SEARCH · PubMed Health

Results for “Proprotein Convertase 5”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 127 records · Page 7Linked to original sources

PubMed2003

Epigenetic regulation of proprotein convertase PACE4 gene expression in human ovarian cancer cells.

Proprotein convertases (PC) are a family of serine endoproteases that play important roles in regulating cell function by converting proproteins to biologically active molecules. Several lines of evidence suggest that overexpression of PCs contributes to tumor formation and progression in various types of cancer. In this study, we examined PC expression in six normal ovarian surface epithelium (OSE) cultures, nine primary ovarian cancer (OC) cultures, and three established OC cell lines (Hey, HeyC2, and OCC-1). Our results show that furin and PC7 expression in OC cells was comparable to that in normal OSE. However, PACE4 expression was greatly reduced in all OC samples studied. PACE4 promoter activity was measured in HeyC2 and OCC-1 cells using transiently transfected luciferase reporter plasmids. Both cell lines supported PACE4 promoter activity, showing that the transcription factors critical for PACE4 expression are present in OC cells. The observation that established OC cell lines have reduced PACE4 expression, but maintained the ability to support PACE4 promoter activity, led to the hypothesis that reduced expression may be due to epigenetic modification of the PACE4 gene, such as DNA methylation and histone deacetylation. Methylation analysis of 79 CpG dinucleotides within the PACE4 promoter and exon I (-196/+340) revealed that the percentage of methylated cytosine nucleotides was 8-9% in normal OSE, but 58-93% in OC cells. Treatment with the demethylating agent 5-aza-2'-deoxycytidine and/or the histone deacetylase inhibitor trichostatin A greatly increased PACE4 expression in OC cells. These data suggest that the reduction of PACE4 expression in OC cells is caused, in part, by DNA hypermethylation and histone deacetylation.

DNA Methylation↗

PubMed2004

Limited redundancy of the proprotein convertase furin in mouse liver.

Furin is an endoprotease of the family of mammalian proprotein convertases and is involved in the activation of a large variety of regulatory proteins by cleavage at basic motifs. A large number of substrates have been attributed to furin on the basis of in vitro and ex vivo data. However, no physiological substrates have been confirmed directly in a mammalian model system, and early embryonic lethality of a furin knock-out mouse model has precluded in vivo verification of most candidate substrates. Here, we report the generation and characterization of an interferon inducible Mx-Cre/loxP furin knock-out mouse model. Induction resulted in near-complete ablation of the floxed fur exon in liver. In sharp contrast with the general furin knock-out mouse model, no obvious adverse effects were observed in the transgenic mice after induction. Histological analysis of the liver did not reveal any overt deviations from normal morphology. Analysis of candidate substrates in liver revealed complete redundancy for the processing of the insulin receptor. Variable degrees of redundancy were observed for the processing of albumin, alpha(5) integrin, lipoprotein receptor-related protein, vitronectin and alpha(1)-microglobulin/bikunin. None of the tested substrates displayed a complete block of processing. The absence of a severe phenotype raises the possibility of using furin as a local therapeutic target in the treatment of pathologies like cancer and viral infections, although the observed redundancy may require combination therapy or the development of a more broad spectrum convertase inhibitor.

Albumins↗

PubMed2004

The melanocortin receptor subtypes in chicken have high preference to ACTH-derived peptides.

1 Melanocortin (MC) receptors are widely distributed throughout the body of chicken, like in mammals, and participate in a wide range of physiological functions. 2 To clarify the pharmacological impact of ligands acting in the MC system, we expressed the chicken MC1, MC2, MC3, MC4 and MC5 (cMC1-5) receptors in eukaryotic cells and performed comprehensive pharmacological characterization of the potency of endogenous and synthetic melanocortin peptides. 3 Remarkably, the cMC receptors displayed high affinity for ACTH-derived peptides and in general low affinity for alpha-MSH. It is evident that not only the cMC2 receptor but also the other cMC receptors interact with ACTH-derived peptide through an epitope beyond the sequence of alpha-MSH. 4 The synthetic ligand MTII was found to be a potent agonist whereas HS024 was a potent antagonist at the cMC4 receptor, indicating that these ligands are suitable for physiological studies in chicken. 5 We also show the presence of prohormone convertase 1 (PC1) and PC2 genes in chicken, and that these peptides are coexpressed with proopiomelanocortin (POMC) in various tissues.

