ABCMdb

ABCC7 p.Asp567Ala

CF databases:	c.1700A>G , p.Asp567Gly (CFTR1) ? ,
Predicted by SNAP2:	A: N (53%), C: D (66%), E: D (63%), F: D (85%), G: D (75%), H: D (80%), I: D (85%), K: D (80%), L: D (85%), M: D (80%), N: D (71%), P: D (85%), Q: D (75%), R: D (63%), S: D (66%), T: D (71%), V: D (80%), W: D (85%), Y: D (85%),
Predicted by PROVEAN:	A: D, C: 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, 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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Sentences [show]
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] COPII machinery cooperates with ER-localized Hsp40... Mol Biol Cell. 2013 Mar;24(5):633-42. doi: 10.1091/mbc.E12-08-0639. Epub 2013 Jan 9. Kakoi S, Yorimitsu T, Sato K
COPII machinery cooperates with ER-localized Hsp40 to sequester misfolded membrane proteins into ER-associated compartments.
Mol Biol Cell. 2013 Mar;24(5):633-42. doi: 10.1091/mbc.E12-08-0639. Epub 2013 Jan 9., [PMID:23303252]

Abstract [show]
Proteins that fail to fold in the endoplasmic reticulum (ER) are subjected to ER-associated degradation (ERAD). Certain transmembrane ERAD substrates are segregated into specialized ER subdomains, termed ER-associated compartments (ERACs), before targeting to ubiquitin-proteasome degradation. The traffic-independent function of several proteins involved in COPII-mediated ER-to-Golgi transport have been implicated in the segregation of exogenously expressed human cystic fibrosis transmembrane conductance regulator (CFTR) into ERACs in Saccharomyces cerevisiae. Here we focus on the properties of COPII components in the sequestration of enhanced green fluorescent protein (EGFP)-CFTR into ERACs. It has been demonstrated that the temperature-sensitive growth defects in many COPII mutants can be suppressed by overexpressing other genes involved in COPII vesicle formation. However, we show that these suppression abilities are not always correlated with the ability to rescue the ERAC formation defect, suggesting that COPII-mediated EGFP-CFTR entry into ERACs is independent of its ER-to-Golgi trafficking function. In addition to COPII machinery, we find that ER-associated Hsp40s are also involved in the sequestration process by directly interacting with EGFP-CFTR. COPII components and ER-associated Hsp40, Hlj1p, act in the same pathway to sequester EGFP-CFTR into ERACs. Our findings point to an as-yet-undefined role of COPII proteins in the formation of ERACs.

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No. Sentence Comment
108 Therefore we examined the ability of the diacidic EGFP-CFTR-D567A mutant to be sequestered into ERACs, but this did not significantly affect its entry into the ERAC (Figure 5). Login to comment
113 Wild-type (ANY21) cells containing the EGFP-CFTR plasmid (top) or EGFP-CFTR-D567A plasmid (bottom) were grown to mid-log phase at 23&#ba;C and incubated with copper-containing medium for 2 h to induce expression. Login to comment
186 The mutation to alanine at position 567 of CFTR was introduced into the gene of EGFP-CFTR in plasmid pEGFP-CFTR using primer-directed mutagenesis, yielding pEGFP-CFTR-D567A. 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
171 CFTR with Mutated Diacidic Exit Code Is Rescued by Low Temperature, but Not by Correctors (A) BHK cell lines stably expressing WT-CFTR or bearing the DAA (D567A) mutation alone or in cis with 4RK were cultured at 37 C, incubated at 26 C for 48 hr or for 24hr with VRT-325, Corr-4a, or VX-809, as indicated. CFTR protein was analyzed by WB. Data are representative of n = 5 experiments. Login to 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