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PubMed2007

Regulation of pancreatic endocrine cell differentiation by sulphated proteoglycans.

AIMS/HYPOTHESIS: Epithelium-mesenchyme interactions play a major role in pancreas development. Recently, we demonstrated that embryonic pancreatic mesenchyme enhanced progenitor cell proliferation but inhibited endocrine cell differentiation. Here, we investigated the role played by sulphated proteoglycans, which are known to be essential to embryonic development, in this inhibitory effect. MATERIALS AND METHODS: We first determined the expression of the genes encoding glypicans, syndecans and the main glycosaminoglycan chain-modifying enzymes in immature embryonic day (E) 13.5 and more differentiated E17.5 rat pancreases. Next, using an in vitro model of pancreas development, we blocked the action of endogenous sulphated proteoglycans by treating embryonic pancreases in culture with chlorate, an inhibitor of proteoglycan sulphation, and examined the effects on pancreatic endocrine cell differentiation. RESULTS: We first showed that expression of the genes encoding glypicans 1, 2, 3 and 5 and heparan sulphate 2-sulfotransferase decreased between E13.5 and E17.5. We next found that alteration of proteoglycan action by chlorate blocked the inhibitory effect of the mesenchyme on endocrine differentiation. Chlorate-treated pancreases exhibited a dramatic increase in beta cell number in a dose-dependent manner (169-and 375-fold increase with 30 mmol/l and 40 mmol/l chlorate, respectively) and in alpha cell development. Insulin-positive cells that developed in the presence of chlorate exhibited a phenotype of mature cells with regard to the expression of the following genes: pancreatic and duodenal homeobox gene 1 (Pdx1), proprotein convertase subtilisin/kexin type 1 (Pcsk1; previously known as pro-hormone convertase 1/3), proprotein convertase subtilisin/kexin type 2 (Pcsk2; previously known as pro-hormone convertase 2) and solute carrier family 2 (facilitated glucose transporter), member 2 (Slc2a1; previously known as glucose transporter 2). Finally, we showed that chlorate activated endocrine cell development by inducing neurogenin 3 (Neurog3) expression in early endocrine progenitor cells. CONCLUSIONS/INTERPRETATION: We demonstrated that sulphated proteoglycans control pancreatic endocrine cell differentiation. Understanding the mechanism by which sulphated proteoglycans affect beta cell development could be useful in the generation of beta cells from embryonic stem cells.

Animals↗

PubMed1999

Rat Chromosome 2: assignment of the genes encoding cyclin B1, interleukin 6 signal transducer, and proprotein convertase 1 to the Mcs1-containing region and identification of new microsatellite markers.

The rat Chromosome (Chr) 2 harbors several genes controlling tumor growth or development, blood pressure, and non-insulin-dependent diabetes mellitus. We report that the region (2q1) containing the mammary susceptibility cancer gene Mcs1 also harbors the genes encoding cyclin B1, interleukin 6 signal transducer (gp130), and proprotein convertase 1. We also generated 13 new anonymous microsatellite markers from Chr 2-sorted DNA. These markers, as well as a microsatellite marker in the cyclin B1 gene, were genetically mapped in combination with known markers. A cyclin B1-related gene was also cytogenetically assigned to rat Chr 11q22-q23.

Animals↗

PubMed2002

Analysis of ten candidate genes in autism by association and linkage.

We studied the possible involvement of ten candidate genes in autism: proenkephalin, prodynorphin, and proprotein convertase subtilisin/kexin type 2 (opioid metabolism); tyrosine hydroxylase, dopamine receptors D2 and D5, monoamine oxidases A and B (monoaminergic system); brain-derived neurotrophic factor, and neural cell adhesion molecule (involved in neurodevelopment). Thirty-eight families with two affected siblings and one family with two affected half-siblings, recruited by the Paris Autism Research International Sibpair Study (PARIS), were tested using the transmission disequilibrium test and two-point affected sib-pair linkage analysis. We found no evidence for association or linkage with intragenic or linked markers. Our family sample has good power for detecting a linkage disequilibrium of 0.80. Thus, these genes are unlikely to play a major role in the families studied, but further studies in a much larger sample would be needed to highlight weaker genetic effects.