Adrenocorticotropic Hormone↗

PubMed1995

Substrate specificity of rabbit liver metalloendopeptidase and its new fluorogenic peptide substrates.

A metalloendopeptidase (MEP) isolated from rabbit liver microsomes with substrate specificity for peptides containing Arg at the P1 and P4 positions has recently proved to be identical to soluble angiotensin-binding protein present in the cytosol. Here we describe the peptide-degrading specificity of MEP, determined using various bioactive peptides and novel fluorogenic substrates for the enzyme. MEP degraded oligopeptides, including bradykinin, alpha-neoendorphin, bovine adrenal medulla dodecapeptide, substance P, bombesin, neurotensin, and alpha-endorphin, but not polypeptides such as reduced lysozyme and histone H4, hence, MEP probably belongs to the family of endo-oligopeptidases. It cleaved most preferentially at the -Phe-Ser- bond of bradykinin (kcat/Km = 2.8 x 10(4) M-1.S-1) but did not cleave high molecular weight and low molecular weight kininogens, the precursors of bradykinin. MEP did not cleave angiotensin I, dynorphin A 1-13, somatostatin, and luteinizing hormone-releasing hormone, some of which are good substrates for metalloendopeptidase-24.15, metalloendopeptidase-24.16, N-arginine dibasic convertase, and yeast endopeptidase-24.15 related peptidase. An active site-directed inhibitor of metalloendopeptidase-24.15, N-[1-(R,S)-carboxyl-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate also had no effects on the amidolytic activity of MEP. Based on the cleavage sites of bioactive peptides and processing sites of vitamin K-dependent proproteins, intramolecularly quenched fluorogenic peptide substrates were newly synthesized. Among the thirteen substrates used, the most reactive was 2-aminobenzoyl-Ala-Arg-Val-Arg-Arg-Ala- Asn-Ser-2,4-dinitroanilinoethylamide (kcat/Km = 9.3 x 10(5) M-1.S-1). An angiotensin antagonist, [Sar1, Ala8]-angiotensin II, inhibited hydrolysis of the substrate by MEP in a competitive manner (Kl = 7.6 microM). MEP cleaved oligopeptides even on the carboxyl side of proline residue and these peptides are resistant to hydrolysis by the cytosol-derived proteasome, therefore MEP may participate in the catabolism of oligopeptides in the cytosol, together with other endo-oligopeptidases.

Amino Acid Sequence↗

PubMed1995

Purification and characteristics of the candidate prohormone processing proteases PC2 and PC1/3 from bovine adrenal medulla chromaffin granules.

