Seven secretory mammalian kexin-like subtilases have been identified that cleave a variety of precursor proteins at monobasic and dibasic residues. The recently characterized pyrolysin-like subtilase SKI-1 cleaves proproteins at nonbasic residues. In this work we describe the properties of a proteinase K-like subtilase, neural apoptosis-regulated convertase 1 (NARC-1), representing the ninth member of the secretory subtilase family. Biosynthetic and microsequencing analyses of WT and mutant enzyme revealed that human and mouse pro-NARC-1 are autocatalytically and intramolecularly processed into NARC-1 at the (Y,I)VV(V,L)(L,M)2 motif, a site that is representative of its enzymic specificity. In vitro peptide processing studies and͞or Ala substitutions of the P1-P5 sites suggested that hydrophobic͞aliphatic residues are more critical at P1, P3, and P5 than at P2 or P4. NARC-1 expression is highest in neuroepithelioma SK-N-MCIXC, hepatic BRL-3A, and in colon carcinoma LoVo-C5 cell lines. In situ hybridization and Northern blot analyses of NARC-1 expression during development in the adult and after partial hepatectomy revealed that it is expressed in cells that have the capacity to proliferate and differentiate. These include hepatocytes, kidney mesenchymal cells, intestinal ileum, and colon epithelia as well as embryonic brain telencephalon neurons. Accordingly, transfection of NARC-1 in primary cultures of embryonic day 13.5 telencephalon cells led to enhanced recruitment of undifferentiated neural progenitor cells into the neuronal lineage, suggesting that NARC-1 is implicated in the differentiation of cortical neurons.cleavage specificity ͉ hypercholesterolemia ͉ neurogenesis ͉ hepatogenesis

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 proprotein convertase PCSK9 gene is the third locus implicated in familial hypercholesterolemia, emphasizing its role in cardiovascular diseases. Loss of function mutations and gene disruption of PCSK9 resulted in a higher clearance of plasma low density lipoprotein cholesterol, likely due to a reduced degradation of the liver low density lipoprotein receptor (LDLR). In this study, we show that two of the closest family members to LDLR are also PCSK9 targets. These include the very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) implicated in neuronal development and lipid metabolism. Our results show that wild type PCSK9 and more so its natural gain of function mutant D374Y can efficiently degrade the LDLR, VLDLR, and ApoER2 either following cellular co-expression or re-internalization of secreted human PCSK9. Such PCSK9-induced degradation does not require its catalytic activity. Membrane-bound PCSK9 chimeras enhanced the intracellular targeting of PCSK9 to late endosomes/lysosomes and resulted in a much more efficient degradation of the three receptors. We also demonstrate that the activity of PCSK9 and its binding affinity on VLDLR and ApoER2 does not depend on the presence of LDLR. Finally, in situ hybridization show close localization of PCSK9 mRNA expression to that of VLDLR in mouse postnatal day 1 cerebellum. Thus, this study demonstrates a more general effect of PCSK9 on the degradation of the LDLR family that emphasizes its major role in cholesterol and lipid homeostasis as well as brain development.Familial hypercholesterolemia is mainly characterized by elevated plasma LDL 2 cholesterol that is highly correlated with cardiovascular diseases (1). The main player in regulating the circulating cholesterol is the low density lipoprotein receptor (LDLR), which is expressed mostly in the liver. Recently, natural mutations in the proprotein convertase PCSK9 (2, 3) have been identified and associated with the third locus implicated in familial hypercholesterolemia (4 -6). The major function of PCSK9 seems to be an enhancement of the degradation of the LDLR (7, 8) in acidic subcellular compartments (3), likely endosomes/lysosomes (9, 10). This can occur either via an extracellular endocytotic route (11), or possibly by a direct cellular circuit not involving cell surface endocytosis of the LDLR (12). The gain of function PCSK9 mutations D374Y (13, 14) or D374H (15) have the highest impact on the development of hypercholesterolemia (16), likely through enhanced binding (17) and degradation of the LDLR (18, 19). The major binding site of LDLR to PCSK9 seems to reside within its first epidermal growth factor-like repeat namely EGF-A (20). Finally, it was recently suggested that the PCSK9-induced degradation of the cell surface LDLR does not require its proteolytic activity (21). One of the unanswered questions is the target specificity of PCSK9, and it is not known, nor obvious, whether other members of the LDLR family are also affected by PCSK9. This family consists of str...