A new method for measurement of total plasma PCSK9: clinical applications

PCSK9 has exploded onto center stage of plasma cholesterol metabolism, raising hopes for a new strategy to treat hypercholesterolemia. PCSK9 in a plasma protein that triggers increased degradation of the LDL receptor. Gain-of-function mutations in PCSK9 reduce LDL receptor levels in the liver, resulting in high levels of LDL cholesterol in the plasma and increased susceptibility to coronary heart disease. Loss-of-function mutations lead to higher levels of the LDL receptor, lower LDL cholesterol levels, and protection from coronary heart disease. Two papers in this issue of the Journal of Lipid Research exemplify the rapid pace of progress in understanding PCSK9 molecular interactions and physiology. Dr. Shilpa Pandit and coworkers from Merck Research Laboratories describe the functional basis for the hypercholesterolemia associated with gain-of-function missense mutations in PCSK9. Dr. Jay Horton's group at UT Southwestern describe the kinetics and metabolism of PCSK9 and the impact of PCSK9 on LDL receptors in the liver and adrenal gland.

The LDL receptor (LDLR) plays an essential role in the regulation of plasma (LDL) cholesterol concentrations by virtue of its ability to clear plasma LDL. Down-regulation of the LDLR by proprotein convertase subtilisin/kexin 9 (PCSK9) has recently emerged as a regulatory mechanism that controls plasma LDL cholesterol concentrations. Studies in which PCSK9 is over-expressed in mice, have demonstrated that PCSK9, by enhancing hepatic LDLR degradation, decreases the availability of the LDLR for LDL uptake, resulting in increased plasma LDL cholesterol levels. However, PCSK9 has also recently been shown to mediate down-regulation of surface receptors other than the LDLR, suggesting that it may have much broader roles than initially thought.

Development of Novel DNA-Encoded PCSK9 Monoclonal Antibodies as Lipid-Lowering Therapeutics.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that regulates the expression of LDL receptor (LDLR) protein. Gain-of-function mutations in PCSK9 cause hypercholesterolemia, and loss-of-function mutations result in lower plasma LDL-cholesterol. Here, we investigate the kinetics and metabolism of circulating PCSK9 relative to tissue levels of LDLRs. The administration of recombinant human PCSK9 (32 microg) to mice by a single injection reduced hepatic LDLRs by approximately 90% within 60 min, and the receptor levels returned to normal within 6 h. The half-life of the PCSK9 was estimated to be approximately 5 min. Continuous infusion of PCSK9 (32 microg/h) into wild-type mice caused a approximately 90% reduction in hepatic LDLRs within 2 h and no associated change in the level of LDLR in the adrenals. Parallel studies were performed using a catalytically inactive form of PCSK9, PCSK9(S386A), and similar results were obtained. Infusion of PCSK9(D374Y), a gain-of-function mutation, resulted in accelerated clearance of the mutant PCSK9 and a greater reduction in hepatic LDLRs. Combined, these data suggest that exogenously administrated PCSK9 in plasma preferentially reduces LDLR protein levels in liver at concentrations found in human plasma and that PCSK9's action on the LDLR is not dependent on catalytic activity in vivo.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low density lipoprotein receptor protein levels by diverting it to lysosomes. Monoclonal antibody therapeutics aimed to neutralize PCSK9 have been shown to successfully lower serum LDL levels; however, we previously found that such therapeutic antibodies are subject to PCSK9-mediated clearance. In this study, we discovered that PCSK9 interacts via its C-terminal domain directly and in a pH-dependent manner with amyloid precursor protein as well as its closely related family member, amyloid precursor protein-like protein 2. Furthermore, we determined that amyloid precursor protein-like protein-2, but not amyloid precursor protein, is involved in mediating postendocytic delivery of PCSK9 to lysosomes and is therefore important for PCSK9 function. Based on our data, we propose a model for a lysosomal transport complex by which a soluble protein can target another protein for degradation from the luminal side of the membrane by bridging it to a lysosomally targeted transmembrane protein.

