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PubMed1994

Proglucagon is processed to glucagon by prohormone convertase PC2 in alpha TC1-6 cells.

Proglucagon is processed differentially in the pancreatic alpha cells and the intestinal L cells to yield either glucagon or glucagon-like peptide 1, respectively, structurally related hormones with opposing metabolic actions. Here, we have studied the processing of proglucagon in alpha TC1-6 cells, an islet-cell line transformed by simian virus 40 large tumor (T) antigen, a model of the pancreatic alpha cell. We found that these cells process proglucagon at certain dibasic cleavage sites to release glucagon and only small amounts of glucagon-like peptide 1, as demonstrated by both continuous and pulse-chase labeling experiments. Both normal islet alpha cells and alpha TC1-6 cells were shown to express the prohormone convertase PC2 at high levels, but not the related protease PC3. Expression of PC2 antisense RNA in alpha TC1-6 cells inhibited both PC2 production and proglucagon processing concomitantly. We conclude that PC2 is the key endoprotease responsible for proglucagon processing in cells with the alpha-cell phenotype.

Animals↗
PubMed1995

The NH2-terminal proregion of peptidylglycine alpha-amidating monooxygenase facilitates the secretion of soluble proteins.

A highly conserved ten amino acid proregion separates the peptidylglycine alpha-hydroxylating monooxygenase (PHM) domain of the bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) protein from the NH2-terminal signal peptide; propeptides with amino acid sequences similar to the PAM proregion have been identified in other secreted proteins. In AtT-20 cells, but not in human embryonic kidney (hEK)-293 cells, an endogenous endoprotease acting at a site distal to the trans-Golgi network efficiently removes the propeptide from stably transfected monofunctional PHM (PHMs). We constructed a mutant PHM protein (delta ProPHMs) in which the proregion was deleted and the signal peptide joined directly to the monooxygenase domain. Newly synthesized, enzymatically active delta ProPHMs was secreted from both AtT-20 cells and hEK-293 cells more slowly than PHMs. In endocrine cells, the proregion was not required for storage in regulated secretory granules. We transferred the PAM proregion to prohormone convertase 2 (PC2), another soluble constituent of secretory granules, to determine whether the effect of the proregion were transferrable. In both AtT-20 cells and hEK-293 cells, the PAM/PC2 fusion molecule was able to exit the endoplasmic reticulum more rapidly than PC2.

Amino Acid Sequence↗
PubMed2000

Extracellular matrix proteins increase the expression of pro-TRH and pro-protein convertase PC1 in fetal hypothalamic neurons in vitro.

External clues for neuron development include extracellular matrix (ECM) molecules. To explore ECM influence on the early development of peptide phenotype in the CNS, we have compared pro-TRH levels in primary cultures of rat hypothalamic cells plated either on poly-lysine (PL) (control) or on PL plus one of various ECM molecules at 10 microgram/ml. Fetal day 17 cells plated at a density of 1250/mm(2) were grown in a serum free medium made of Neurobasal medium supplemented with B27 (GIBCO). Cultures, consisting mainly of neurons, were analyzed at DIV 2. ECM proteins induced morphological effects in agreement with previously published studies. The amount of pro-TRH per dish, quantified by Western blotting, was increased to 275% for laminin, 191% for fibronectin and 173% for tenascin-C (control=100%); there was no effect of vitronectin. Laminin or fibronectin did not change pro-TRH mRNA or TRH levels but enhanced levels of the pro-protein convertase PC1 suggesting that the ECM molecules did regulate the translational status of pro-TRH. In conclusion, we have shown that some ECM proteins increased pro-TRH level in vitro; this may contribute to the enhancement of pro-TRH levels observed early in vivo in the hypothalamus.

Animals↗
PubMed1996

Processing of pro-opiomelanocortin in GH3 cells: inhibition by prohormone convertase 2 (PC2) antisense mRNA.

The processing of pro-opiomelanocortin (POMC) was examined in GH3 cells, a rat sommatomammotrope cell line, by transiently-transfecting the cells with mouse POMC cDNA. The peptide products were extracted, chromatographed on HPLC and identified by specific radioimmunoassay. POMC was processed to generate ACTH-related peptides, beta-endorphin and Lys-gamma 3- MSH, with complete disappearance of the POMC precursor. The ACTH-related molecules were identified as ACTH1-14, ACTH1-15, ACTH1-17, as well as ACTH1-39. GH3 cells which were not transfected with POMC cDNA did not contain endogenous POMC-related peptides. RT-PCR demonstrated that GH3 cells contain prohormone convertase 2 (PC2) mRNA but no PC1 mRNA. To determine if PC2 was the enzyme responsible for POMC processing in this cell line, GH3 cells were stably-transfected with PC2 antisense cDNA. A cell line was obtained which showed an absence of PC2 protein compared to control untransfected GH3 cells, indicating successful hybridization of PC2 antisense mRNA to the endogenous PC2 mRNA. When this cell line was then transiently-transfected with POMC cDNA, POMC was not processed. The results from these experiments suggest that PC2 alone can correctly process POMC to biologically active smaller peptides in vivo. Additionally, the GH3 cell line with and without incorporation of PC2 antisense cDNA can be used as a model system to study the role of PC2 in the post-translational processing of other prohormones and proproteins in vivo.

