ABCC7 p.Tyr517Ala
| CF databases: |
c.1549T>C
,
p.Tyr517His
(CFTR1)
?
, 1540del10 mutation was found in the other allele, which was inherited from her healthy Japanese mother.
c.1550A>G , p.Tyr517Cys (CFTR1) ? , The mutation was detected by SSCP analysis and identified by direct DNA sequencing and confirmed by restriction site generated PCR: a modified primer creates a RsaI site, destroyed by the mutation. |
| Predicted by SNAP2: | A: D (80%), C: D (66%), D: D (91%), E: D (91%), F: D (63%), G: D (85%), H: D (80%), I: D (66%), K: D (91%), L: D (53%), M: D (80%), N: D (91%), P: D (95%), Q: D (85%), R: D (91%), S: D (85%), T: D (85%), V: D (66%), W: D (75%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, |
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[hide] Identification of molecular determinants that modu... Cell Biochem Biophys. 2005;42(1):41-53. Tsigelny I, Hotchko M, Yuan JX, Keller SH
Identification of molecular determinants that modulate trafficking of DeltaF508 CFTR, the mutant ABC transporter associated with cystic fibrosis.
Cell Biochem Biophys. 2005;42(1):41-53., [PMID:15673927]
Abstract [show]
Cystic fibrosis is a life-shortening inherited disorder associated primarily with a three-base in frame deletion that eliminates Phe508 in the ABC transporter, cystic fibrosis transmembrane conductance regulator (CFTR). Mutant CFTR, designated deltaF508 CFTR, is misprocessed and retained intracellularly. It is unclear what causes the trafficking impairment despite extensive investigative effort and the disease's prevalence. We hypothesize that the trafficking impairment is mediated by "receptors" of the cellular trafficking machinery that at three sequential "trafficking checkpoints" govern (1) exit from the endoplasmic reticulum (ER), (2) Golgi to the ER retrieval, and (3) targeting from post-Golgi compartments to lysosomes. We propose that, because of the Phe508 deletion and polypeptide misfolding: (1) a forward-directing signal recognized by the sec24 component of the COPII complex that mediates ER exit is eliminated; (2) a basic amino acid signal recognized by the COPI machinery involved in Golgi to ER retrieval becomes activated; and (3) a tyrosine-based sorting signal that targets to the lysosomes likewise becomes activated. We employed recently reported crystal structures of CFTR nucleotide binding domain 1 and sec24 in computational docking models to identify the most plausible CFTR-sec24 recognition domain. Site-directed mutagenesis and heterologous expression were also used to identify amino acid sequences that operate in Golgi to ER and post-Golgi to lysosome targeting. The importance of considering a multiple checkpoint model for trafficking is that rationale design of pharmaceutical interventions would require abrogation of all major checkpoints to deliver deltaF508 CFTR to the cell surface.
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No. Sentence Comment
172 The putative basic amino acid and tyrosine-based trafficking signals were altered by site-directed mutagenesis to eliminate the canonical trafficking signal sequences at the 516-520 site by changing the Tyr-517 and Arg-518 residues to Ala (this variant designated Y517A:R518A ∆F508 CFTR).
X
ABCC7 p.Tyr517Ala 15673927:172:203
status: NEWX
ABCC7 p.Tyr517Ala 15673927:172:264
status: NEW190 Of the modifications examined, only the double mutation Y517A:R518A in the ∆F508 CFTR template (designated Y517A:R518A ∆F508 CFTR) appeared to enhance the cell surface expression (Fig. 7A).
X
ABCC7 p.Tyr517Ala 15673927:190:56
status: NEWX
ABCC7 p.Tyr517Ala 15673927:190:114
status: NEW191 In cells transfected with Y517A:R518A ∆F508 CFTR, both intracellular and the cell boundary regions revealed immunofluorescent stain (Fig. 7A).
X
ABCC7 p.Tyr517Ala 15673927:191:26
status: NEW192 Fluorescent emission at the cell boundary is consistent with the interpretation that Y517A:R518A ∆F508 CFTR was delivered to the cell surface.
