Catalytic Activity Is Not Required for Secreted PCSK9 to Reduce Low Density Lipoprotein Receptors in HepG2 Cells
ABSTRACT Proprotein convertase subtilisin/kexin type 9 (PCSK9), a member of the proteinase K subfamily of subtilases, promotes internalization
and degradation of low density lipoprotein receptors (LDLRs) after binding the receptor on the surface of hepatocytes. PCSK9
has autocatalytic activity that releases the prodomain at the N terminus of the protein. The prodomain remains tightly associated
with the catalytic domain as the complex transits the secretory pathway. It is not known whether enzymatic activity is required
for the LDLR-reducing effects of PCSK9. Here we expressed the prodomain together with a catalytically inactive protease domain
in cells and purified the protein from the medium. The ability of the catalytically inactive PCSK9 to bind and degrade LDLRs
when added to culture medium of human hepatoma HepG2 cells at physiological concentrations was similar to that seen using
wild-type protein. Similarly, a catalytic-dead version of a gain-of-function mutant, PCSK9(D374Y), showed no loss of activity
compared with a catalytically active counterpart; both proteins displayed ∼10-fold increased activity in degradation of cell
surface LDLRs compared with wild-type PCSK9. We conclude that the ability of PCSK9 to degrade LDLRs is independent of catalytic
activity and suggest that PCSK9 functions as a chaperone to prevent LDLR recycling and/or to target LDLRs for lysosomal degradation.
- SourceAvailable from: Nicola Ferri
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- "A, leads to the secretion of PCSK9 . is evidence further demonstrated the requirement of the autocatalytic processing and a correct association between the prodomain and the catalytic domain of PCSK9 for a proper folding and secretion of the protein. "
ABSTRACT: The identification of the HMG-CoA reductase inhibitors, statins, has represented a dramatic innovation of the pharmacological modulation of hypercholesterolemia and associated cardiovascular diseases. However, not all patients receiving statins achieve guideline-recommended low density lipoprotein (LDL) cholesterol goals, particularly those at high risk. There remains, therefore, an unmet medical need to develop additional well-tolerated and effective agents to lower LDL cholesterol levels. The discovery of proprotein convertase subtilisin/kexin type 9 (PCSK9), a secretory protein that posttranscriptionally regulates levels of low density lipoprotein receptor (LDLR) by inducing its degradation, has opened a new era of pharmacological modulation of cholesterol homeostasis. This paper summarizes the current knowledge of the basic molecular mechanism underlying the regulatory effect of LDLR expression by PCSK9 obtained from in vitro cell-cultured studies and the analysis of the crystal structure of PCSK9. It also describes the epidemiological and experimental evidences of the regulatory effect of PCSK9 on LDL cholesterol levels and cardiovascular diseases and summarizes the different pharmacological approaches under development for inhibiting PCSK9 expression, processing, and the interaction with LDLR.09/2012; 2012:927352. DOI:10.6064/2012/927352
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- "Since the discovery of its role in cholesterol metabolism and LDLR regulation and the realization that PCSK9 is a promising therapeutic target for LDL-C lowering, the domains and structures important for PCSK9 function have been under intense investigation. The protease activity has been shown to be necessary for self-cleavage, folding, and secretion of the mature PCSK9 protein, but it is not involved in LDLR degradation (Li et al., 2007; McNutt et al., 2007). "
ABSTRACT: Proprotein convertase substilisin/kexin type 9 (PCSK9) promotes the degradation of low-density lipoprotein (LDL) receptor (LDLR) and thereby increases serum LDL-cholesterol (LDL-C). We have developed a humanized monoclonal antibody that recognizes the LDLR binding domain of PCSK9. This antibody, J16, and its precursor mouse antibody, J10, potently inhibit PCSK9 binding to the LDLR extracellular domain and PCSK9-mediated down-regulation of LDLR in vitro. In vivo, J10 effectively reduces serum cholesterol in C57BL/6 mice fed normal chow. J16 reduces LDL-C in healthy and diet-induced hypercholesterolemic cynomologous monkeys, but does not significantly affect high-density lipoprotein-cholesterol. Furthermore, J16 greatly lowered LDL-C in hypercholesterolemic monkeys treated with the HMG-CoA reductase inhibitor simvastatin. Our data demonstrate that anti-PCSK9 antibody is a promising LDL-C-lowering agent that is both efficacious and potentially additive to current therapies.Journal of Pharmacology and Experimental Therapeutics 02/2012; 340(2):228-36. DOI:10.1124/jpet.111.187419 · 3.86 Impact Factor
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- "Downregulation of the receptor is not associated with significant changes of mRNA levels, suggesting a post-transcriptional regulatory mechanism, and is elicited also by the catalytic inactive PCSK9 mutant, indicating that the LDLR downregulation is not a result of the proteolytic activity of PCSK9. These results are in agreement to previous studies conducted with HepG2 cells  . Moreover, the incubation with the acidotropic agent ammonium chloride, significantly increased the LDLR levels in J774 cells (data not shown) as previously reported by Breslow et al. , indicating that the degradation of the LDLR by PCSK9 may occur in a pH sensitive compartment like the lysosome. "
ABSTRACT: Proprotein convertase subtilisin kexin type 9 (PCSK9) is an important regulator of hepatic low-density lipoprotein (LDL)-cholesterol levels. Although PCSK9 is mainly of hepatic origin, extra-hepatic tissues significantly contribute to PCSK9 production and, potentially, local regulation of LDL receptor expression. In the present study we show that, among vascular cells, PCSK9 is expressed in smooth muscle cells (SMCs) but not in endothelial cells, macrophages and monocytes. PCSK9 was also detectable in human atherosclerotic plaques. Conditioned media from SMCs significantly reduced LDLR expression in human macrophage and in the macrophage cell line J774. Co-culture experiments also demonstrated the influence of SMCs on LDLR expression in J774. PCSK9 released from SMCs directly regulated LDLR expression in macrophages as demonstrated by retroviral overexpression or knockdown of PCSK9 with small interfering RNA and by using recombinant PCSK9. Moreover, the proteolytic activity of PCSK9 was not required for LDLR downregulation since cultured media containing either the catalytic inactive PCSK9 or PCSK9 WT had a similar effect on LDLR in J774. Finally, conditioned media from SMCs affected β-VLDL cholesterol uptake and PCSK9 expression reduced both LDLR and LDL uptake in J774. Taken together our data indicate that PCSK9 secreted by human SMCs is functionally active and capable of reducing LDLR expression in macrophages. A possible direct role for this protein in foam cell formation and atherogenesis is suggested.Atherosclerosis 11/2011; 220(2):381-6. DOI:10.1016/j.atherosclerosis.2011.11.026 · 3.97 Impact Factor