Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the proteinase K subfamily of subtilases that reduces the number of LDL receptors (LDLRs) in liver through an undefined posttranscriptional mechanism. We show that purified PCSK9 added to the medium of HepG2 cells reduces the number of cellsurface LDLRs in a dose-and time-dependent manner. This activity was approximately 10-fold greater for a gain-of-function mutant, PCSK9(D374Y), that causes hypercholesterolemia. Binding and uptake of PCSK9 were largely dependent on the presence of LDLRs. Coimmunoprecipitation and ligand blotting studies indicated that PCSK9 and LDLR directly associate; both proteins colocalized to late endocytic compartments. Purified PCSK9 had no effect on cell-surface LDLRs in hepatocytes lacking autosomal recessive hypercholesterolemia (ARH), an adaptor protein required for endocytosis of the receptor. Transgenic mice overexpressing human PCSK9 in liver secreted large amounts of the protein into plasma, which increased plasma LDL cholesterol concentrations to levels similar to those of LDLR-knockout mice. To determine whether PCSK9 was active in plasma, transgenic PCSK9 mice were parabiosed with wild-type littermates. After parabiosis, secreted PCSK9 was transferred to the circulation of wild-type mice and reduced the number of hepatic LDLRs to nearly undetectable levels. We conclude that secreted PCSK9 associates with the LDLR and reduces hepatic LDLR protein levels.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes degradation of hepatic low density lipoprotein receptors (LDLR), the major route of clearance of circulating cholesterol. Gain-of-function mutations in PCSK9 cause hypercholesterolemia and premature atherosclerosis, whereas loss-of-function mutations result in hypocholesterolemia and protection from heart disease. Recombinant human PCSK9 binds the LDLR on the surface of cultured hepatocytes and promotes degradation of the receptor after internalization. Here we localized the site of binding of PCSK9 within the extracellular domain of the LDLR and determined the fate of the receptor after PCSK9 binding. Recombinant human PCSK9 interacted in a sequence-specific manner with the first epidermal growth factor-like repeat (EGF-A) in the EGF homology domain of the human LDLR. Similar binding specificity was observed between PCSK9 and purified EGF-A. Binding to EGF-A was calcium-dependent and increased dramatically with reduction in pH from 7 to 5.2. The addition of PCSK9, but not heat-inactivated PCSK9, to the medium of cultured hepatocytes resulted in redistribution of the receptor from the plasma membrane to lysosomes. These data are consistent with a model in which PCSK9 binding to EGF-A interferes with an aciddependent conformational change required for receptor recycling. As a consequence, the LDLR is rerouted from the endosome to the lysosome where it is degraded.Genetic variation in proprotein convertase subtilisin/kexin type 9 (PCSK9) 4 contributes to differences in plasma levels of low density lipoprotein (LDL) cholesterol (1, 2), the primary cholesterol-carrying lipoprotein in humans. Selected missense mutations in PCSK9 cause dominant hypercholesterolemia and premature atherosclerosis (1, 3, 4), whereas loss-of-function mutations in PCSK9 reduce plasma LDL levels and protect against coronary heart disease (5-7). Studies in mice suggest that the major metabolic effect of PCSK9 is to reduce the amount of hepatic LDL receptor (LDLR), the primary conduit for the clearance of LDL from the circulation (8). Expression of recombinant PCSK9 in the livers of mice causes a reduction in hepatic LDLR protein (but not mRNA) and produces severe hypercholesterolemia (9 -11). Conversely, mice lacking PCSK9 manifest increased levels of LDLR protein in the liver and accelerated clearance of circulating LDL (12). PCSK9 is expressed predominantly in the liver, small intestine, kidney, and brain (13) and is present in human plasma (14,15). Introduction of PCSK9 into the circulation of mice through parabiosis reduces hepatic LDLR levels, which is consistent with PCSK9 interacting with the LDLR on the cell surface (14). In cultured cells, the addition of recombinant PCSK9 to the medium results in LDLR degradation, providing further evidence that PCSK9 can promote the degradation of the LDLR by acting at the cell surface (14, 16). Autocatalytic cleavage is required for PCSK9 maturation and secretion (10), but whether the catalytic activity of PCSK9 is required for LDLR d...

