Lagace, T.A. et al. Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice. J. Clin. Invest. 116, 2995-3005

Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
Journal of Clinical Investigation (Impact Factor: 13.22). 12/2006; 116(11):2995-3005. DOI: 10.1172/JCI29383
Source: PubMed


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 cell-surface 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.

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    • "PCSK9 protein is expressed primarily in the liver and secreted into circulation (Seidah et al., 2003), where it binds LDLR on the liver cell surface. The resulting LDLR:PCSK9 complex enters the cell and is transported to the lysosome compartment and degraded (Lagace et al., 2006). "
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    ABSTRACT: Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as an attractive therapeutic target for cardiovascular disease. Monoclonal antibodies (mAbs) that bind PCSK9 and prevent PCSK9:LDLR complex formation reduce serum LDL-C in vivo. PCSK9-mediated lysosomal degradation of bound mAb, however, dramatically reduces mAb exposure and limits duration of effect. Administration of high affinity mAb1:PCSK9 complex (1:2) to mice resulted in significantly lower mAb1 exposure compared to mAb1 dosed alone in normal mice or in PCSK9 knock-out mice lacking antigen. To identify mAb binding characteristics that minimize lysosomal disposition, the pharmacokinetic behavior of four mAbs representing a diverse range of PCSK9 binding affinities at neutral (serum) and acidic (endosomal) pH was evaluated in cynomolgus monkeys. Results revealed an inverse correlation between affinity and both mAb exposure and duration of LDL-C lowering. High affinity mAb1 exhibited the lowest exposure and shortest duration of action (6 days), while mAb2 displayed prolonged exposure and LDL-C reduction (51 days) as a consequence of lower affinity and pH-sensitive PCSK9 binding. mAbs with shorter endosomal PCSK9:mAb complex dissociation half-lives (<20 seconds) produced optimal exposure-response profiles. Interestingly, incorporation of previously reported Fc-region amino acid substitutions or novel "loop-insertion" peptides that enhance in vitro FcRn binding led to only modest PK improvements for mAbs with pH-dependent PCSK9 binding, with only limited augmentation of PD activity relative to native mAbs. A pivotal role for PCSK9 in mAb clearance was demonstrated, more broadly suggesting that therapeutic mAb binding characteristics require optimization based on target pharmacology. The American Society for Pharmacology and Experimental Therapeutics.
    No preview · Article · Feb 2015 · Journal of Pharmacology and Experimental Therapeutics
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    • "First, missense mutations in PCSK9, later determined to confer a gain-of-function, were found to cause a rare form of autosomal dominant hypercholesterolemia [2]; second, loss-of-function PCSK9 mutations identified at relatively high frequencies (2–4%) in certain ethnic populations were associated with lowered plasma LDL-C levels and significant protection from CHD [3,4]. Mechanistic studies determined that PCSK9 binds to the LDL receptor (LDLR) and promotes its lysosomal degradation in cells [5–7]. The molecular mapping of the PCSK9-LDLR binding interface aided the development of therapeutic anti-PCSK9 monoclonal antibodies (mAbs) that effectively block this interaction at the cell surface [8,9]. "
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    ABSTRACT: Purpose of review Proprotein convertase subtilisin/kexin type-9 (PCSK9) binds to LDL receptor (LDLR) and targets it for lysosomal degradation in cells. Decreased hepatic clearance of plasma LDL-cholesterol is the primary gauge of PCSK9 activity in humans; however, PCSK9's evolutionary role may extend to other lipoprotein classes and processes. This review highlights studies that are providing novel insights into physiological regulation of PCSK9 transcription and plasma PCSK9 activity. Recent findings Recent studies indicate that circulating PCSK9 binds to apolipoprotein B100 on LDL particles, which in turn inhibits PCSK9's ability to bind to cell surface LDLRs. Negative feedback of secreted PCSK9 activity by LDL could serve to increase plasma excursion of triglyceride-rich lipoproteins and monitor lipoprotein remodeling. Recent findings have identified hepatocyte nuclear factor-1α as a key transcriptional regulator that cooperates with sterol regulatory element-binding protein-2 to control PCSK9 expression in hepatocytes in response to nutritional and hormonal inputs, as well as acute inflammation. Summary PCSK9 is an established target for cholesterol-lowering therapies. Further study of PCSK9 regulatory mechanisms may identify additional control points for pharmacological inhibition of PCSK9-mediated LDLR degradation. PCSK9 function could reflect ancient roles in the fasting-feeding cycle and in linking lipoprotein metabolism with innate immunity.
    Full-text · Article · Aug 2014 · Current Opinion in Lipidology
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    • "Human PCSK9 Transgenic Mouse Models. Human PCSK9 overexpresser transgenic mice originally described by Lagace et al. (2006) were obtained from Dr. Jay Horton (University of Texas South Western Medical School, Dallas, TX) and "
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    ABSTRACT: Proprotein convertase subtilisin kexin-9 (PCSK9) is an important pharmacological target for decreasing low density lipoprotein (LDL) in cardiovascular disease, though seemingly inaccessible to small molecule approaches. Compared with therapeutic IgG antibodies currently in development, targeting circulating PCSK9 with smaller molecular scaffolds could offer different profiles and reduced dose burdens. This inspired genesis of PCSK9-binding Adnectins, a protein family derived from human fibronectin-10th-type-III-domain and engineered for high affinity target binding. BMS-962476, an ~11 kilodalton polypeptide conjugated to polyethylene glycol to enhance pharmacokinetics, binds with sub-nanomolar affinity to human. The X-ray co-crystal structure of PCSK9 with a progenitor Adnectin shows ~910 Å2 of PCSK9 surface covered next to the LDLR binding site, largely by residues of a single loop of the Adnectin. In hypercholesterolemic, overexpressing human PCSK9 transgenic mice, BMS-962476 rapidly lowered cholesterol and free PCSK9 levels. In normal-expressing genomic transgenic mice, BMS-962476 potently reduced free human PCSK9 (ED50 ~0.01 mg/kg) followed by ~2-fold increases in total PCSK9 before return to baseline. Treatment of cynomolgus monkeys with BMS-962476 rapidly suppressed free PCSK9 >99% and LDL-cholesterol ~55% with subsequent 6-fold increase in total PCSK9, suggesting reduced clearance of circulating complex. Liver sterol response genes were consequently down-regulated, following which LDL and total PCSK9 returned to baseline. These studies highlight the rapid dynamics of PCSK9 control over LDL and liver cholesterol metabolism and characterize BMS-962476 as a potent and efficacious PCSK9 inhibitor.
    Full-text · Article · Jun 2014 · Journal of Pharmacology and Experimental Therapeutics
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