Abstract
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. The mechanisms of this action, however, remain to be defined. We show here that recombinant human PCSK9 expressed in HEK293 cells was readily secreted into the medium, with the prosegment associated with the C-terminal domain. Secreted PCSK9 mediated cell surface LDLR degradation in a concentration- and time-dependent manner when added to HEK293 cells. Accordingly, cellular LDL uptake was significantly reduced as well. When infused directly into C57B6 mice, purified human PCSK9 substantially reduced hepatic LDLR protein levels and resulted in increased plasma LDL cholesterol. When added to culture medium, fluorescently labeled PCSK9 was endocytosed and displayed endosomal-lysosomal intracellular localization in HepG2 cells, as was demonstrated by colocalization with DiI-LDL. PCSK9 endocytosis was mediated by LDLR as LDLR deficiency (hepatocytes from LDLR null mice), or RNA interference-mediated knockdown of LDLR markedly reduced PCSK9 endocytosis. In addition, RNA interference knockdown of the autosomal recessive hypercholesterolemia (ARH) gene product also significantly reduced PCSK9 endocytosis. Biochemical analysis revealed that the LDLR extracellular domain interacted directly with secreted PCSK9; thus, overexpression of the LDLR extracellular domain was able to attenuate the reduction of cell surface LDLR levels by secreted PCSK9. Together, these results reveal that secreted PCSK9 retains biological activity, is able to bind directly to the LDLR extracellular domain, and undergoes LDLR-ARH-mediated endocytosis, leading to accelerated intracellular degradation of the LDLR.
Highlights
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels
To address whether secreted PCSK9 reduces cellular LDLR protein levels, we transferred the medium from PCSK9-overexpressing cells to control HEK293 cells expressing endogenous LDLR
Concentration titration experiments indicated that ?10 mg/ml PCSK9 virtually completely abolished DiI-LDL uptake, with a calculated EC50 of 0.8 mg/ml in this experimental paradigm (Fig. 2E), consistent with the LDLR Western blot data. These results indicated that secreted PCSK9 was functional in degrading LDLR in cultured cells, even though its prosegment was tightly associated with the C-terminal domain
Summary
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. When infused directly into C57B6 mice, purified human PCSK9 substantially reduced hepatic LDLR protein levels and resulted in increased plasma LDL cholesterol. The molecular basis has yet to be determined, multiple mutations in the PCSK9 gene have been described to result in reduced cellular LDLR levels and significantly increased plasma LDL cholesterol [5,6,7,8]. Opposite to these “gainof-function” mutations, Cohen et al [9,10,11,12,13] found apparent loss-of-function mutations that are presumed to lead to increased cellular LDLR protein levels In humans carrying these mutations, plasma LDL cholesterol is reduced by 30–40% compared with controls. Plasma LDL cholesterol levels and provides evidence that loss of PCSK9 function in humans is not associated with apparent deleterious effects [12]
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