In:
Circulation, Ovid Technologies (Wolters Kluwer Health), Vol. 116, No. suppl_16 ( 2007-10-16)
Abstract:
Mutations within proprotein convertase subtilisin/kexin type 9 (PCSK9) are associated with dominant forms of familial hypercholesterolemia. Recent studies have shown that PCSK9 directly binds the LDL receptor (LDLR) and that addition of PCSK9 to cells promotes degradation of LDLR. Mutations associated with hypercholesterolemia (S127R and D374Y) bind the LDLR stronger and are more potent in decreasing LDL-uptake. Based on these data, we sought to better understand the mechanism by which mutations at both the S127 and D374 residues of PCSK9 affect PCSK9 function. A limited vertical scanning mutagenesis was done, resulting in the following mutants: S127(R, A, D, K, L, T) and D374(Y, A, E, F, K, L). Expression of several S127 alleles in HEK293 cells resulted in less efficient processing of PCSK9, with S127L severely defective in processing. In contrast, all D374 alleles tested had no effect on PCSK9 processing. In a cell-based functional assay of PCSK9 activity, both S127R and S127K proteins were significantly more potent (2– 4 fold) in decreasing LDL-uptake than wild-type PCSK9, while all other S127 mutants were similar to wild-type. Each D374 mutant tested was more potent than wild-type PCSK9 in reducing LDL-uptake (D374Y, F 〉 A, L 〉 E, K 〉 wild-type). To determine if the potencies of S127 and D374 mutations in lowering LDL-uptake correlated with their ability to interact with the LDLR, each mutant was tested in a PCSK9-LDLR time-resolved FRET assay. Results indicate that potency in the cell-based LDL-uptake assay correlates with PCSK9’s binding affinity for the LDLR. Combination of S127R and D374Y was also found to have an additive effect in enhancing PCSK9’s ability to reduce LDL-uptake. Molecular modeling indicates that mutations at S127 and D374 which enhance PCSK9 function stabilize or destabilize the protein, respectively. In conclusion, these results suggest a model in which mutations at S127 and D374 residues modulate PCSK9’s ability to regulate LDLR function through distinct mechanisms.
Type of Medium:
Online Resource
ISSN:
0009-7322
,
1524-4539
DOI:
10.1161/circ.116.suppl_16.II_161-b
Language:
English
Publisher:
Ovid Technologies (Wolters Kluwer Health)
Publication Date:
2007
detail.hit.zdb_id:
1466401-X
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