The gene encoding the proprotein convertase subtilisin/kexin type 9 (PCSK9) is linked to familial hypercholesterolemia, as are those of the low-density lipoprotein receptor (LDLR) and apolipoprotein B. PCSK9 enhances LDLR degradation, resulting in low-density lipoprotein accumulation in plasma. To analyze the role of hepatic PCSK9, total and hepatocyte-specific knockout mice were generated. They exhibit 42% and 27% less circulating cholesterol, respectively, showing that liver PCSK9 was responsible for two thirds of the phenotype. We also demonstrated that, in liver, PCSK9 is exclusively expressed in hepatocytes, representing the main source of circulating PCSK9. The data suggest that local but not circulating PCSK9 regulates cholesterol levels. Although transgenic mice overexpressing high levels of liver and circulating PCSK9 led to the almost complete disappearance of the hepatic LDLR, they did not recapitulate the plasma cholesterol levels observed in LDLRdeficient mice. Single LDLR or double LDLR/PCSK9 knockout mice exhibited similar cholesterol profiles, indicating that PCSK9 regulates cholesterol homeostasis exclusively through the LDLR. Finally, the regenerating liver of PCSK9-deficient mice exhibited necrotic lesions, which were prevented by a high-cholesterol diet. However, lipid accumulation in hepatocytes of these mice was markedly reduced under both chow and high-cholesterol diets, revealing that PCSK9 deficiency confers resistance to liver steatosis. Conclusion: Although PCSK9 is a target for controlling hypercholesterolemia, our data indicate that upon hepatic damage, patients lacking PCSK9 could be at risk. (HEPATOLOGY 2008;48:646-654.) P roprotein convertase subtilisin/kexin type 9 (PCSK9) 1 is the ninth member of the proprotein convertase family. 2 The first seven members, including furin, cleave protein precursors of hormones, growth factors, receptors, or surface glycoproteins at basic sites (after Arg or Lys residues). The eighth member, SKI-1 3 or S1P, 4 is known to cleave membrane-bound transcription factors such as the SREBPs 5 in their luminal domains, resulting in the release of their DNA-binding domain. Proprotein convertases can also inactivate secreted substrates, such as endothelial lipase 6 and PCSK9. 7 PCSK9 is synthesized as a precursor that undergoes autocatalytic cleavage of its N-terminal prosegment in the ER, 1 a step required for its exit from this compartment and its efficient secretion. Secreted PCSK9 remains associated with its prosegment. 1 Different from the other proprotein convertases, this serine protease has no known substrate other than itself. In addition, the tight association of the prosegment with the active site 8 raises the question of the existence of an in trans PCSK9 protease
The discovery of autosomal dominant hypercholesterolemic patients with mutations in the PCSK9 gene, encoding the proprotein convertase NARC-1, resulting in the missense mutations suggested a role in low density lipoprotein (LDL) metabolism. We show that the endoplasmic reticulum-localized proNARC-1 to NARC-1 zymogen conversion is Ca 2؉ -independent and that within the zymogen autocatalytic processing site SSVFAQ2SIP Val at P4 and Pro at P3 are critical. The S127R and D374Y mutations result in ϳ50 -60% and >98% decrease in zymogen processing, respectively. In contrast, the double [D374Y ؉ N157K], F216L, and R218S natural mutants resulted in normal zymogen processing. The cell surface LDL receptor (LDLR) levels are reduced by 35% in lymphoblasts of S127R patients. The LDLR levels are also reduced in stable HepG2 cells overexpressing NARC-1 or its natural mutant S127R, and this reduction is abrogated in the presence of 5 mM ammonium chloride, suggesting that overexpression of NARC-1 increases the turnover rate of the LDLR. Adenoviral expression of wild type human NARC-1 in mice resulted in a maximal ϳ9-fold increase in circulating LDL cholesterol, while in LDLR(؊/؊) mice a delayed ϳ2-fold increase in LDL cholesterol was observed. In conclusion, NARC-1 seems to affect both the level of LDLR and that of circulating apoB-containing lipoproteins in an LDLR-dependent and -independent fashion.The mammalian proprotein convertases constitute a family of 9 serine proteinases related to bacterial subtilisin. These include the 7 basic amino acid-specific convertases known as PC1/PC3, PC2, furin, PC4, PACE4, PC5/PC6, PC7/LPS (1, 2) and the two enzymes cleaving at nonbasic residues SKI-1/S1P (3, 4) and NARC-1/PCSK9 (5). These proteases are implicated in the limited proteolysis of precursors of secretory proteins that regulate a variety of cellular functions, including cellular growth, adhesion, differentiation, cell to cell communications, and endocrine/paracrine functions (6, 7). Published gene knockout analyses (reviewed in Ref. 8) revealed that only furin (9) and SKI-1/S1P (10) are embryonic lethal. So far, nothing is known about the phenotype consequences of NARC-1 1 knockout in mice. The cDNA of the enzyme NARC-1 was cloned during pharmaceutical screening of mRNAs up-regulated following induction of neural apoptosis by serum withdrawal, and the encoded protein was called "neural apoptosis regulated convertase 1" (NARC-1) (11). We characterized this enzyme, and we showed that it is highly expressed in liver and small intestine and that specific mutations in the prosegment of NARC-1 completely abrogated its autocatalytic processing (5). We further showed that overexpression of NARC-1 enhances neurogenesis of progenitor brain telencephalic cells. The sustained expression of NARC-1 in liver and small intestine and its transient expression in telencephalon, kidney, and cerebellum beg for the identification of its physiological substrates, which are still unknown.Human genetic point mutations resulting in pathology have been rep...
The risk of atherosclerosis is inversely associated with plasma levels of high-density lipoprotein cholesterol (HDL-C). However, HDL metabolism is incompletely understood, and there are few effective approaches to modulate HDL-C levels. Here we show that inhibition in the liver of the classical proprotein convertases (PCs), but not the atypical PCs S1P and PCSK9, decreases plasma HDL-C levels. This metabolic effect of hepatic PCs is critically dependent on expression of endothelial lipase (EL), an enzyme that directly hydrolyzes HDL phospholipids and promotes its catabolism. Hepatic PCs reduce EL function through direct inactivating cleavage of EL as well as through activating cleavage of angiopoietin-like protein 3 (ANGPTL3), an endogenous inhibitor of EL. Thus, inhibition of hepatic PCs results in increased EL activity, leading to reduced HDL-C as well as impaired reverse cholesterol transport. The hepatic PC-ANGPTL3-EL-HDL pathway is therefore a novel mechanism controlling HDL metabolism and cholesterol homeostasis.
Processing of LDL and HDL differed notably after initial binding via EL to the cell surface. More than 90% of the surface-bound125 I-LDL was destined for internalization and degradation, whereas about 70% of the surfacebound 125 I-HDL 3 was released back into the medium. These differences were significantly attenuated after HDL clustering was promoted using antibody against apolipoprotein A-I. At equal protein concentration of added lipoproteins the ratio of HDL 3 to VLDL bridging via EL was 0.092 compared with 0.174 via HL and 0.002 via LpL. In summary, EL mediates binding and uptake of plasma lipoproteins via a process that is independent of its enzymatic activity, requires cellular heparan sulfate proteoglycans, and is regulated by ligand clustering.
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