Alleles↗

PubMed2003

Synthetic peptides derived from the prosegments of proprotein convertase 1/3 and furin are potent inhibitors of both enzymes.

Proprotein convertases (PCs) are Ca(2+)-dependent serine proteases of the subtilisin/kexin family which are known specifically to cleave propeptide and proprotein substrates at the C-terminal of R-X-(K/R)-R/ to generate the relevant biologically active peptides. PCs are initially synthesized as enzymically inactive proenzyme forms where the prosegments play an important inhibitory role to the respective enzymes. Here we investigated whether synthetic peptides derived from the pro-region could also represent specific and potent inhibitors. Based upon sequence alignment, secondary structure analysis and hydrophilicity plot, a number of peptides ranging from 8 to 33 residues were selected. These included segments encompassing residues 55-62, 50-62, 39-62, 50-83, 55-83, 64-83 and 74-83 in the pro-mouse PC1/3 sequence and residues 54-62, 48-62 and 39-62 of the pro-human furin sequence. All peptides were prepared by solid-phase FastMoc chemistry, purified by reversed-phase HPLC and characterized by MS and amino acid analysis. These peptides were tested in vitro for inhibitory activity towards recombinant mouse PC1/3 and human furin. Progress-curve and end-time kinetic analysis demonstrated that a number of these peptides, particularly those containing both the primary and the secondary processing sites, displayed strong inhibition of both enzymes with inhibition constants (K (i)) in the high nanomolar range. Unlike the whole propeptide, these small synthetic peptide inhibitors exhibited either true competitive or mixed competitive inhibition, depending on the sequence. Our data revealed further the critical role of the last two basic amino acid residues (e.g. Lys(82)-Arg(83) for the mouse PC1/3 sequence) of the prodomain in imparting a strong anti-convertase activity. The study also establishes the inhibitory potential of certain regions contained within the prosegment of the two convertases.

Amino Acid Sequence↗

PubMed2004

Pathogenic factors in vascular dementia and Alzheimer's disease. Multiple actions of heparin that probably are beneficial.

The following areas are discussed in this review: atherogenesis; cerebrovascular factors; hypoperfusion; beta-amyloid production; beta-amyloid fibril formation; beta-sheets; metal cations; reactive oxygen species/free radicals; chronic inflammatory factors; endogenous plasma heparin; lipoprotein lipase; polyamines; protein kinase C; casein kinases; phospholipase A2; serine proteases; myeloperoxidase; cyclooxygenase 2; cysteine proteases; caspases; proprotein convertases; aspartic proteases; cyclin proteinases; thrombin; tau hyperphosphorylation; advanced glycosylation end products; activator protein 1; calcium; apolipoprotein E epsilon4; histamine; blood-brain barrier; glutamate; transglutaminase; insulin-like growth factor 1.

Aged↗

PubMed2001

Biosynthetic processing of the pro-alpha 1(V)2pro-alpha 2(V) collagen heterotrimer by bone morphogenetic protein-1 and furin-like proprotein convertases.

The low abundance fibrillar collagen type V is incorporated into and regulates the diameters of type I collagen fibrils. Bone morphogenetic protein-1 (BMP-1) is a metalloprotease that plays key roles in regulating formation of vertebrate extracellular matrix; it cleaves the C-propeptides of the major fibrillar procollagens I-III and processes precursors to produce the mature forms of the cross-linking enzyme prolysyl oxidase, the proteoglycan biglycan, and the basement membrane protein laminin 5. Here we have successfully produced recombinant pro-alpha1(V)(2)pro-alpha2(V) heterotrimers, and we have used these to characterize biosynthetic processing of the most prevalent in vivo form of type V procollagen. In addition, we have compared the processing of endogenous pro-alpha1(V) chains by wild type mouse embryo fibroblasts and by fibroblasts derived from embryos doubly homozygous null for the Bmp-1 gene and for a gene encoding the closely related metalloprotease mammalian Tolloid-like 1. Together, results presented herein indicate that within pro-alpha1(V)(2)pro-alpha2(V) heterotrimers, pro-alpha1(V) N-propeptides and pro-alpha2(V) C-propeptides are processed by BMP-1-like enzymes, and pro-alpha1(V) C-propeptides are processed by furin-like proprotein convertases in vivo.

