ABCC7 p.Glu726Lys
| Predicted by SNAP2: | A: D (71%), C: D (75%), D: N (53%), F: D (85%), G: D (71%), H: D (85%), I: D (75%), K: D (63%), L: D (85%), M: D (80%), N: D (85%), P: D (91%), Q: D (63%), R: D (85%), S: D (80%), T: D (85%), V: D (80%), W: D (91%), Y: D (85%), |
| Predicted by PROVEAN: | A: N, C: D, D: N, F: D, G: N, H: N, I: D, K: N, L: D, M: D, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: D, Y: N, |
[switch to compact view]
Comments [show]
None has been submitted yet.
[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.
Comments [show]
None has been submitted yet.
No. Sentence Comment
48 The E725K/E726K mutant was included to test if alteration of charges would affect the function of the PEST sequence.
X
ABCC7 p.Glu726Lys 12361483:48:10
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.
X
ABCC7 p.Glu726Lys 12361483:109:506
status: NEW134 Mature Immature WT CFTR Mature Immature ∆F508 CFTR WT/∆F508 Poly-Valine S728A E725K/E726K T717A/E725K A. B.
X
ABCC7 p.Glu726Lys 12361483:134:98
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].
X
ABCC7 p.Glu726Lys 12361483:135:125
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.
X
ABCC7 p.Glu726Lys 12361483:153:80
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.
X
ABCC7 p.Glu726Lys 12361483:154:122
status: NEW156 Hours 0 2 4 6 8 12 24 E725K/E726K Mature Immature T717A/E725KMature Immature WT Mature Immature A.
X
ABCC7 p.Glu726Lys 12361483:156:28
status: NEW157 Hours 0 2 4 6 8 12 24 ∆∆∆∆F508Mature Immature ∆∆∆∆F508/ E725K/E726K Mature Immature ∆∆∆∆F508/ T717A/E725K Mature Immature Mature Immature ∆∆∆∆F508/ Poly-Valine B. mutant protein to not mature suggests otherwise.
X
ABCC7 p.Glu726Lys 12361483:157:118
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].
X
ABCC7 p.Glu726Lys 12361483:177:332
status: NEW