ABCMdb

PMID: 12361483

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 [PubMed]

Sentences

No. Mutations Sentence Comment

44 ABCC7 p.Ser728Ala 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). Login to comment 48 ABCC7 p.Glu725Lys ABCC7 p.Glu726Lys The E725K/E726K mutant was included to test if alteration of charges would affect the function of the PEST sequence. Login to comment 50 ABCC7 p.Ser728Ala S728 and T717 were both altered to alanine, a small, neutral residue; S728A has PEST score of +4.07. Login to comment 51 ABCC7 p.Glu725Lys ABCC7 p.Ser728Ala ABCC7 p.Thr717Ala ABCC7 p.Thr717Ala ABCC7 p.Thr717Ala 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. Login to comment 109 ABCC7 p.Glu725Lys ABCC7 p.Glu725Lys ABCC7 p.Ser728Ala ABCC7 p.Glu726Lys ABCC7 p.Thr717Ala 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. Login to comment 134 ABCC7 p.Glu725Lys ABCC7 p.Glu725Lys ABCC7 p.Ser728Ala ABCC7 p.Glu726Lys ABCC7 p.Thr717Ala Mature Immature WT CFTR Mature Immature ∆F508 CFTR WT/∆F508 Poly-Valine S728A E725K/E726K T717A/E725K A. B. Login to comment 135 ABCC7 p.Glu725Lys ABCC7 p.Glu725Lys ABCC7 p.Ser728Ala ABCC7 p.Glu726Lys ABCC7 p.Thr717Ala 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]. Login to comment 153 ABCC7 p.Glu725Lys ABCC7 p.Glu725Lys ABCC7 p.Ser728Ala ABCC7 p.Glu726Lys ABCC7 p.Thr717Ala 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. Login to comment 154 ABCC7 p.Glu725Lys ABCC7 p.Glu725Lys ABCC7 p.Ser728Ala ABCC7 p.Glu726Lys 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. Login to comment 156 ABCC7 p.Glu725Lys ABCC7 p.Glu726Lys ABCC7 p.Thr717Ala Hours 0 2 4 6 8 12 24 E725K/E726K Mature Immature T717A/E725KMature Immature WT Mature Immature A. Login to comment 157 ABCC7 p.Glu725Lys ABCC7 p.Glu725Lys ABCC7 p.Glu726Lys ABCC7 p.Thr717Ala 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. Login to comment 177 ABCC7 p.Glu725Lys ABCC7 p.Glu725Lys ABCC7 p.Glu725Lys ABCC7 p.Ser728Ala ABCC7 p.Glu726Lys ABCC7 p.Thr717Ala ABCC7 p.Thr717Ala 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]. Login to comment 202 ABCC7 p.Glu725Lys ABCC7 p.Thr717Ala WT/∆F508Mature Immature Poly-ValineMature Immature S728AMature Immature E725K/E726KMature Immature T717A/E725KMature Immature - + - + WT CFTR ∆∆∆∆F508 CFTR 2µµµµM MG-132 tion at 44,000 × g for 45 min. at 4°C and resuspended with 300 µl of TBS (Tris-buffered saline; 10 mM Tris-HCl, pH 7.5, 150 mM NaCl). Login to comment