTR mutant is not degraded completely and there exists a relationship between CFTR's fragmentation fingerprint and the CFTR sequence through putative CK2-interactive sites that lie near F508.
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No. Sentence Comment
44 Cell culture, lysis, protein solubilization and Western blotting The cell culture methods to create the stable CFTR-expressing cell lines (WT, F, WT S422A, WT S422D, WT S511A, WT S511D, WT T1471A and WT T1471D) and their culture protocols have been described recently [25].
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ABCC7 p.Thr1471Asp 23067305:44:204
status: NEW187 The most dramatic effect on synthesis was seen after the T1471D mutation (augmenting the negative charge at the regulatory acid stretch, nine amino acids from the C-terminus of wt-CFTR), which abolished the mature 'band C` form of CFTR reminiscent of the findings when F508delCFTR fails to mature in the Golgi from band B (in the ER) to band C (in the plasma membrane).
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ABCC7 p.Thr1471Asp 23067305:187:57
status: NEW205 Compare similarities and differences in fragmentation between lanes 8 and 9 which both reduce the amount of full-length CFTR (T1471D or F508del).
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ABCC7 p.Thr1471Asp 23067305:205:126
status: NEW216 In contrast T1471D displays gross attenuation of all fragments (only Q4 remains visible with the NBD1 and N-terminal antibodies).
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ABCC7 p.Thr1471Asp 23067305:216:12
status: NEW217 The absence of other bands may be a false-negative artefact of low fragment abundance because we find that the full-length wt-CFTR with the T1471D mutation is also attenuated and thus (from an abundance perspective) is indistinguishable from the pattern seen with F508delCFTR, despite the wt background.
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ABCC7 p.Thr1471Asp 23067305:217:140
status: NEW218 This confirms recent data [25] showing that T1471D abolished the formation of any fully glycosylated band C CFTR in the same cell line and is consistent with the importance of the acid cluster surrounding T1471 towards the relative rates of opening and closing of CFTR [26].
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ABCC7 p.Thr1471Asp 23067305:218:44
status: NEW220 In summary, the two S511A/S511D and T1471A/T1471D pairs manifested very different CFTR cleavage patterns and abundance of full-length CFTR when present on a wt background.
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ABCC7 p.Thr1471Asp 23067305:220:43
status: NEW