The prohormone-processing proteases PC1/3 and PC2 belong to the family of mammalian subtilisin-related proprotein convertases (PC) possessing homology to the yeast Kex2 protease. The presence of PC1/3 and PC2 in secretory vesicles of bovine adrenal medulla (chromaffin granules) implicates their role in the processing the precursors of enkephalin, neuropeptide Y, somatostatin, and other neuropeptides that are present in chromaffin granules. In this study, PC1/3 and PC2 were purified to apparent homogeneity from the soluble fraction of chromaffin granules by chromatography on concanavalin A-Sepharose, Sephacryl S-200, pepstatin A-agarose, and anti-PC1/3 or anti-PC2 immunoaffinity resins. PC1/3 and PC2 were monitored during purification by measuring proteolytic activities with 35S-enkephalin precursor and Boc-Arg-Val-Arg-Arg-methylcoumarin amide (MCA) substrates and by following PC1/3 and PC2 immunoreactivity with specific anti-PC1/3 and anti-PC2 sera generated in this study. Purified PC1/3 and PC2 on SDS-polyacrylamide gels each show a molecular mass of 66 kDa. PC2 in the soluble fraction of chromaffin granules was present at 5- and 10-fold higher enzyme protein and activity, respectively, compared with that of PC1/3. PC1/3 and PC2 cleaved paired basic and monobasic sites within peptide-MCA substrates, with Boc-Arg-Val-Arg-Arg-MCA and pGlu-Arg-Thr-Lys-Arg-MCA as the most effectively cleaved peptides tested. PC1/3 and PC2 showed pH optima of 6.5 and 7.0, respectively. Kinetic studies indicated apparent Km values for hydrolysis of Boc-Arg-Val-Arg-Arg-MCA as 66 and 40 microM, with Vmax values of 255 and 353 nmol/h/mg for PC1/3 and PC2, respectively. Specificity of the PC enzymes for dibasic sites was confirmed by potent inhibition by the active site-directed peptide inhibitors (D-Tyr)-Glu-Phe-Lys-Arg-CH2Cl and Ac-Arg-Arg-CH2Cl. Inhibition by EGTA and activation by Ca2+ indicated PC1/3 and PC2 as Ca(2+)-dependent proteases. In addition, PC enzymes were activated by dithiothreitol and inhibited by thiol-blocking reagents, p-hydroxymercuribenzoate and mercuric chloride. These results illustrate the properties of endogenous PC1/3 and PC2 as prohormone-processing enzymes.

Adrenal Medulla↗

PubMed1997

Post-translational processing of human procorticotrophin-releasing factor in transfected mouse neuroblastoma and Chinese hamster ovary cell lines.

The mouse neuroblastoma cell line (Neuro 2 A) has been shown to contain the mRNA of a prohormone converting enzyme, PC2. The Chinese hamster ovary cell line (CHO) does not express PC2 mRNA, but is thought to contain the ubiquitous protease, furin. The enzyme(s) responsible for releasing corticotrophin-releasing hormone (CRH) from its precursor (proCRH) have not been identified, therefore to investigate the possible function(s) of PC2 or furin in the processing of proCRH, stable Neuro 2 A and CHO cell lines that express the 21 kDa human (h)proCRH were established. A specific two-site IRMA for CRH demonstrated that the hpreproCRH-expressing Neuro 2 A cell line cleaved the CRH precursor to the CRH peptide, and was able to release the mature peptide into cell medium at levels that were 4-fold higher than produced by the hproCRH-expressing CHO cells. RIA showed that the CHO cells secreted levels of CRH-containing peptides that were 10-fold higher than produced by the Neuro 2 A cells. Medium from the transfected CHO and Neuro 2 A cells was analysed by HPLC; this showed that CHO cells released a single protein corresponding to the unprocessed CRH precursor, whereas Neuro 2 A cells secreted two peptides, which could be identified as the 5 kDa CRH(1-41) and residual 16 kDa CRH peptides. These results suggest that Neuro 2 A cells, which contain PC2, can process proCRH to the mature peptide.

Animals↗

PubMed1992

The cDNA sequence of the human pro-hormone and pro-protein convertase PC1.

Using a probe consisting of the full-length cDNA sequence of the mouse pro-hormone convertase PC1 (mPC1), we isolated from a lambda gt10 human pituitary cDNA library a number of contiguous clones, of which composite sequence of 3.3-kb defined the complete coding sequence of human PC1 (hPC1). The cDNA sequence of hPC1 encodes a protein containing 753 amino acids and potentially two N-glycosylation sites, one carboxy-terminal amidation site, a cAMP-dependent protein kinase Ser phosphorylation site, a tyrosine kinase phosphorylation site, and an ArgGlyAsp (RGD) sequence. Like mPC1, the carboxy-terminal sequence of hPC1 exhibits an amphipathic domain potentially involved in membrane association. The coding region of hPC1 exhibits an overall 92.6% protein sequence identity to the mouse mPC1 sequence, with the highest homology (98%) found in the catalytic segment of the molecule (residues 84-399). Whereas Northern blot analysis of tissues obtained from mouse, rat and porcine demonstrated the presence of two mRNAs of 3 kb and 5 kb, Northern blots of human tissues and cells demonstrated the presence of a dominant transcript of 6.2 kb and the presence of smaller transcripts in some tissues. The major site of production of hPC1 seems to be the pituitary and brain, although detection was also possible in pancreas and heart.

