Functional analysis of sites within PCSK9 responsible for hypercholesterolemia

Abstract

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 wildtype 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, S

Structural and Biochemical Characterization of the Wild Type PCSK9-EGF(AB) Complex and Natural Familial Hypercholesterolemia Mutants

“…Thus, formation of an H-bond at neutral pH between Asp-374 and EGF(A) Tyr-306, or between Tyr-374 or His-374 and the carbonyl oxygen of EGF(A) Cys-319, and formation of a salt bridge at low pH between Asp-374 and EGF(A) His-306 have similar effects in increasing the affinity of the complex. Nevertheless, we note that mutation of Asp-374 into Ala or Phe has also been reported to result in an increased affinity for the LDLR (27) suggesting that other factors, such as electrostatic effects, might also modulate the affinity of the complex.…”

“…These additional interactions with EGF(A) may explain the enhanced affinity of the PCSK9 D374Y(H) -LDLR interactions reported previously (10,17) and indeed the severe hypercholesterolemia in carriers of such mutations. Previously we reported a mild GOF effect for PCSK9 D374A (27). Not surprisingly the EGF(AB)-PCSK9 D374A structure (Table 2) does not reveal additional interactions of Ala-374 with EGF(AB).…”

“…The only experimental difference is that Alexa Fluor 546-labeled LDL, rather than LDL labeled with 3,3-dioctadecylindocarbocyanine (Dil), was used to follow cellular LDL uptake; labeling was performed as described previously (27). The inhibition of PCSK9 activity was assessed in titrations in which putative competitor proteins, in the nanomolar to micromolar concentration range, were added to the growth medium at the same time as PCSK9 followed by a 6.5-h incubation at 37°C.…”

PCSK9 is not involved in the degradation of LDL receptors and BACE1 in the adult mouse brain

“…Wild-type PCSK9 protein was purifi ed from HEK293 generated media as described ( 29,30 ). Recombinant human LDLR, mouse VLDLR, and human ApoER2 used on surface plasmon resonance (SPR) experiments were purchased from R and D Systems.…”

Errata. PCSK9 function and physiology

“…In this issue of the Journal of Lipid Research ( JLR), two leading research groups describe their recent efforts. Dr. Shilpa Pandit et al (12) from Merck Research Laboratories describe the functional basis for the hypercholesterolemia associated with gain-of-function missense mutations in PCSK9. Grefhorst et al (13) at UT Southwestern describe the kinetics and metabolism of recombinant PCSK9 and the impact of PCSK9 on LDL receptors in the liver and adrenal gland.…”