The proprotein convertase PCSK9 gene is the third locus implicated in familial hypercholesterolemia, emphasizing its role in cardiovascular diseases. Loss of function mutations and gene disruption of PCSK9 resulted in a higher clearance of plasma low density lipoprotein cholesterol, likely due to a reduced degradation of the liver low density lipoprotein receptor (LDLR). In this study, we show that two of the closest family members to LDLR are also PCSK9 targets. These include the very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) implicated in neuronal development and lipid metabolism. Our results show that wild type PCSK9 and more so its natural gain of function mutant D374Y can efficiently degrade the LDLR, VLDLR, and ApoER2 either following cellular co-expression or re-internalization of secreted human PCSK9. Such PCSK9-induced degradation does not require its catalytic activity. Membrane-bound PCSK9 chimeras enhanced the intracellular targeting of PCSK9 to late endosomes/lysosomes and resulted in a much more efficient degradation of the three receptors. We also demonstrate that the activity of PCSK9 and its binding affinity on VLDLR and ApoER2 does not depend on the presence of LDLR. Finally, in situ hybridization show close localization of PCSK9 mRNA expression to that of VLDLR in mouse postnatal day 1 cerebellum. Thus, this study demonstrates a more general effect of PCSK9 on the degradation of the LDLR family that emphasizes its major role in cholesterol and lipid homeostasis as well as brain development.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with familial autosomal dominant hypercholesterolemia and is a natural inhibitor of the LDL receptor (LDLr). PCSK9 is degraded by other proprotein convertases: PC5/6A and furin. Both PCSK9 and the LDLr are up-regulated by the hypocholesterolemic statins. Thus, inhibitors or repressors of PCSK9 should amplify their beneficial effects. In the present study, we showed that PPARalpha activation counteracts PCSK9 induction by statins by repressing PCSK9 promoter activity and by increasing PC5/6A and furin expression. Quantification of mRNA and protein levels showed that various fibrates decreased PCSK9 and increased PC5/6A and furin expression. Fenofibric acid (FA) reduced PCSK9 protein content in immortalized human hepatocytes (IHH) as well as its cellular secretion. FA suppressed PCSK9 induction by statins or by the liver X receptor agonist TO901317. PCSK9 repression is occurring at the promoter level. We showed that PC5/6A and furin fibrate-mediated up-regulation is PPARalpha-dependent. As a functional test, we observed that FA increased by 30% the effect of pravastatin on the LDLr activity in vitro. In conclusion, fibrates simultaneously decreased PCSK9 expression while increasing PC5/6A and furin expression, indicating a broad action of PPARalpha activation in proprotein convertase-mediated lipid homeostasis. Moreover, this study validates the functional relevance of a combined therapy associating PCSK9 repressors and statins.

The proprotein convertase subtilisin/kexin-type 9 (PCSK9), which promotes degradation of the hepatic low density lipoprotein receptor (LDLR), is now recognized as a major player in plasma cholesterol metabolism. Several gain-of-function mutations in PCSK9 cause hypercholesterolemia and premature atherosclerosis, and thus, inhibition of PCSK9-induced degradation of the LDLR may be used to treat this deadly disease. Herein, we discovered an endogenous PCSK9 binding partner by Far Western blotting, co-immunoprecipitation, and pull-down assays. Following two-dimensional gel electrophoresis and mass spectrometry analysis, we demonstrated that PCSK9 binds to a approximately 33-kDa protein identified as annexin A2 (AnxA2) but not to the closely related annexin A1. Furthermore, our functional LDLR assays and small hairpin RNA studies show that AnxA2 and the AnxA2.p11 complex could prevent PCSK9-directed LDLR degradation in HuH7, HepG2, and Chinese hamster ovary cells. Immunocytochemistry revealed that PCSK9 and AnxA2 co-localize at the cell surface, indicating a possible competition with the LDLR. Structure-function analyses demonstrated that the C-terminal cysteine-histidine-rich domain of PCSK9 interacts specifically with the N-terminal repeat R1 of AnxA2. Mutational analysis of this 70-amino acid-long repeat indicated that the RRTKK81 sequence of AnxA2 is implicated in this binding because its mutation to AATAA81 prevents its interaction with PCSK9. To our knowledge, this work constitutes the first to show that PCSK9 activity on LDLR can be regulated by an endogenous inhibitor. The identification of the minimal inhibitory sequence of AnxA2 should pave the way toward the development of PCSK9 inhibitory lead molecules for the treatment of hypercholesterolemia.