Amino Acid Sequence↗

PubMed2001

Implication of the proprotein convertases furin, PC5 and PC7 in the cleavage of surface glycoproteins of Hong Kong, Ebola and respiratory syncytial viruses: a comparative analysis with fluorogenic peptides.

Fluorogenic peptides encompassing the processing sites of envelope glycoproteins of the infectious influenza A Hong Kong virus (HKV), Ebola virus (EBOV) and respiratory syncytial virus (RSV) were tested for cleavage by soluble recombinants of the proprotein convertases furin, PC5 and PC7. Kinetic studies with these intramolecularly quenched fluorogenic peptides revealed selective cleavages at the physiological dibasic sites. The HKV peptide is cleaved by both furin and PC5 with similar efficacy; in comparison, PC7 cleaves this substrate poorly. In contrast with the basic tetrapeptide insertion within the haemagglutinin sequence of HKV, two other dipeptide insertions revealed a poorer cleavage with a similar rank order of potency. These results demonstrate that the N-terminal RERR insertion to the wild-type avian RKKR downward arrow sequence is functionally significant, and suggest that the approx. 5-fold increase in cleavage efficacy contributes to the high infectivity of the H5N1 virus subtype. With regard to RSV peptide processing, PC7 is twice as effective as PC5 and furin. The EBOV peptide was processed with similar efficiency by the three enzymes. Our observations that all of these cleavages can be effectively inhibited by a plant andrographolide derivative at 250 microM or less might aid in the design of potent convertase inhibitors as alternative antiviral therapies.

Amino Acid Sequence↗

PubMed1995

Characterization of the endopeptidase PC2 activity towards secretogranin II in stably transfected PC12 cells.

To study the processing of secretogranin II (SgII) by the prohormone convertase PC2 we have generated a stable PC12 cell line which expresses mouse PC2. We here present the characteristics of the PC12/PC2 cell line and demonstrate that the exogenous PC2 is sorted and stored in secretory granules in the PC12/PC2 cell line as efficiently as the endogenous granins. By indirect immunofluorescence with antibodies specific for chromogranin B (CgB) and PC2 we were able to establish that the PC2 is stored in secretory granules in the PC12/PC2 cell line. After subcellular fractionation, followed by immunoblotting, the mature 68 kDa form of PC2 was found co-sedimented with SgII in fractions containing secretory granules. Two-dimensional gel electrophoresis was used to characterize a secretory granule fraction obtained from the PC12/PC2 cells, and a comparison was done of the electrophoretic pattern obtained from the PC12/PC2 cells with the parent cell line PC12. The products derived from the processing of SgII by PC2 were identified by immunoblotting with a panel of antibodies directed against SgII. Using [35S]sulphate to label the newly synthesized SgII, we performed a time course to monitor the appearance of the lower-molecular-mass fragments of SgII: beginning 15 min after a 5 min pulse of [35S]sulphate we were able to detect the first proteolytic fragment of SgII. Our results demonstrate that SgII is proteolytically processed by PC2 in the immature secretory granule into several lower-molecular-mass proteins, the major ones being an 18 kDa sulphated fragment and a 28 kDa fragment.

Amino Acid Sequence↗

PubMed1994

A Kex2-related endopeptidase activity present in rat liver specifically processes the insulin proreceptor.