Multiple metabolic and genetic factors contribute to variation in plasma levels of PCSK9 in the general population. Although levels of PCSK9 correlate with plasma levels of LDL-C, they account for only a small proportion of the variation in the levels of this lipoprotein.

Elevated levels of circulating low-density lipoprotein cholesterol (LDL-C) play a central role in the development of atherosclerosis. Mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9) that are associated with lower plasma levels of LDL-C confer protection from coronary heart disease. Here, we show that four severe loss-of-function mutations prevent the secretion of PCSK9 by disrupting synthesis or trafficking of the protein. In contrast to recombinant wild-type PCSK9, which was secreted from cells into the medium within 2 hours, the severe loss-of-function mutations in PCSK9 largely abolished PCSK9 secretion. This finding predicted that circulating levels of PCSK9 would be lower in individuals with the loss-of-function mutations. Immunoprecipitation and immunoblotting of plasma for PCSK9 provided direct evidence that the serine protease is present in the circulation and identified the first known individual who has no immunodetectable circulating PCSK9. This healthy, fertile college graduate, who was a compound heterozygote for two inactivating mutations in PCSK9, had a strikingly low plasma level of LDL-C (14 mg/dL). The very low plasma level of LDL-C and apparent good health of this individual demonstrate that PCSK9 plays a major role in determining plasma levels of LDL-C and provides an attractive target for LDL-lowering therapy.

hypercholesterolemia ͉ proprotein convertase ͉ crystal structure P roprotein convertase (PC) subtilisin/kexin type 9 (PCSK9) is a serine protease of the PC family that has profound effects on plasma low-density lipoprotein (LDL)-cholesterol (LDL-C) levels through its ability to mediate LDL receptor (LDLR) protein degradation (1, 2). The link between PCSK9 and plasma LDL-C levels was first established by the discovery of missense mutations in PCSK9 that were present in patients with an autosomal dominant form of familial hypercholesterolemia (FH) (3). These mutations were speculated to result in a gain-offunction of PCSK9 owing to their mode of inheritance. Subsequently, loss-of-function mutations in PCSK9 were discovered that are associated with low plasma LDL-C levels and significantly reduced coronary heart disease (CHD) (4, 5).The overall domain structure of PCSK9 is similar to other PC family members. It includes a signal peptide, followed by a prodomain, a subtilisin-like catalytic domain, and a variable C-terminal domain (6). The prodomain serves a dual role as a chaperone for folding and as an inhibitor of catalytic activity (7-9). Autocatalysis between Gln-152 and Ser-153 (VFAQ:SIP) separates the prodomain from the catalytic domain, but the prodomain remains bound, occluding the catalytic site (10, 11). For other PC family members, a second catalytic cleavage is required to release the prodomain, which unmasks the catalytic site, resulting in an active protease (7). No site of secondary cleavage has been identified that activates PCSK9.The crystal structure of apo-PCSK9 revealed a tightly bound prodomain that is predicted to render the active site inaccessible to exogenous substrates (12-14). The structure of the PCSK9 prodomain and catalytic domain is similar to that of other subtilisin-like serine proteases. The C-terminal domain of PCSK9 contains three six-stranded ␤-sheet subdomains arranged with quasi-threefold symmetry. This domain shares structural homology to the adipokine resistin and has been speculated to mediate protein-protein interactions (12-14).Recent studies have provided insights into the site and mode of PCSK9's action. Addition of recombinant PCSK9 to the medium of cultured hepatocytes results in a dramatic reduction in LDLR number. PCSK9 binds directly to the LDLR on the cell surface and PCSK9-stimulated degradation of the LDLR requires autosomal recessive hypercholesterolemia (ARH), an adaptor protein needed for internalization of LDLRs (11). The affinity of PCSK9 binding to the LDLR is enhanced at acidic pH, suggesting that PCSK9 binds more avidly to LDLRs in the lysosomal/endosomal compartments (12,15,16). One gain-offunction mutant, Asp-374-Tyr, was Ϸ10-fold more active than wild-type PCSK9 in mediating degradation of LDLRs, owing to an Ϸ5-to 30-fold increased affinity of PCSK9 for the LDLR (11,12,15). Although autocatalysis of PCSK9 is required for proper folding and secretion, catalytic activity was not required for PCSK9-mediated LDLR degradation when added to cultured cells (...