ABCC4 p.Ser794Leu
| Predicted by SNAP2: | A: N (82%), C: D (53%), D: D (80%), E: D (80%), F: D (80%), G: N (78%), H: D (80%), I: D (80%), K: D (85%), L: D (80%), M: D (75%), N: D (71%), P: D (85%), Q: D (75%), R: D (85%), T: D (53%), V: D (75%), W: D (91%), Y: D (85%), |
| Predicted by PROVEAN: | A: N, 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, T: N, V: D, W: D, Y: D, |
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[hide] Crucial role for phylogenetically conserved cytopl... J Biol Chem. 2013 Aug 2;288(31):22207-18. doi: 10.1074/jbc.M113.476218. Epub 2013 Jun 13. Cheepala SB, Bao J, Nachagari D, Sun D, Wang Y, Zhong TP, Naren AP, Zheng J, Schuetz JD
Crucial role for phylogenetically conserved cytoplasmic loop 3 in ABCC4 protein expression.
J Biol Chem. 2013 Aug 2;288(31):22207-18. doi: 10.1074/jbc.M113.476218. Epub 2013 Jun 13., [PMID:23766510]
Abstract [show]
The ABC transporter ABCC4 is recognized as an ATP-dependent exporter of endogenous substances as well as an increasing variety of anionic chemotherapeutics. A loss-of-function variant of zebrafish Abcc4 was identified with a single amino acid substitution in the cytoplasmic loop T804M. Because this substituted amino acid is highly conserved among ABCC4 orthologs and is located in cytoplasmic loop 3 (CL3), we investigated the impact of this mutation on human and zebrafish Abcc4 expression. We demonstrate that zebrafish Abcc4 T804M or human ABCC4 T796M exhibit substantially reduced expression, coupled with impaired plasma membrane localization. To understand the molecular basis for the localization defect, we developed a homology model of zebrafish Abcc4. The homology model suggested that the bulky methionine substitution disrupted side-chain contacts. Molecular dynamic simulations of a fragment of human or zebrafish CL3 containing a methionine substitution indicated altered helicity coupled with reduced thermal stability. Trifluoroethanol challenge coupled with circular dichroism revealed that the methionine substitution disrupted the ability of this fragment of CL3 to readily form an alpha-helix. Furthermore, expression and plasma membrane localization of these mutant ABCC4/Abcc4 proteins are mostly rescued by growing cells at subphysiological temperatures. Because the cystic fibrosis transmembrane conductance regulator (ABCC7) is closely related to ABCC4, we extended this by engineering certain pathogenic CFTR-CL3 mutations, and we showed they destabilized human and zebrafish ABCC4. Altogether, our studies provide the first evidence for a conserved domain in CL3 of ABCC4 that is crucial in ensuring its proper plasma membrane localization.
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No. Sentence Comment
199 Notably, disease-related point mutations in CFTR CL3 at positions S945L and H949Y (Fig. 5A) (44) affected maturation of CFTR (26).
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ABCC4 p.Ser794Leu 23766510:199:277
status: NEW200 Because these two amino acids are adjacent to Thr-796 in ABCC4 and mostly conserved with CFTR, we hypothesized that this region of CL3 might be generally important to ABCC protein stability. Using site-directed mutagenesis, we generated the analogous mutations in human ABCC4 (S794L and H798Y) and ZF Abcc4 (A802L and H806Y).
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ABCC4 p.Ser794Leu 23766510:200:277
status: NEW202 ABCC4 S794L and H796Y as well as ZF Abcc4 A802L and H806Y substitutions affected both protein expression and highly reduced the levels of the mature band c (Fig. 5, C and E).
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ABCC4 p.Ser794Leu 23766510:202:6
status: NEW249 E, immunoblot of whole cell lysates (100 òe;g of protein per lane) prepared from NIH3T3 cells transfected with human ABCC4 and ABCC4 mutants (T796M, S794L, and H798Y), and these blots were probed with anti-GFP antibody.
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ABCC4 p.Ser794Leu 23766510:249:153
status: NEW201 ABCC4 S794L and H796Y as well as ZF Abcc4 A802L and H806Y substitutions affected both protein expression and highly reduced the levels of the mature band c (Fig. 5, C and E).
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ABCC4 p.Ser794Leu 23766510:201:6
status: NEW248 E, immunoblot of whole cell lysates (100 òe;g of protein per lane) prepared from NIH3T3 cells transfected with human ABCC4 and ABCC4 mutants (T796M, S794L, and H798Y), and these blots were probed with anti-GFP antibody.
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ABCC4 p.Ser794Leu 23766510:248:153
status: NEW