Functional analysis of sites within PCSK9 responsible for hypercholesterolemia.
Mutations within proprotein convertase subtilisin/kexin type 9 (PCSK9) are associated with dominant forms of familial hypercholesterolemia. PCSK9 binds the LDL receptor (LDLR), and addition of PCSK9 to cells promotes degradation of LDLR. PCSK9 mutant proteins associated with hypercholesterolemia (S127R and D374Y) are more potent in decreasing LDL uptake than is wild-type PCSK9. To better understand the mechanism by which mutations at the Ser127 and Asp374 residues of PCSK9 influence PCSK9 function, a limited vertical scanning mutagenesis was performed at both sites. S127R and S127K proteins were more potent in decreasing LDL uptake than was wild-type PCSK9, and each D374 mutant tested was more potent in reducing LDL uptake when the proteins were added exogenously to cells. The potencies of D374 mutants in lowering LDL uptake correlated with their ability to interact with LDLR in vitro. Combining S127R and D374Y was also found to have an additive effect in enhancing PCSK9's ability to reduce LDL uptake. Modeling of PCSK9 S127 and D374 mutations indicates that mutations that enhance PCSK9 function stabilize or destabilize the protein, respectively. In conclusion, these results suggest a model in which mutations at Ser127 and Asp374 residues modulate PCSK9's ability to regulate LDLR function through distinct mechanisms.
Pandit
Shilpa
S
Division of Cardiovascular Diseases, Merck Research Laboratories, Rahway, NJ 07065, USA.
Wisniewski
Doug
D
Santoro
Joseph C
JC
Ha
Sookhee
S
Ramakrishnan
Vijayalakshmi
V
Cubbon
Rose M
RM
Cummings
Richard T
RT
Wright
Samuel D
SD
Sparrow
Carl P
CP
Sitlani
Ayesha
A
Fisher
Timothy S
TS
D001224:Aspartic Acid / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D006801:Humans; D006937:Hypercholesterolemia / Q000503:physiopathology*; D008077:Lipoproteins, LDL / Q000378:metabolism; D016296:Mutagenesis; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D011973:Receptors, LDL / Q000502:physiology; D012694:Serine / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D017931:DNA Primers; D008077:Lipoproteins, LDL; D011973:Receptors, LDL; D001224:Aspartic Acid; D012694:Serine; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases