Autosomal dominant hypercholesterolemia (ADH; OMIM144400), a risk factor for coronary heart disease, is characterized by an increase in low-density lipoprotein cholesterol levels that is associated with mutations in the genes LDLR (encoding low-density lipoprotein receptor) or APOB (encoding apolipoprotein B). We mapped a third locus associated with ADH, HCHOLA3 at 1p32, and now report two mutations in the gene PCSK9 (encoding proprotein convertase subtilisin/kexin type 9) that cause ADH. PCSK9 encodes NARC-1 (neural apoptosis regulated convertase), a newly identified human subtilase that is highly expressed in the liver and contributes to cholesterol homeostasis.

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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

A recombinant vaccinia virus vector was used to coexpress the two candidate mouse prohormone convertases, PC1 and PC2, together with mouse proopiomelanocortin (POMC) in the constitutively secreting cell line BSC-40 and in the endocrine tissue-derived cell lines PC12 and AtT-20, which exhibit regulated secretion. Monitoring of POMC processing demonstrated the distinct cleavage specificities of PCi and PC2, since in the cell lines analyzed (i) PCi cleaves POMC into corticotropin and fB-lipotropin, (ii) PC2 cleaves POMC into (3-endorphin, an N-terminally extended corticotropin containing the joining peptide, and either aMSH or desacetyl-aMSH, and (iii) PC2 cleaves POMC at the five pairs of basic residues analyzed, whereas PC1 cleaves two of them preferentially, suggesting that PC2 has a broader spectrum of activity than PC1. These data are consistent with our hypothesis on the physiological role of PCi and PC2 as distinct proprotein convertases acting alone or together to produce a set of tissuespecific maturation products in the brain and in peripheral tissues.Limited proteolysis of inactive precursors at pairs of basic residues or, less frequently, at single basic amino acids (1, 2) is a general mechanism by which the cell produces a variety of active proteins and peptides. Until recently, the molecular nature of the processing enzyme(s) was not identified except for the yeast pro-a mating factor precursor, where a subtilisin-like serine proteinase called KEX2 was proven to be the physiological convertase (3). In the last year, different groups obtained the complete cDNA structures of three distinct candidate mammalian convertases, furin (4), PC1 (5, 6), and PC2 (5, 7). The putative convertase function of these proteins was largely based on sequence homology with KEX2 (8, 9) and on their tissue and cellular distribution, especially for PC1 and PC2 (5, 6). Here we show that the expression of PC1 or PC2 by vaccinia virus (VV) recombinants results in the production of distinct proteolytic enzymes in three cell lines: the mouse pituitary corticotroph line AtT-20 and two that do not express these enzymes endogenously, the African green monkey kidney epithelial cell line BSC-40 and the rat pheochromocytoma cell line PC12. These proteinases cleave proopiomelanocortin (POMC) into the same set of distinct peptides known to be produced in vivo in the hypothalamus, adenohypophysis (AP), and pars intermedia of the pituitary, including adrenocorticotropin (ACTH), p-lipotropin (,8LPH), ,f-endorphin (f3End), and a-melanotropin (aMSH) or desacetyl-aMSH.MATERIALS AND METHODS Ws. Purified recombinant VVs using the full-length mouse (i) PC1 and mPC2 cDNA inserts (5, 6) (VV:mPC1 and VV:mPC2) were prepared as described (10). VV:POMC (mouse) was a gift of G. Thomas (Vollum Institute, Portland, OR).Northern Blot Analysis. Total RNA was prepared as described (11). The [a-32P]UTP-labeled cRNA probes were generated from cDNA sequences of mPC1 (6) and mPC2 (5) inserted in plasmid Rc/CMV vector (Invitrogen, San Diego) and linearized...

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...

Using reverse transcriptase-PCR and degenerate oligonucleotides derived from the active-site residues of subtilisin͞kexin-like serine proteinases, we have identified a highly conserved and phylogenetically ancestral human, rat, and mouse type I membrane-bound proteinase called subtilisin͞kexin-isozyme-1 (SKI-1). Computer databank searches reveal that human SKI-1 was cloned previously but with no identified function. In situ hybridization demonstrates that SKI-1 mRNA is present in most tissues and cells. Cleavage specificity studies show that SKI-1 generates a 28-kDa product from the 32-kDa brain-derived neurotrophic factor precursor, cleaving at an RGLT2SL bond. In the endoplasmic reticulum of either LoVo or HK293 cells, proSKI-1 is processed into two membrane-bound forms of SKI-1 (120 and 106 kDa) differing by the nature of their N-glycosylation. Late along the secretory pathway some of the membrane-bound enzyme is shed into the medium as a 98-kDa form. Immunocytochemical analysis of stably transfected HK293 cells shows that SKI-1 is present in the Golgi apparatus and within small punctate structures reminiscent of endosomes. In vitro studies suggest that SKI-1 is a Ca 2؉ -dependent serine proteinase exhibiting a wide pH optimum for cleavage of pro-brainderived neurotrophic factor.

Using a 796-basepair cDNA fragment obtained from a mouse pituitary library we have screened two mouse insulinoma libraries and isolated a full-length cDNA clone (2516 basepairs; 753 amino acids), designated mPC1. The cDNA sequence of mPC1 codes for a protein containing 753 amino acids and three potential N-glycosylation sites. This cDNA encodes a putative novel subtilisin-like proteinase, exhibiting within its presumed catalytic domain 64%, 55%, and 47% amino acid sequence identity to the recently characterized candidate prohormone convertases human Furin, mouse PC2, and yeast Kex2 gene products, respectively. An identical sequence to mPC1 was derived from a cDNA library of mouse corticotroph AtT-20 tumor cells. An ArgGlyAsp tripeptide identical to the recognition sequence of integrins was observed in the structures of the mammalian PC1, PC2, and Furin. In situ hybridization results demonstrated a distinct localization of the mPC1 and mPC2 transcripts in pituitary and brain. Thus, whereas both mPC1 and mPC2 are found in the intermediate lobe of the pituitary, only mPC1 is easily detected in the anterior lobe. In extrahypothalamic regions of the brain, including cortex, hippocampus, thalamus, and spinal cord, mPC2 transcripts predominate over mPC1. Both mRNAs are found in only a fraction of hypothalamic neurons, with greater abundance of mPC1 over mPC2 in the supraoptic nucleus. The genes coding for mPC1 and mPC2 map to the murine chromosomes 13 (band 13c) and 2 (2F3-2H2 region), respectively.

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...