The insulin proreceptor is cleaved by limited proteolysis post-translationally at an Arg-Lys-Arg-Arg site to generate its mature alpha- and beta-subunit form. An 35S-labelled insulin proreceptor substrate preparation and a 15-mer peptide substrate that mimics the amino acid sequence around and including the insulin proreceptor processing site (IRP-peptide) has revealed an endopeptidase activity that catalyses insulin proreceptor cleavage in a rat liver subcellular fraction. Under optimal conditions, normal 35S-labelled insulin proreceptor substrate processing by this fraction was quantitative. This fraction was not able to process an 35S-labelled insulin proreceptor variant substrate (where the Arg-1 of the tetrabasic cleavage site had been replaced by Ala-1), similarly to previous in vivo observations, suggesting that this endopeptidase activity has physiological relevance. Biochemical characterization of the insulin proreceptor/IRP-peptide processing revealed this rat liver endopeptidase activity to have a broad pH range (> 70% maximal activity between pH 5.5 and 10.0) and a pH optimum of pH 8-10. It was Ca(2+)-dependent activity, maximally active between 0.5 and 5 mM Ca2+ and half-maximally activated between 50 and 90 microM Ca2+. Endoproteolytic activity was not inhibited by group-specific inhibitors of serine-, cysteinyl or aspartyl proteinases or by 1,10-phenanthroline; however, EDTA and 1,2-cyclohexanediaminetetraacetic acid did inhibit the activity, but this was accounted for by Ca2+ chelation. The IRP-peptide substrate assay enabled measurement of an apparent Km of 22 microM and a Vmax of 18.6 pmol/min for this endopeptidase activity. These biochemical characteristics suggest that insulin proreceptor processing endopeptidase activity to be a legitimate member of the Kex2-related proprotein convertase family. Immunoblotting detected furin and PACE4 proteins (both members of this family) to be present in the rat liver subcellular fraction containing insulin proreceptor processing activity. Since the biochemical characteristics of the insulin proreceptor processing endopeptidase activity mostly resembled those of furin activity, it is likely that insulin proreceptor proteolytic maturation can be catalysed by furin in the liver.

3T3 Cells↗

PubMed1997

Regulation of human prohormone convertase 2 promoter activity by the transcription factor EGR-1.

Prohormone convertases are involved in the tissue-specific endoproteolytic processing of prohormones and neuropeptide precursors within the secretory pathway. In the present study, we have isolated genomic clones comprising the 5'-terminal region of the human prohormone convertase 2 (PC2) gene and established characteristics of the PC2 promoter region. The proximal promoter region is very G+C-rich and does not contain a canonical TATA box or a CAAT box. Transient expression assays with a set of human PC2 gene fragments containing progressive 5' deletions demonstrate that the proximal promoter region is capable of directing high levels of neuroendocrine-specific expression of reporter gene constructs. In addition, we show that the transcription factor EGR-1 interacts with two distinct elements within the proximal human PC2 promoter region. Transfection experiments also demonstrate that EGR-1 is able to enhance PC2 promoter activity.

Animals↗

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↗

PubMed1995

Neuroendocrine-specific expression of the human prohormone convertase 1 gene. Hormonal regulation of transcription through distinct cAMP response elements.

Prohormone convertases are involved in the tissue-specific endoproteolytic processing of prohormones and neuropeptide precursors within the secretory pathway. In the present study, we have isolated genomic clones comprising the 5'-terminal region of the human prohormone convertase 1 (PC1) gene and identified and characterized the PC1 promoter region. We found multiple transcription start sites located within a 15-base pair region, 205 base pairs upstream of the translation start codon. The promoter region is not G+C-rich and does not contain a canonical TATA box nor a CAAT box. Transient expression assays with a set of human PC1 gene fragments containing progressive 5' deletions demonstrate that the proximal promoter region is capable of directing high levels of neuroendocrine-specific expression of reporter gene constructs. In addition, the proximal promoter region confers both basal and hormone-regulated promoter activity. Site-specific mutagenesis experiments demonstrate that two closely spaced cAMP response elements within the proximal promoter region direct cAMP-mediated hormonal regulation of transcription of the PC1 gene.