Amino Acid Sequence↗

PubMed2006

Recombinant expression, purification, and kinetic and inhibitor characterisation of human site-1-protease.

Human site-1-protease (S1P, MEROPS S08.8063), also widely known as subtilisin/kexin isozyme 1 (SKI-1), is a membrane bound subtilisin-related serine protease, that belongs to a group of nine mammalian proprotein convertases. Among these proteases, S1P displays unique substrate specificity, by showing preferred cleavage after non-basic amino acids. S1P plays a key role in a proteolytic pathway that controls the cholesterol content of membranes, cells and blood. S1P also participates in the activation of viral coat glycoproteins of the lassa virus, the lympocytic choriomeningitis virus and the crimean congo hemorrhagic fever virus. We expressed recombinant human S1P using the baculovirus expression vector system and characterized the highly purified enzyme. Featuring a new chromogenic substrate (Acetyl-Arg-Arg-Leu-Leu-p-nitroanilide) we show that the enzymatic activity of S1P is not calcium dependent, but can be modulated by a variety of mono- and divalent cations. S1P displayed pronounced positive cooperativity with a substrate derived from the viral coat glycoprotein of the lassa virus. The screening of a limited number of protease inhibitors showed that S1P was not inhibited by specific inhibitors of other proprotein convertases or by Pefabloc SC (4-(2-aminoethyl) benzene sulphonyl fluoride, AEBSF). We found 3,4-dichloroisocoumarin (DCI) to be a potent slow binding inhibitor of human S1P, with a K(iapp) = 6.8 microM, thus representing a new small molecule inhibitor of S1P. These findings show that S1P differs significantly from other proprotein convertases with respect to kinetics, co-factor requirement and inhibition.

Amino Acid Sequence↗

PubMedDate not supplied

Proteomics as a tool for discovery: proteins implicated in Alzheimer's disease are highly expressed in normal pancreatic islets.

A proteomic analysis of islets was undertaken to determine the protein constituents of normal adult mouse islets. Unexpectedly, we identified several islet proteins that are associated with the pathogenesis of Alzheimer's disease. Some of these proteins had chaperone activity that is integral to proper protein folding. This group includes GRP78, valosin-containing protein, calreticulin, protein disulfide isomerase, DnaK, HSP70, HSP60, and TCP-1. Additionally, neuronal proteins key to coordinated neuronal guidance and survival were also identified in islets. This group includes proprotein convertase subtilisin, collapsin response mediator protein 2, ubiquinol-cytochrome c reductase core protein, L-3-hydroxyacyl-Coenzyme A dehydrogenase, glutamine synthetase, peroxiredoxin, and secretogogin. An important subset of the proteins identified here has not been reported previously in pancreatic islets. Abnormal activity of these proteins in brain may contribute to the pathogenesis of Alzheimer's disease, a neurodegenerative condition characterized by focal amyloid deposits with neurofibrillary tangles. The putative role of these proteins in Alzheimer's pathogenesis is intriguing given the possible clinical relationship and pathological similarity of Alzheimer's disease to type 2 diabetes. These findings have therefore led to the hypothesis that these proteins may also play a role in type 2 diabetes.

Alzheimer Disease↗

PubMed2006

Differential effects of GnRH and androgens on Cres mRNA and protein in male mouse anterior pituitary gonadotropes.

