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Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and inlivers of parabiotic mice

Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
Journal of Clinical Investigation (Impact Factor: 13.77). 12/2006; 116(11):2995-3005. DOI: 10.1172/JCI29383
Source: PubMed

ABSTRACT 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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Available from: Robert E Hammer, Jul 31, 2015
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    • "sin - like catalytic domain and a C - terminal cysteine / histidine rich domain ( CTD ) ( Figure 1A & B ) ( Henrich et al . , 2005 ) . PCSK9 undergoes autocatalytic cleavage , but the 14 kDa prodomain remains non - covalently attached to the catalytic domain and renders the protease inactive ( Benjannet et al . , 2004 ; Cunningham et al . , 2007 ; Lagace et al . , 2006 ; Seidah et al . , 2003 ) . It is believed that the prodomain acts as a chaperone and assists in folding of the protein ( Kwon et al . , 2008 ) whereas the autocatalytic processing is crucial for the secretion of PCSK9 ( Seidah et al . , 2003 ) . The LDL - R is a multidomain protein comprising an extracellular domain with an N - termina"
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    ABSTRACT: Disrupting the binding interaction between proprotein convertase (PCSK9) and the epidermal growth factor-like domain A (EGF-A domain) in the low-density lipoprotein receptor (LDL-R) is a promising strategy to promote LDL-R recycling and thereby lower circulating cholesterol levels. In this study, truncated 26 amino acid EGF-A analogs were designed and synthesized, and their structures were analyzed in solution and in complex with PCSK9. The most potent peptide had an increased binding affinity for PCSK9 (KD = 0.6 μM) compared with wild-type EGF-A (KD = 1.2 μM), and the ability to increase LDL-R recycling in the presence of PCSK9 in a cell-based assay.
    Chemistry & biology 01/2014; DOI:10.1016/j.chembiol.2013.11.014 · 6.59 Impact Factor
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    • "Functional studies showed that PCSK9 does not degrade LDLR but binds to it on the surface of hepatocytes. In the presence of the endocytic adaptor protein ARH (autosomal recessive hypercholesterolemia ) that binds LDLR in the cytosol, the whole PCSK9-bound receptor complex is internalized and undergoes lysosomal degradation [17] [18]. "
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    ABSTRACT: PCSK9 (Proprotein Convertase Subtilisin Kexin type 9) is associated with autosomal dominant hypercholesterolemia (ADH), a state of elevated levels of LDL cholesterol. ADH can result in severe implications like stroke and coronary heart disease. The inhibition of PCSK9 function by therapeutic antibodies that block interaction of PCSK9 with the EGF-A domain of LDL receptor (LDLR) was shown to successfully lower LDL cholesterol levels in clinical studies. Here we present data on the identification, structural and biophysical characterization and in vitro and in vivo pharmacology of a PCSK9 antibody (mAb1). The X-ray structure shows that mAb1 binds the module 1 of the C-terminal domain (CTD) of PCSK9. It blocks access to an area bearing several naturally occurring gain and loss of function mutations. Although the antibody does not inhibit binding of PCSK9 to EGF-A, it partially reverses PCSK9 induced reduction of the LDLR and LDL cholesterol uptake in a cellular assay. mAb1 is also effective in lowering serum levels of LDL cholesterol in cynomolgus monkeys in vivo. Complete loss of PCSK9 is associated with insufficient liver regeneration and increased risk of hepatitis C infections. Blocking of the CTD is sufficient to partially inhibit PCSK9 function. Antibodies binding the CTD of PCSK9 may thus be advantageous in patients that do not tolerate complete inhibition of PCSK9.
    Journal of Molecular Biology 11/2013; 426(4). DOI:10.1016/j.jmb.2013.11.011 · 4.33 Impact Factor
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    • "Population studies have shown that PCSK9 gain of function variants associate with high LDL-C levels and autosomal dominant hypercholesterolemia [5] [6], whereas loss of function variants associate with low LDL- C levels [7] [8]. In addition, there is a positive correlation between serum PCSK9 and LDL-C levels [9] [10] [11] [12], which is not surprising given that secreted PCSK9, acting as an escort protein, binds to low density lipoprotein receptors (LDLR) on the cell surface of the liver, thereafter interfering with its natural course of recycling [13] [14] and routing the LDLR to the lysosome for degradation [15] [16], resulting in accumulation of LDL particles in circulation. "
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    ABSTRACT: Background. Serum lipids including total cholesterol (TC), triglycerides (TG), and low density lipoprotein cholesterol (LDL-C) are increased in pregnancy. Serum proprotein convertase subtilisin kexin 9 (PCSK9) is a significant player in lipoprotein metabolism. Circulating PCSK9 downregulates the LDL receptor on the surface of the liver, inhibiting clearance of LDL-C. Therefore, our study assessed serum PCSK9 concentrations at parturition (Maternal) compared to a nonpregnant (Control) cohort, as well as between mother and newborn (Maternal and Newborn). Methods. Blood was collected from women at parturition and from umbilical cords. Serum lipids and PCSK9 were measured and data were analysed for significance by Mann-Whitney U test at P < 0.05 and presented as median levels. Spearman's correlations were made at a 95% confidence interval. Results. Serum PCSK9 was significantly higher in Maternal versus Control cohorts (493.1 versus 289.7 ng/mL; P < 0.001, resp.), while the Newborn cohort was significantly lower than Maternal (278.2 versus 493.1 ng/mL; P < 0.0001, resp.). PCSK9 was significantly correlated with TC and HDL-C in Maternal and with TC, LDL-C, and HDL-C in Newborn cohorts. Conclusions. Our study provides the first quantitative report on PCSK9 in pregnancy (at parturition) and in umbilical cord blood. Further research will determine how these changes may affect lipoprotein levels during this physiological state.
    06/2013; 2013:341632. DOI:10.1155/2013/341632
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