Exploring Alternate Confirmations of PCSK9: Effects on LDL, LDL Receptor Binding, and Inflammation

Abstract

Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a central regulator of circulating levels of low-density lipoprotein-cholesterol (LDL-C) and related cardiovascular disease (CVD) risk. PCSK9 is a sol uble secreted protein expressed predominantly in the liver, where it mediates lysosomal degradation of cell surface LDL receptor (LDLR), the primary conduit for LDL-C plasma clearance. Cell culture and mouse in vivo studies support the notion that PCSK9 mediates pro-inflammatory signaling, and therapeutic monoclonal antibodies (e.g., alirocumab) that bind PCSK9 also exert anti-inflammatory proper ties in addition to LDL-C lowering. Plasma PCSK9 exists in intact (full-length) and truncated forms. A significant number of the former are LDL-bound. An N-terminal intrinsically disordered region (IDR) within the PCSK9 prodomain (aa 31-52) - an area rich in negatively charged amino acids - is critical in this association and has also been shown to exert a negative allosteric effect on PCSK9-LDLR binding affinity. Herein, we showed that mutagenesis to change amino acid residues within this region leads to a PCSK9 variant with altered LDL and LDLR association. Specifically, inserting a disordered Gly-Ser Gly-Gly (GSGG) linker to replace acidic Ser47-Glu-Glu-Asp increased LDLR- dependent uptake to the same extent as a complete deletion of the IDR, whereas individual amino acid replacements within the IDR showed similar LDL and LDLR binding as wild-type (WT)- PCSK9. The major truncated PCSK9 variant found in human plasma is a proteolytic product of furin (f-PCSK9), which is considered inert because it cannot bind LDLR. However, f-PCSK9 interaction with native LDL (nLDL) and oxidatively modified LDL (oxLDL) was increased by 1.5-fold and ~10-fold, respectively, compared with intact PCSK9. F-PCSK9 also showed preferential binding to artificial liposomes mimicking the outer lipid membrane of LDL. The endocytic uptake of f-PCSK9 in HEK293 cells transiently overexpressing the scavenger receptors CD36 or LOX1 was increased compared to intact PCSK9. OxLDL co-incubation increased f-PCSK9 uptake in M1-polarized THP-1 human macrophages; pro-inflammatory cytokines such as IL-1β and TNF-α significantly increased, as determined by qPCR. F-PCSK9 was increased in the serum of LPS-treated female mice 24 hours post-treatment. The therapeutic anti-PCSK9 monoclonal antibody alirocumab blocked furin proteolysis of PCSK9. These results suggest that the f-PCSK9 may function as an inflammatory signaling molecule and mediate oxLDL interactions with scavenger recep tors in immune cells. Overall, these findings suggest a new paradigm in PCSK9 biology whereby furin proteolysis reveals novel immune functions and could aid in designing future PCSK9 inhibitors directed at different sub species of PCSK9.