The Cres gene defines a new subgroup in the family 2 cystatins of cysteine protease inhibitors. However, unlike typical cystatins, CRES does not inhibit cysteine proteases but rather inhibits the serine protease prohormone convertase 2, an enzyme with roles in proprotein processing in the neuroendocrine system. Cres is expressed in the gonadotropes and colocalizes with LHbeta, suggesting a role in the regulation of gonadotrope secretion. Our present studies were carried out to examine the regulation of Cres mRNA and protein expression by GnRH and steroid hormones, thus providing clues regarding its role in gonadotropes. Castration profoundly reduced Cres mRNA, while replacement with estradiol (E(2)), testosterone (T), or dihydrotestosterone (DHT) further decreased Cres, suggesting negative regulation by GnRH or steroid hormones. The administration of Antide, a GnRH antagonist, resulted in a 3-fold increase in Cres mRNA, supporting a negative regulation by GnRH. Because all hormonal manipulations in vivo resulted in alterations in steroid hormones, organ culture was used to assess the effects of GnRH independent of steroids. Mouse pituitaries cultured in the absence of GnRH or steroids showed high Cres mRNA levels, while DHT or E(2) resulted in decreases of 25% and 68%, respectively. However, an 85% decrease in Cres mRNA occurred following the administration of GnRH, demonstrating that GnRH, and to a lesser degree E(2), negatively regulate Cres mRNA in gonadotropes. Examination of CRES protein by immunohistochemistry showed that levels were profoundly reduced following castration, while DHT and in part T, but not E(2), restored CRES levels. Castrated mice treated with Antide showed little effect. However, castrated mice treated with Antide + DHT showed a dramatic recovery of CRES, suggesting that androgens act directly at the level of the gonadotrope to regulate CRES protein. Together, our studies suggest that Cres mRNA and protein are low at peak gonadotrope secretory activity, possibly as a means to allow proprotein processing events to occur that are integral to gonadotrope function.

Androgens↗

PubMed2002

The proprotein convertase PACE4 is upregulated by PDGF-BB in megakaryocytes: gene expression of PACE4 and furin is regulated differently in Dami cells.

The differentiation of megakaryocytes into platelets is highly regulated by many cytokines and growth factors. PACE4 and furin are Ca(2+)-dependent serine endoproteases belonging to the subtilisin-like proprotein convertase (SPC) family. These enzymes are involved in the proteolytic activation of proteins that play essential roles in cell growth and differentiation. In this study, we examined the expression of PACE4 and furin during the differentiation of megakaryoblastic cell lines, Dami and HEL cells, induced by phorbol 12-myristate 13-acetate (PMA). PMA stimulates not only the expression of platelet-derived growth factor-B (PDGF-B) mRNA, but also PACE4 mRNA in these cell lines. The expression of PACE4 transcripts (both the PACE4A and PACE4C/CS isoforms) was upregulated more than 4-fold by PMA. Moreover, direct treatment with PDGF-BB also resulted in an increase in the level of PACE4 mRNA. Further, the effect of PDGF-BB on PACE4 expression was confirmed by promoter assay of the PACE4 gene. Although the furin mRNA level was increased by TGF-beta1 in Dami cells, it was not affected by PDGF-BB. These results indicate for the first time that PACE4 expression is specifically upregulated by PDGF-BB in differentiated megakaryoblasts, suggesting a unique role for PACE4 in platelet production.

Becaplermin↗

PubMed2003

Barley serine proteinase inhibitor 2-derived cyclic peptides as potent and selective inhibitors of convertases PC1/3 and furin.

Proprotein convertases (PCs) are serine proteases containing a subtilisin-like catalytic domain that are involved in the conversion of hormone precursors into their active form. This study aims at designing small cyclic peptides that would specifically inhibit two members of this family of enzymes, namely, the neuroendocrine PC1/3 and the ubiquitously expressed furin. We studied peptide sequences related to the 18-residue loop identified as the active site of the 83 amino acid barley serine protease inhibitor 2 (BSPI-2). Peptides incorporating mutations at various positions in the sequence were synthesized on solid phase and purified by HPLC. Cyclization was achieved by the introduction of a disulfide bridge between the two Cys residues located at both the N- and C-terminal extremities. Peptides VIIA and VIIB incorporating P4Arg, P2Lys, P1Arg, and P2'Lys were the most potent inhibitors with K(i) around 4 microM for furin and around 0.5 microM for PC1/3. Whereas peptide VIIB behaved as a competitive inhibitor of furin, peptide VIIA acted as a noncompetitive one. However, all peptides were eventually cleaved after variable incubation times by PC1/3 or furin. To avoid this problem, we incorporated at the identified cleavage site a nonscissile aminomethylene bond (psi[CH(2)-NH]). Those pseudopeptides, in particular peptide VIID, were shown not to be cleaved and to inhibit potently furin. Conversely, they were not able to inhibit PC1/3 at all. Those results show the validity of this approach in designing new effective PC inhibitors showing a certain level of discrimination between PC1/3 and furin.