X
ABCC7 p.Tyr517Ala 15673927:192:85
status: NEW197 Because expression of ∆F508 CFTR and single alterations in the ∆F508 CFTR template did not correspond with fluorescent emission at the cell periphery, whereas expression of wild-type CFTR and Y517A:R518A ∆F508 CFTR was reflected in detection of fluorescent emission at the cell boundary, we conclude that elimination of the specific tyrosine-based and basic amino acid signals elevated delivery of ∆F508 CFTR to the plasma membrane.
X
ABCC7 p.Tyr517Ala 15673927:197:206
status: NEW203 Western Blot Analysis We expressed wild-type CFTR, ∆F508 CFTR, and Y517A:R518A ∆F508 CFTR in HEK cells using equivalent amount of plasmids and cells, and processed the cell lysates identically for Western blotting.
X
ABCC7 p.Tyr517Ala 15673927:203:74
status: NEW205 A B-band was associated with ∆F508 CFTR (Fig. 7B, lane 1) and Y517A:R518A ∆F508 CFTR (Fig. 7B, lane 3).
X
ABCC7 p.Tyr517Ala 15673927:205:69
status: NEW207 Although ∆F508 CFTR lacked a C-band (Fig. 7B, lane 1), Y517A:R518A ∆F508 CFTR revealed a C-band (Fig. 7B, lane 3), albeit significantly fainter than the C-band associated with wild-type CFTR (Fig. 7B, lane 2).
X
ABCC7 p.Tyr517Ala 15673927:207:62
status: NEW208 The appearance of the C-band associated with Y517A:R518A ∆F508 CFTR indicated that the sequence alteration elevated the upstream trafficking of ∆F508 CFTR.
X
ABCC7 p.Tyr517Ala 15673927:208:45
status: NEW209 Since the Y517A:R518A alterations did not quantitatively restore trafficking levels comparable to wild-type CFTR (Fig. 7B, compare lanes and 3), addi- 50 Tsigelny et al. Cell Biochemistry and Biophysics Volume 42, 2005 Fig. 6.
X
ABCC7 p.Tyr517Ala 15673927:209:10
status: NEW215 (A) Confocal-immunofluorescent images of ∆F508 CFTR, wild-type, and Y517A:R518A ∆F508 CFTR expressed transiently in human embryonic kidney cells.
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ABCC7 p.Tyr517Ala 15673927:215:75
status: NEW217 Immunofluorescent stain at the cell surface (CS) is apparent in the cells expressing wild-type and Y517A:R518A ∆F508 CFTR.
X
ABCC7 p.Tyr517Ala 15673927:217:99
status: NEW219 (B) Western blot of lysates from cells expressing ∆F508 CFTR, wild-type, and Y517A:R518A ∆F508 CFTR.
X
ABCC7 p.Tyr517Ala 15673927:219:84
status: NEW225 Lane 3, Y517A:R518A ∆F508 CFTR.
X
ABCC7 p.Tyr517Ala 15673927:225:8
status: NEW[hide] The short apical membrane half-life of rescued {De... J Biol Chem. 2005 Nov 4;280(44):36762-72. Epub 2005 Aug 30. Swiatecka-Urban A, Brown A, Moreau-Marquis S, Renuka J, Coutermarsh B, Barnaby R, Karlson KH, Flotte TR, Fukuda M, Langford GM, Stanton BA
The short apical membrane half-life of rescued {Delta}F508-cystic fibrosis transmembrane conductance regulator (CFTR) results from accelerated endocytosis of {Delta}F508-CFTR in polarized human airway epithelial cells.
J Biol Chem. 2005 Nov 4;280(44):36762-72. Epub 2005 Aug 30., 2005-11-04 [PMID:16131493]
Abstract [show]
The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in individuals with cystic fibrosis, DeltaF508, causes retention of DeltaF508-CFTR in the endoplasmic reticulum and leads to the absence of CFTR Cl(-) channels in the apical plasma membrane. Rescue of DeltaF508-CFTR by reduced temperature or chemical means reveals that the DeltaF508 mutation reduces the half-life of DeltaF508-CFTR in the apical plasma membrane. Because DeltaF508-CFTR retains some Cl(-) channel activity, increased expression of DeltaF508-CFTR in the apical membrane could serve as a potential therapeutic approach for cystic fibrosis. However, little is known about the mechanisms responsible for the short apical membrane half-life of DeltaF508-CFTR in polarized human airway epithelial cells. Accordingly, the goal of this study was to determine the cellular defects in the trafficking of rescued DeltaF508-CFTR that lead to the decreased apical membrane half-life of DeltaF508-CFTR in polarized human airway epithelial cells. We report that in polarized human airway epithelial cells (CFBE41o-) the DeltaF508 mutation increased endocytosis of CFTR from the apical membrane without causing a global endocytic defect or affecting the endocytic recycling of CFTR in the Rab11a-specific apical recycling compartment.
