ABCC7 p.Ser728Ala
| Predicted by SNAP2: | A: N (87%), C: N (82%), D: N (82%), E: N (78%), F: N (82%), G: N (82%), H: N (93%), I: N (78%), K: N (72%), L: N (87%), M: N (82%), N: N (87%), P: N (66%), Q: N (93%), R: N (82%), T: N (87%), V: N (87%), W: N (57%), Y: N (87%), |
| 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, T: N, V: N, W: D, Y: N, |
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[hide] The PEST sequence does not contribute to the stabi... BMC Biochem. 2002 Oct 2;3:29. Epub 2002 Oct 2. Chen EY, Clarke DM
The PEST sequence does not contribute to the stability of the cystic fibrosis transmembrane conductance regulator.
BMC Biochem. 2002 Oct 2;3:29. Epub 2002 Oct 2., 2002-10-02 [PMID:12361483]
Abstract [show]
BACKGROUND: Endoplasmic reticulum retention of misfolded cystic fibrosis transmembrane conductance regulator (CFTR) mutants and their rapid degradation is the major cause of cystic fibrosis (CF). An important goal is to understand the mechanism of how the misfolded proteins are recognized, retained, and targeted for degradation. RESULTS: Using a web-based algorithm, PESTFind, we found a PEST sequence in the regulatory (R) domain of CFTR. The PEST sequence is found in many short-lived eukaryotic proteins and plays a role in their degradation. To determine its role in the stability and degradation of misprocessed CFTR, we introduced a number of site-directed mutations into the PEST sequence in the cDNA of DeltaF508 CFTR, the most prevalent misprocessed mutation found in CF patients. Analysis of these mutants showed that the disruption of the PEST sequence plays a minor role in the degradation of the CFTR mutants. Multiple mutations to the PEST sequence within the R domain of CFTR inhibit maturation of CFTR and prevent the formation of a 100 kDa degradation product. The mutations, however, do not improve the stability of the mutant DeltaF508 CFTR. CONCLUSION: These observations show that disruption of the structure of the R domain of CFTR can inhibit maturation of the protein and that the predicted PEST sequence plays no significant role in the degradation of CFTR.
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No. Sentence Comment
44 The mutants' PEST scores range from being poor PEST candidates (+4.07 for S728A and -26.19 for poly-valine) to being invalid PEST candidates (i.e. no score available).
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ABCC7 p.Ser728Ala 12361483:44:74
status: NEW50 S728 and T717 were both altered to alanine, a small, neutral residue; S728A has PEST score of +4.07.
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ABCC7 p.Ser728Ala 12361483:50:70
status: NEW51 However, since both S728A and T717A mutant have a high PEST score (T717A mutant alone has PEST score of +3.87), we added an additional E725K mutation to the T717A mutant just to further disrupt the PEST sequence.
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ABCC7 p.Ser728Ala 12361483:51:20
status: NEW109 Misfolded or misassembled proteins, such as processing defective ∆F508 mutant CFTR, are recognized and retained in the ER by the quality control system in the ER; although the exact mechanism for recognition is yet to be elucidated, evidence indicate that the prolonged association with mo- Table 1: Mutations introduced into the predicted PEST sequence of CFTR Mutant Sequence PEST Score Wild-type (WT) 716 - KTPLQMNGIEEDSDEPLER - 734 +6.91 Poly-Valine 716 - KTPLQMNGIVVVVVVPLER - 734 -26.19 E725K/E726K 716 - KTPLQMNGIKKDSDEPLER - 734 N/A S728A 716 - KTPLQMNGIEEDADEPLER - 734 +4.07 T717A/E725K 716 - KAPLQMNGIKEDSDEPLER - 734 N/A * Residues in bold are the mutations introduced.
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ABCC7 p.Ser728Ala 12361483:109:548
status: NEW134 Mature Immature WT CFTR Mature Immature ∆F508 CFTR WT/∆F508 Poly-Valine S728A E725K/E726K T717A/E725K A. B.
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ABCC7 p.Ser728Ala 12361483:134:86
status: NEW135 205 130 90 kDa 100 kD 81 kD Immature ∆F508 Poly-Valine ∆F508/Poly-Valine ∆F508/S728A ∆F508/E725K/E726K ∆F508/T717A/E725K volved in protein-protein interactions: direct interaction with ubc9 [33] and ligand recognition [34,35].
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ABCC7 p.Ser728Ala 12361483:135:100
status: NEW153 A) Pulse-chase radiograph for non-processing defective constructs: WT, WT/E725K/E726K, and WT/T717A/E725K; results for WT/S728A not shown, but is similar to that of WT.
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ABCC7 p.Ser728Ala 12361483:153:122
status: NEW154 B) Pulse-chase radiograph for processing defective constructs: ∆F508, ∆F508/poly-valine, ∆F508/E725K/E726K, and ∆F508/T717/E725K; results for WT/Poly-valine and ∆F508/S728A not shown but they show similar results as ∆F508.
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ABCC7 p.Ser728Ala 12361483:154:202
status: NEW177 0 0.1 1 10 100 1000 [Trypsin] (µg/ml) T717A/E725KMature Immature E725K/E726KMature Immature S728AMature Immature WTMature Immature Poly-ValineMature Immature ∆F508Mature Immature 0 0.1 1 10 100 1000 [Trypsin] (µg/ml) ∆F508/ Poly-Valine Mature Immature ∆F508/ S728A Mature Immature ∆F508/ E725K/E726K Mature Immature ∆F508/ T717A/E725K Mature Immature the PEST region of another nuclear SUMO-1 target protein, HIPK2 [48].
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ABCC7 p.Ser728Ala 12361483:177:290
status: NEW