Amino Acid Sequence↗

PubMed2003

The EGL-21 carboxypeptidase E facilitates acetylcholine release at Caenorhabditis elegans neuromuscular junctions.

Proneuropeptides are packaged into dense-core vesicles in which they are processed into active peptides by copackaged enzymes. Proprotein convertases (PCs) cleave precursors after dibasic residues, and carboxypeptidases remove basic residues from the C terminals. We show here that the Caenorhabditis elegans egl-21 gene encodes a protein that is very similar to carboxypeptidase E (CPE) and is broadly expressed in the nervous system. Mutants lacking either egl-21 CPE or egl-3, which encodes the C. elegans ortholog of PC type 2 (PC2), were defective for processing endogenously expressed FMRFamide (Phe-Met-Arg-Phe-NH2)-related peptides (FaRPs). Mutants lacking the unc-104 kinesin motor protein were defective for anterograde movement of dense-core vesicle components, including egl-3 PC2, egl-21 CPE, and FaRPs. We provide evidence that egl-3 PC2 and egl-21 CPE mutants have diminished acetylcholine release at neuromuscular junctions (NMJs). Taken together, these results suggest that egl-21 CPE and egl-3 PC2 process endogenous neuropeptides that facilitate acetylcholine release at C. elegans NMJs.

Acetylcholine↗

PubMed1998

Post-translational processing of the insulin-like growth factor-2 precursor. Analysis of O-glycosylation and endoproteolysis.

Insulin-like growth factor-2 (IGF-2) is expressed in most embryonic tissues and is required for normal development during gestation. After birth IGF-2 expression is extinguished in most tissues, but the gene is often reactivated during tumorigenesis. Tumors secrete high molecular weight forms of IGF-2 that result from aberrant post-translational processing of pro-IGF-2. As a first step toward understanding how high molecular weight IGF-2 peptides might contribute to tumor progression, we have characterized the biosynthesis of IGF-2 in a human embryonic cell line. We have found that pro-IGF-2 can initially form two disulfide isomers that undergo rearrangement to a single conformation in vivo. The addition of N-acetylgalactosamine to Ser71, Thr72, Thr75, and Thr139 likely occurs in the cis- Golgi apparatus. Sialic acid addition begins in the trans- Golgi apparatus, but IGF-2 peptides must reach the trans-Golgi network for oligosaccharide maturation to be completed. Endoproteolysis occurs concomitant to or slightly after oligosaccharide maturation. Cleavage was observed only at Arg104, resulting in the secretion of IGF-2-(1-104) and free E-peptide. Proteolysis required basic residues in the P1 (Arg104) and P4 (Arg101) positions, was completely blocked by a furin inhibitor, and was enhanced by coexpression with furin, PACE4, PC6A, PC6B, and LPC. These data suggest that members of the subtilisin-related proprotein convertase family mediate processing of pro-IGF-2 at Arg104. We did not detect the IGF-2 peptides that are most abundant in normal serum, mature IGF-2, and IGF-2-(1-87), in this expression system, which indicates that novel endoproteases are responsible for generating these products.

Arginine↗

PubMed2005

Furin-like proprotein convertases are central regulators of the membrane type matrix metalloproteinase-pro-matrix metalloproteinase-2 proteolytic cascade in atherosclerosis.

