Characterization of the Non-Proteolytic Mechanism and Cellular Site of Action of PCSK9-Mediated Degradation of the Low-Density Lipoprotein Receptor

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) is a serine protease that has emerged as a central regulator of plasma low-density lipoprotein cholesterol levels. Here, it is demonstrated that PCSK9 is secreted into the blood and that the secreted PCSK9 binds and degrades LDLRs in liver. To determine if PCSK9 catalytic activity was involved in the degradation of the LDLR, a mutant PCSK9 was engineered that is catalytically inactive. Studies with catalytically inactive PCSK9 demonstrated that the protein degraded LDLRs in a manner that was indistinguishable from wild-type PCSK9, suggesting that proteolytic activity is not required for PCSK9-mediated degradation of the LDLR. Crystallographic analysis of a PCSK9:LDLR complex supported the experimental findings that PCSK9 does not catalytically cleave LDLRs. These studies also suggested that one therapeutic approach for treating hypercholesterolemia might be to disrupt the PCSK9:LDLR interaction at the cell surface. Recombinant LDLR subfragments were synthesized and added to the medium of cells that overexpressed PCSK9. These sub-fragments restored LDLRs to levels found in the control cells. These experiments confirmed that the disruption of PCSK9:LDLR at the cell surface can inhibit PCSK9 activity and suggested that the majority of PCSK9 activity is extracellular. Further structural analysis of the PCSK9:LDLR co-crystal predicted that an LDLR mutation (His306Tyr) might increase the affinity of PCSK9 for the LDLR, and thus could be associated with familial hypercholesterolemia (FH). A search of the LDLR mutation database revealed that the LDLR mutation (His306Tyr) had been reported in a kindred with FH. Structure/function studies with the mutant LDLR(H306Y) protein showed that this mutation mimics a conformation change in the wild-type LDLR that occurs at low pH, which results in increased binding. The increased affinity between PCSK9 and LDLR(H306Y) promoted enhanced LDLR degradation. Thus, this mutation represents a previously unrecognized class (Class VI) of FH mutants. Finally, two high-throughput assays were developed to discover new small molecule inhibitors of intracellular PCSK9 autocleavage and to identify previously unrecognized protein activators of PCSK9 action. Use of these assays could provide additional avenues for modulating PCSK9 activity and lead to new therapeutic options for the treatment of hypercholesterolemia.