Animals↗

PubMed2003

Furin and prohormone convertase 1/3 are major convertases in the processing of mouse pro-growth hormone-releasing hormone.

We investigated the proteolytic processing of mouse pro-GHRH [84 amino acids (aa)] by furin, PC1/3, PC2, and PC5/6A. We created six point mutations in the N- and C-terminal cleavage sites, RXXR decreased and RXRXXR decreased, respectively. The following results were obtained after transient transfection/cotransfection and metabolic pulse-chase labeling studies in several neuroendocrine cells. 1) Furin was the most efficient convertase in cleaving the N-terminal RXXR/RXRR site to generate intermediate I, 12-84aa, whereas PC1/3 was the most potent in processing the C-terminal RXRXXR site to yield mature GHRH, 12-53aa. 2) Both PC1/3 and PC5/6A also processed the N-terminal site but less efficiently than furin. 3) PC2 was much weaker in cleaving the C-terminal site relative to PC1/3 to generate mature GHRH. 4) The Q10R mutant was significantly more susceptible to furin cleavage at the N-terminal site than the wild-type pro-GHRH. And 5) the N- and C-terminal P1 Arg residues, R11 and R54, respectively, were essential for mature GHRH production. We also showed localization of the GHRH immunoreactive peptides in Golgi and secretory granules in neuroendocrine cells by an immunofluorescence assay. We conclude that the efficient production of mature GHRH from pro-GHRH is a stepwise process mediated predominantly by furin at the N-terminal cleavage site followed by PC1/3 at the C terminus.

Animals↗

PubMed1996

Purification and enzymatic characterization of recombinant prohormone convertase 2: stabilization of activity by 21 kDa 7B2.

Although previous efforts to produce significant quantities of purified prohormone convertase 2 from either recombinant or natural sources have been unsuccessful, our recent finding that the neuroendocrine polypeptide 7B2 is necessary for the biosynthesis of enzymatically active prohormone convertase 2 (PC2) has enabled us to obtain active recombinant enzyme from the conditioned medium of PC2-producing CHO cells supertransfected with cDNA coding for 21 kDa 7B2. The recombinant enzyme was purified to apparent homogeneity, with a 40% recovery, in milligram quantities. Two protein bands of Mrs 71 and 75 kDa were observed after SDS-PAGE followed by either Coomassie staining or Western blotting with PC2 antiserum. Spontaneous conversion of the 71- and 75-kDa species to the 66-kDa form occurred during incubation at pH 5.0; the degree of conversion correlated with a dramatic increase in activity. Kms of 124 and 131 microM and Kcats of 0.49 and 0.81 s(-1) were obtained for the substrates Cbz-Arg-Ser-Lys-Arg-AMC and Pyr-Arg-Thr-Lys-Arg-AMC, respectively. The pH optimum was 5.0, and the enzyme was inhibited by h7B2(155-185') p-CMS, and EDTA but not by other inhibitors tested. Interestingly, 21 kDa 7B2 was observed to copurify with the enzyme in a molar ratio of about 1:100 (7B2:PC2). Prior addition of recombinant 21 kDa 7B2 to activated 66 kDa PC2 provided significant protection against thermal denaturation. When coassociated 7B2 was mostly removed from activated PC2 through gel filtration, subsequent addition of recombinant 7B2 exerted a significant stabilizing effect on enzyme activity. Millimolar Ca2+ and pHs between 5 and 6 were required to observe this effect. Since these conditions resemble those thought to occur within secretory granules, and since 21 kDa 7B2 represents a stored secretory granule protein, our data suggest a physiological role for 21 kDa 7B2 in the stabilization of PC2 activity.

Amino Acid Sequence↗

PubMed1997

Obesity and impaired prohormone processing associated with mutations in the human prohormone convertase 1 gene.