BACKGROUND: Accumulation of macrophages and their in situ expression of matrix metalloproteinases (MMPs) are important determinants of plaque stability. Activation of membrane-bound MT1-MMP, the major activator of pro-MMP-2, requires intracellular endoproteolytic cleavage of its precursor protein. This type of activation typically requires suitable furin-like proprotein convertases (PCs), specifically furin and PC5. The present study was done to investigate the function of MT1-MMP as well as furin-like PCs in mononuclear inflammatory cells. METHODS AND RESULTS: Macrophage differentiation of human monocytic THP-1 cells was accompanied by increased expression of furin, PC5, and MT1-MMP. Some pro-MMP-2 activation was found in macrophages, but pro-MMP-2 level or activation was not enhanced after stimulation with the proinflammatory mediators tumor necrosis factor-alpha or lipopolysaccharide. However, culturing of macrophages in conditioned medium from serum-starved vascular smooth muscle cells, which constitutively secrete pro-MMP-2, resulted in a strong pro-MMP-2 activation. Inhibition of furin-like PCs with the specific pharmacological inhibitor decanoyl-RVKR-chloromethylketone (dec-CMK) inhibited MT1-MMP activation in macrophages. Dec-CMK or furin-specific small interfering RNA significantly inhibited macrophage MT1-MMP-dependent activation of vascular smooth muscle cell-derived pro-MMP-2. Flow cytometry demonstrated that human circulating monocytes express furin and PC5, and MT1-MMP and immunohistochemistry revealed their colocalization in macrophages in advanced human atherosclerotic lesions. CONCLUSIONS: Furin-like PCs (furin and PC5) play a central role in a MT-MMP-MMP-2 proteolytic cascade, involving provision of macrophage MT1-MMP for the activation of pro-MMP-2 synthesized by other cells. Furin and PC5 are expressed in human peripheral blood mononuclear cells and colocalize with MT1-MMP in macrophages in the atherosclerotic plaque, supporting the hypothesis that they are potential targets in atherosclerosis.

Arteriosclerosis↗

PubMed1995

Mutational analysis of the insulin-like growth factor I prohormone processing site.

Insulin-like growth factor I (IGF-I) is a mitogenic peptide that is produced in most tissues and cell lines and plays an important role in embryonic development and postnatal growth. IGF-I is initially synthesized as a prohormone precursor that is converted to mature IGF-I by endoproteolytic removal of the carboxyl-terminal E-domain. Regulation of the conversion of proIGF-I to mature IGF-I is a potential mechanism by which the biological activity of this growth factor might be modulated. Endoproteolysis of the IGF-I prohormone occurs at the unique pentabasic motif Lys-X-X-Lys-X-X-Arg71-X-X-Arg-X-X-Arg. Recently, a family of enzymes which cleave prohormone precursors at sites containing multiple basic residues has been discovered. The goals of this study were 1) to determine which basic residues in the pentabasic proIGF-I processing site were necessary for proper cleavage and 2) to examine the role that subtilisin-related proprotein convertase 1 (SPC1/furin) might play in proIGF-I processing. We have shown that an expression vector coding for an epitope-tagged proIGF-I directs synthesis and secretion of mature IGF-I-(1-70), extended IGF-I-(1-76), proIGF-I, and N-glycosylated proIGF-I in human embryonic kidney 293 cells. Extended IGF-I-(1-76) is produced by cleavage at Arg77 and requires both Arg74 (P4) and Arg77 (P1). Cleavage at Arg77 does not occur in the SPC1-deficient cell lines RPE.40 and LoVo, suggesting that processing at this site is mediated by SPC1. Mature IGF-I-(1-70) is produced by cleavage at Arg71 and requires both Lys68 (P4) and Arg71 (P1). Lys65 in the P7 position is important for efficient cleavage. SPC1 is not required for processing at Arg71 since this cleavage occurs in RPE.40 and LoVo cells. These data suggest the existence of a processing enzyme which is specific for the Lys-X-X-Arg motif of proIGF-I.

Amino Acid Sequence↗

PubMed1998

Molecular diversity in neurosecretion: reflections on the hypothalamo-neurohypophysial system.