Furin-cleaved Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Is Active and Modulates Low Density Lipoprotein Receptor and Serum Cholesterol Levels

Hepatocyte Rap1a contributes to obesity- and statin-associated hyperglycemia.

Long-term Physiologically Regulated Expression of the Low-density Lipoprotein Receptor In Vivo Using Genomic DNA Mini-gene Constructs

More than Cholesterol Transporters: Lipoprotein Receptors in CNS Function and Neurodegeneration

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake. We have identified a fragment antigen-binding (Fab) 1D05 which binds PCSK9 with nanomolar affinity. The fully human antibody 1D05-IgG2 completely blocks the inhibitory effects of wild-type PCSK9 and two gain-of-function human PCSK9 mutants, S127R and D374Y. The crystal structure of 1D05-Fab bound to PCSK9 reveals that 1D05-Fab binds to an epitope on the PCSK9 catalytic domain which includes the entire LDLr EGF(A) binding site. Notably, the 1D05-Fab CDR-H3 and CDR-H2 loops structurally mimic the EGF(A) domain of LDLr. In a transgenic mouse model (CETP/LDLr-hemi), in which plasma lipid and PCSK9 profiles are comparable to those of humans, 1D05-IgG2 reduces plasma LDL cholesterol to 40% and raises hepatic LDLr protein levels approximately fivefold. Similarly, in healthy rhesus monkeys, 1D05-IgG2 effectively reduced LDL cholesterol 20%-50% for over 2 weeks, despite its relatively short terminal half-life (t(1/2) = 3.2 days). Importantly, the decrease in circulating LDL cholesterol corresponds closely to the reduction in free PCSK9 levels. Together these results clearly demonstrate that the LDL-lowering effect of the neutralizing anti-PCSK9 1D05-IgG2 antibody is mediated by reducing the amount of PCSK9 that can bind to the LDLr.

Tumor necrosis factor (TNF) family members are initially synthesized as type II transmembrane proteins, but some of these proteins are substrates for proteolytic enzymes that generate soluble cytokines with biological activity. TWEAK (TNF-like weak inducer of apoptosis), a member of the TNF family, is a multifunctional cytokine that acts via binding to a cell surface receptor named Fn14 (fibroblast growth factor-inducible 14). Studies conducted to date indicate that TWEAK-producing cells can co-express both membrane-anchored and soluble TWEAK isoforms, but there is little information on TWEAK proteolytic processing. Also, it is presently unclear whether membrane-anchored TWEAK, like soluble TWEAK, is biologically active. Here we show that full-length human TWEAK is processed intracellularly by the serine protease furin and identify TWEAK amino acid residues 90-93 as the predominant furin recognition site. In addition, we report that full-length, membrane-anchored TWEAK can bind the Fn14 receptor on neighboring cells and activate the NF-kappaB signaling pathway. Thus, TWEAK can act in a juxtacrine manner to initiate cellular responses, and this property may be important for TWEAK function during physiological wound repair and disease pathogenesis.

Processing of polypeptide precursors by proprotein convertases (PCs) such as furin typically occurs within the trans-Golgi network. Here, we show in a variety of cell types that the propeptide of ADAMTS9 is not excised intracellularly. Pulse-chase analysis in HEK293F cells indicated that the intact zymogen was secreted to the cell surface and was subsequently processed there before release into the medium. The processing occurred via a furin-dependent mechanism as shown using PC inhibitors, lack of processing in furin-deficient cells, and rescue by furin in these cells. Moreover, down-regulation of furin by small interference RNA reduced ADAMTS9 processing in HEK293F cells. PC5A could also process pro-ADAMTS9, but similarly to furin, processed forms were absent intracellularly. Cell-surface, furin-dependent processing of pro-ADAMTS9 creates a precedent for extracellular maturation of endogenously produced secreted proproteins. It also indicates the existence of a variety of mechanisms for processing of ADAMTS proteases.