Abstract
BackgroundProprotein convertase subtilisin/kexin type 9 (PCSK9) post-transcriptionally degrades the low density lipoprotein receptors (LDLR). However, it is unknown whether PCSK9 acts directly on the LDLR or if PCSK9 activates another protein that in turn causes degradation of the LDLR.ResultsWe have transiently transfected HepG2 cells with wild-type and mutant D374Y-PCSK9 plasmids to study the effect of the conditioned medium on the LDLR of untransfected HepG2 cells. The ability of the conditioned medium to reduce the internalization of LDL was abolished by removal of recombinant PCSK9 from the conditioned medium by affinity chromatography. Thus, PCSK9 is the only factor in the conditioned medium able to mediate degradation of the LDLR. Moreover, fractionation of the conditioned medium by gel filtration showed that the ability of the fractions to reduce the internalization of LDL, closely paralleled the amount of D374Y-PCSK9 in the fractions. Incubation of a secreted, truncated LDLR without cytoplasmic and transmembrane domains, as well as membrane fractions from HepG2 cells, with conditioned medium containing PCSK9, did not reduce the amount of LDLR as determined by western blot analysis. Thus, the LDLR is not degraded by PCSK9 on the cell surface. The LDLR of HepG2 cells incubated with conditioned medium was protected from PCSK9-mediated degradation by the addition of nocodazole or ammonium chloride, but was not protected when the conditioned medium was made hypertonic. These findings indicate that the intracellular degradation of the LDLR involves intracellular transport along microtubules, an acidic intracellular compartment and that it occurs even when endocytosis through clathrin-coated pits has been blocked.ConclusionDegradation of the LDLR by PCSK9 is not mediated by a secreted protein acted upon by PCSK9 extracellularly. Also the PCSK9-mediated degradation of the LDLR does not take place on the cell surface. Rather, the PCSK9-mediated degradation of the LDLR appears to take place intracellularly and occurs even when endocytosis through clathrin-coated pits is blocked by hypertonic medium.
Highlights
Proprotein convertase subtilisin/kexin type 9 (PCSK9) post-transcriptionally degrades the low density lipoprotein receptors (LDLR)
We have previously shown that conditioned medium from HepG2 cells transiently transfected with a PCSK9-containing plasmid, reduces the amount of cell surface LDLR and internalization of LDL of untransfected HepG2 cells [21]
Western blot analysis showed that all WT-PCSK9-His and D374Y-PCSK9His of the conditioned medium, had been removed from the conditioned medium by the affinity chromatography (Fig. 1)
Summary
Proprotein convertase subtilisin/kexin type 9 (PCSK9) post-transcriptionally degrades the low density lipoprotein receptors (LDLR). It is unknown whether PCSK9 acts directly on the LDLR or if PCSK9 activates another protein that in turn causes degradation of the LDLR. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a proprotein convertase of the subtilase family [1,2] It is synthesized as a soluble zymogen which undergoes autocatalytic intramolecular cleavage in the endoplasmic reticulum [1,2]. Even though the substrate for PCSK9 has not been identified, PCSK9 has been shown to play a role in cholesterol metabolism by regulating the number of cell surface low density lipoprotein receptors (LDLR) [6,7,8,9]. Because PCSK9 reduces the number of LDLR without reducing the amount of LDLR mRNA [6,7], degradation of LDLR by PCSK9 is apparently a post-transcriptional event
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