1. The diversity of molecules involved in various aspects of neurosecretion, such as proprotein processing, axonal transport of large dense core vesicles (LDCVs), and regulated secretion, is discussed in the context of the hypothalamo-neurohypophysial system (HNS). 2. Recent studies have uncovered a family of at least seven processing enzymes known as proprotein convertases (PCs) which are involved in proteolytically cleaving protein precursors at paired basic amino acid motifs to yield biologically active peptides. Three of these, PC1(3), 2, and 5, are found in neurons and are involved in producing regulated secretory peptide products. 3. The axonal transport of LDCVs occurs on microtubule tracks by still unknown mechanisms. There are over 11 distinct kinesin-related molecules that have now been identified as possible microtubule motor candidates. 4. Calcium channels in the nervous system are known to be derived from at least five alpha-subunit and four beta-subunit genes with multiple alternatively spliced isoforms in each case. These could account, in part, for the varied calcium currents found in the HNS. 5. The large number of proteins and isoforms now demonstrated to be involved in regulated secretion are discussed, with a focus on LDCV compositions and the synaptotagmin gene family.

Animals↗

PubMed2004

Structure and expression of Furin mRNA in the ovary of the medaka, Oryzias latipes.

A cDNA for furin was cloned from the ovary of the medaka, Oryzias latipes, by a combination of cDNA library screening, 5'-rapid amplification of cDNA ends (RACE), and 3'- RACE. The cDNA sequence codes for a protein of 814 amino acid residues highly homologous to other vertebrate furins, Ca(2+)-dependent serine proteases belonging to the subtilysin-like proprotein convertase family. The medaka preprofurin consists of a leader sequence, a propeptide with autoactivation sites, a Kex2-like catalytic domain, a P domain, a cysteine-rich domain, a putative transmembrane domain, and a cytoplasmic domain. The catalytic triad residues (Asp-164, His-205, and Ser-379) were all conserved. Furin mRNA was expressed in many tissues of this, including the ovary. In the ovary, the greatest expression of furin mRNA occurred in oocytes of small growing follicles, as demonstrated by Northern blotting, RT-PCR, and in situ hybridization analysis. Temporary and spatial expression patterns of the medaka fish furin were similar to those of stromelysin-3 and MT5-MMP during oocyte growth and postnatal development.

Amino Acid Sequence↗

PubMed2006

Implication of the proprotein convertase NARC-1/PCSK9 in the development of the nervous system.

Neural apoptosis-regulated convertase-1/proprotein convertase subtilisin-kexin like-9 (NARC-1/PCSK9) is a proprotein convertase recently described to play a major role in cholesterol homeostasis through enhanced degradation of the low-density lipoprotein receptor (LDLR) and possibly in neural development. Herein, we investigated the potential involvement of this proteinase in the development of the CNS using mouse embryonal pluripotent P19 cells and the zebrafish as models. Time course quantitative RT-PCR analyses were performed following retinoic acid (RA)-induced neuroectodermal differentiation of P19 cells. Accordingly, the mRNA levels of NARC-1/PCSK9 peaked at day 2 of differentiation and fell off thereafter. In contrast, the expression of the proprotein convertases subtilisin kexin isozyme 1/site 1 protease and Furin was unaffected by RA, whereas that of PC5/6 and PC2 increased within and/or after the first 4 days of the differentiation period respectively. This pattern was not affected by the cholesterogenic transcription factor sterol regulatory element-binding protein-2, which normally up-regulates NARC-1/PCSK9 mRNA levels in liver. Furthermore, in P19 cells, RA treatment did not affect the protein level of the endogenous LDLR. This agrees with the unique expression pattern of NARC-1/PCSK9 in the rodent CNS, including the cerebellum, where the LDLR is not significantly expressed. Whole-mount in situ hybridization revealed that the pattern of expression of zebrafish NARC-1/PCSK9 is similar to that of mouse both in the CNS and periphery. Specific knockdown of zebrafish NARC-1/PCSK9 mRNA resulted in a general disorganization of cerebellar neurons and loss of hindbrain-midbrain boundaries, leading to embryonic death at approximately 96 h after fertilization. These data support a novel role for NARC-1/PCSK9 in CNS development, distinct from that in cholesterogenic organs such as liver.

Animals↗