Human obesity has an inherited component, but in contrast to rodent obesity, precise genetic defects have yet to be defined. A mutation of carboxypeptidase E (CPE), an enzyme active in the processing and sorting of prohormones, causes obesity in the fat/fat mouse. We have previously described a women with extreme childhood obesity (Fig. 1), abnormal glucose homeostasis, hypogonadotrophic hypogonadism, hypocortisolism and elevated plasma proinsulin and pro-opiomelanocortin (POMC) concentrations but a very low insulin level, suggestive of a defective prohormone processing by the endopeptidase, prohormone convertase 1 (PC1; ref. 4). We now report this proband to be a compound heterozygote for mutations in PC1. Gly-->Arg483 prevents processing of proPC1 and leads to its retention in the endoplasmic reticulum (ER). A-->C+4 of the intro-5 donor splice site causes skipping of exon 5 leading to loss of 26 residues, a frameshift and creation of a premature stop codon within the catalytic domain. PC1 acts proximally to CPE in the pathway of post-translational processing of prohormones and neuropeptides. In view of the similarity between the proband and the fat/fat mouse phenotype, we infer that molecular defects in prohormone conversion may represent a generic mechanism for obesity, common to humans and rodents.

Amino Acid Sequence↗

PubMed1992

Expression in human lung tumor cells of the proprotein processing enzyme PC1/PC3. Cloning and primary sequence of a 5 kb cDNA.

Northern blot analysis of human lung tumors indicated that the gene, which encodes the subtilisin-like proprotein processing enzyme PC1/PC3, was highly expressed in almost all carcinoid tumors tested. In small cell lung carcinomas (SCLCs), expression varied. In non-SCLCs and normal lung, no expression was found. Analysis of SCLC cell lines revealed that expression was restricted preferentially to cell lines of the classical type. In lung tumor cells expressing the PC1/PC3 gene, transcripts of 3 kb and 5 kb were detected, the 5 kb mRNA always being the most abundant species. We isolated a cDNA corresponding to the 5 kb human PC1/PC3 transcript, determined the nucleotide sequence of it and deduced the amino acid sequence of the corresponding protein. Furthermore, we conclude that the two PC1/PC3 transcripts have 3' non-coding regions of different size and encode the same protein.

Amino Acid Sequence↗

PubMed1992

Distribution and regulation of the prohormone convertases PC1 and PC2 in the rat pituitary.

PC1 and PC2 are enzymes involved in the activation of prohormones via the cleavage of pairs of basic amino acids. The expression levels of each of these enzymes were evaluated in the rat anterior and neurointermediate pituitary lobes by in situ hybridization and Northern gel analysis and after various pharmacological manipulations. All intermediate lobe melanotrophs expressed high levels of PC2 mRNA and lower levels of PC1 mRNA. PC1 mRNA was highly expressed throughout the anterior lobe; however, appreciable PC2 mRNA levels were also found. Based on colocalization studies, anterior lobe corticotrophs were found to express PC1 mRNA, but very little PC2 mRNA. Neurointermediate lobe levels of PC1, PC2, and POMC mRNA increased 2- to 6-fold in rats treated with haloperidol, while they decreased to 10-25% of their control values after bromocriptine treatment. These results indicate that in the intermediate lobe, dopamine is involved in the regulation of PC1 and PC2. In the anterior lobe, haloperidol had a strong effect on PC2 mRNA, increasing its levels by 8- to 12-fold compared to the control value, while PC1 mRNA was unaffected. Both PC1 and PC2 mRNA levels were increased 5- to 9-fold in animals made hypothyroid by treatment with 6-n-propyl-2-thiouracil. Adrenalectomy had no significant effect on anterior lobe PC1 mRNA levels. However, both PC1 and PC2 mRNA levels were responsive to dexamethasone treatment in the AtT-20 cell lines. Our results indicate that dopamine, thyroid hormones, and corticosteroids are involved in PC1 and/or PC2 gene expression. These data are also consistent with the role of PC1 and PC2 as prohormone-processing enzymes.

Amino Acid Sequence↗