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No. Sentence Comment
52 To construct the GFP-⌬F508-CFTR Y517A mutant, the GFP-⌬F508-CFTR cDNA sequence in pcDNA3.1 was mutated using the QuikChangeTM XL site-directed mutagenesis kit (Stratagene; La Jolla, CA).
X
ABCC7 p.Tyr517Ala 16131493:52:39
status: NEW54 Transient transfection of the GFP-tagged CFTR cDNAs into parental CFBE41o-cells was performed using LipofectamineTM 2000 according to the manufacturer`s instructions. CFBE41o-cells grown on 40-mm tissue culture plates were incubated for 24 h with the transfection mixture (2 g of cDNA and 4 l of LipofectamineTM 2000 per plate) at 37 °C. Subsequently, cells were cultured in fresh medium at 27 °C for 36 h to increase the expression of GFP-⌬F508-CFTR and GFP-⌬F508-CFTR Y517A in the plasma membrane.
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ABCC7 p.Tyr517Ala 16131493:54:510
status: NEW64 Parental CFBE41o-cells transiently transfected with either GFP- ⌬F508-CFTR or GFP-⌬F508-CFTR Y517A were grown on plastic tissue culture plates.
X
ABCC7 p.Tyr517Ala 16131493:64:107
status: NEW161 To test this hypothesis, we transiently expressed the GFP-tagged ⌬F508-CFTR or the GFP-⌬F508-CFTR Y517A mutant in parental CFBE41o-cells.
X
ABCC7 p.Tyr517Ala 16131493:161:112
status: NEW164 The apical membrane expression of GFP-⌬F508-CFTR and GFP-⌬F508-CFTR Y517A was similar at steady state (Fig. 7A).
X
ABCC7 p.Tyr517Ala 16131493:164:82
status: NEW165 Furthermore, as illustrated in Figs. 7, B and C, the apical membrane half-life of GFP-⌬F508-CFTR Y517A (1.0 h) did not differ from that of GFP-⌬F508-CFTR (1.1 h).
X
ABCC7 p.Tyr517Ala 16131493:165:104
status: NEW194 FIGURE 7. Summary of experiments performed to determine the effects of the Y517A mutation in ⌬F508-CFTR on the apical membrane expression at steady state and the apical membrane half-life of rescued ⌬F508-CFTR.
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ABCC7 p.Tyr517Ala 16131493:194:75
status: NEW195 Parental CFBE41o-cells grown on plastic tissue culture plates were transiently transfected with either GFP-⌬F508-CFTR or the GFP-⌬F508-CFTR Y517A mutant.
X
ABCC7 p.Tyr517Ala 16131493:195:154
status: NEW197 A, summary of biotinylation experiments demonstrating that the apical membrane expression of GFP-⌬F508-CFTR and GFP-⌬F508-CFTR Y517A at steady state did not differ.
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ABCC7 p.Tyr517Ala 16131493:197:141
status: NEW198 B, summary of biotinylation experiments demonstrating that the apical membrane half-life of GFP-⌬F508-CFTR (1.0 h) and GFP-⌬F508-CFTR Y517A (1.1 h) did not differ in CFBE41o-cells.
X
ABCC7 p.Tyr517Ala 16131493:198:148
status: NEW199 Disappearance of GFP-⌬F508-CFTR and GFP-⌬F508-CFTR Y517A from the apical membrane was monitored over time in the presence of 20 g/ml cyclohexamide (CHX) at 37 °C.
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ABCC7 p.Tyr517Ala 16131493:199:65
status: NEW201 "C,representativeWesternblotsdemonstratingthedisappearance of GFP-⌬F508-CFTR and GFP-⌬F508-CFTR Y517A from the apical membrane over time in CFBE41o-cells.
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ABCC7 p.Tyr517Ala 16131493:201:110
status: NEW