ABCMdb

ABCC7 p.Asp565Ala

ClinVar:	c.1694A>G , p.Asp565Gly ? , not provided
CF databases:	c.1694A>G , p.Asp565Gly (CFTR1) ? , Additional consequence: splicing mutation (mRNA analysis proves that mutation causes exon 12 skipping)
Predicted by SNAP2:	A: D (71%), C: D (66%), E: D (59%), F: D (80%), G: D (75%), H: D (75%), I: D (80%), K: N (53%), L: D (85%), M: D (80%), N: D (59%), P: D (85%), Q: N (72%), R: D (75%), S: D (63%), T: D (66%), V: D (75%), W: D (85%), Y: D (80%),
Predicted by PROVEAN:	A: D, C: D, E: N, F: D, G: D, H: N, I: D, K: N, L: D, M: D, N: N, P: D, Q: N, R: N, S: D, T: D, V: D, W: D, Y: D,

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Publications
[hide] Correctors promote folding of the CFTR in the endo... Biochem J. 2008 Jul 1;413(1):29-36. Loo TW, Bartlett MC, Clarke DM
Correctors promote folding of the CFTR in the endoplasmic reticulum.
Biochem J. 2008 Jul 1;413(1):29-36., 2008-07-01 [PMID:18361776]

Abstract [show]
Cystic fibrosis (CF) is most commonly caused by deletion of a residue (DeltaF508) in the CFTR (cystic fibrosis transmembrane conductance regulator) protein. The misfolded mutant protein is retained in the ER (endoplasmic reticulum) and is not trafficked to the cell surface (misprocessed mutant). Corrector molecules such as corr-2b or corr-4a are small molecules that increase the amount of functional CFTR at the cell surface. Correctors may function by stabilizing CFTR at the cell surface or by promoting folding in the ER. To test whether correctors promoted folding of CFTR in the ER, we constructed double-cysteine CFTR mutants that would be retained in the ER and only undergo cross-linking when the protein folds into a native structure. The mature form, but not the immature forms, of M348C(TM6)/T1142C(TM12) (where TM is transmembrane segment), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) mutants were efficiently cross-linked. Mutations to the COPII (coatamer protein II) exit motif (Y(563)KDAD(567)) were then made in the cross-linkable cysteine mutants to prevent the mutant proteins from leaving the ER. Membranes were prepared from the mutants expressed in the absence or presence of correctors and subjected to disulfide cross-linking analysis. The presence of correctors promoted folding of the mutants as the efficiency of cross-linking increased from approx. 2-5% to 22-35%. The results suggest that correctors interact with CFTR in the ER to promote folding of the protein into a native structure.

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No. Sentence Comment
49 The COPII exit motif (Y563 KDAD567 ) was disrupted by introducing the Y563N or D565A/D567A changes into wild-type CFTR [21] or into the double-cysteine mutants. Login to comment
63 Cell-surface labelling of CFTR Wild-type CFTR or COPII exit motif mutant Y563N or D565A/ D567A was transiently expressed in HEK-293 cells. Login to comment
145 Tyr563 , Asp565 and Asp567 in the exit motif are evolutionarily Figure 5 Effect of COPII mutations on cross-linking of cysteine mutants (A) Whole-cell SDS extracts of HEK-293 cells expressing wild-type CFTR and wild-type CFTR containing Y563N or D565A/D567A mutation were subjected to immunoblot analysis. Login to comment
146 (B) HEK293 cells expressing wild-type CFTR or wild-type CFTR containing Y563N or D565A/D567A mutant were surface-labelled with sulfo-NHS-SS-biotin. Login to comment
158 Accordingly, we introduced the Y563N or D565A/D567A mutations in the COPII exit motif of wild-type CFTR. Login to comment
161 Cells expressing wild-type CFTR or Y563N or D565A/D567A mutant were treated with sulfo-NHS-SS-biotin, and then lysed with detergent. 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.

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No. Sentence Comment
174 (C) BHK cell lines stably expressing WT-CFTR or bearing the DD/AA mutations (D565A, D567A) alone or in cis with 4RK were cultured at 37 C, incubated at 26 C for 48 hr or for 24 hr with VRT-325, Corr-4a, or VX-809, as indicated. CFTR protein was analyzed by WB. Data are representative of n = 6 experiments. 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