Animals↗
PubMed1999

Secreted site-1 protease cleaves peptides corresponding to luminal loop of sterol regulatory element-binding proteins.

We describe a permanent line of Chinese hamster ovary cells transfected with a cDNA encoding a truncated form of Site-1 protease (S1P) that is secreted into the culture medium in an enzymatically active form. S1P, a subtilisin-like protease, normally cleaves the luminal loop of sterol regulatory element-binding proteins (SREBPs). This cleavage initiates the two-step proteolytic process by which the NH(2)-terminal domains of SREBPs are released from cell membranes for translocation to the nucleus, where they activate transcription of genes involved in the biosynthesis and uptake of cholesterol and fatty acids. Truncated S1P (amino acids 1-983), produced by the transfected Chinese hamster ovary cells, lacks the COOH-terminal membrane anchor. Like native S1P, this truncated protein undergoes normal autocatalytic processing after residue 137 to release an NH(2)-terminal propeptide, thereby generating an active form, designated S1P-B. Prior to secretion, truncated S1P-B, like native S1P-B, is cleaved further after residue 186 to generate S1P-C, which is the only form that appears in the culture medium. The secreted enzyme, designated S1P(983)-C, cleaves a synthetic peptide that terminates in a 7-amino-4-methyl-coumarin fluorochrome. This peptide, RSLK-MCA, corresponds to the internal propeptide cleavage site that generates S1P-B as described in the accompanying paper (Espenshade, P. J., Cheng, D., Goldstein, J. L., and Brown, M. S. (1999), J. Biol. Chem. 274, 22795-22804). The secreted enzyme does not cleave RSVL-MCA, a peptide corresponding to the physiologic cleavage site in SREBP-2. However, S1P(983)-C does cleave after this leucine when the RSVL sequence is contained within a 16-residue peptide corresponding to the central portion of the SREBP-2 luminal loop. The catalytic activity of S1P(983)-C differs from that of furin/prohormone convertases, two related proteases, in its more alkaline pH optimum (pH 7-8), its relative resistance to calcium chelating agents, and its ability to cleave after lysine or leucine rather than arginine. These data provide direct biochemical evidence that S1P is the protease that cleaves SREBPs and thereby functions to control lipid biosynthesis and uptake in animal cells.

Amino Acid Sequence↗
PubMed2002

Processing of proendothelin-1 by members of the subtilisin-like pro-protein convertase family.

Endothelial cells (ECs) secrete numerous bioactive peptides that are initially synthesized as inactive precursor proteins. One of these, proendothelin-1 (proET-1), undergoes proteolysis at specific pairs of basic amino acids. Here, we wished to examine the role of mammalian convertases in this event. Northern blot analysis shows that only furin and PC7 are expressed in ECs. In vitro cleavage of proET-1 by furin or PC7 demonstrated that both enzymes efficiently and specifically process proET-1. These data reveal that furin and PC7 have similar specificities towards proET-1 and suggest that both enzymes may participate in the maturation of proET-1 in ECs.

Animals↗
PubMed2000

Pro-opiomelanocortin-related peptides, prohormone convertases 1 and 2 and the regulatory peptide 7B2 are present in melanosomes of human melanocytes.

Recently, it has been shown that alpha-melanocyte stimulating hormone can directly activate tyrosinase by removing the allosteric regulator 6(R)-L-erythro 5,6,7,8 tetrahydrobiopterin resulting in a stable alpha-melanocyte stimulating hormone/6(R)-L-erythro 5,6,7,8 tetrahydrobiopterin complex. As melanin production occurs in the melanosome, a specific organelle of the melanocyte, it seemed important to investigate whether these organelles themselves actually produce pro-opiomelanocortin-related peptides in their acidic environment. The presence of alpha-melanocyte stimulating hormone and adrenocorticotropin in the epidermis and melanocytes has been shown by several investigators. In order to follow possible pro-opiomelanocortin processing in the melanosome, human melanocytes were established in MCDB 153 medium and utilized for immunohistochemistry, immunogold electron microscopy, and western blotting. For this purpose antibodies against alpha-melanocyte stimulating hormone, adrenocorticotropin, prohormone convertases 1 and 2 (PC1 and PC2) and the PC2 regulatory protein 7B2 were used. Our results demonstrated the presence of the entire system for pro-opiomelanocortin processing in the melanosome. Considering the pH optima of these convertases, the results are in agreement with an autocrine intramelanosomal production of pro- opiomelanocortin-related peptides and an autocrine production and recycling of the cofactor 6(R)-L- erythro 5,6,7,8 tetrahydrobiopterin in melanocytes. Based on these novel observations, we would like to propose that the pigmentation process may not necessarily involve a melanocortin-1 receptor-mediated mechanism.

Blotting, Western↗
PubMed2000

The Kex2p proregion is essential for the biosynthesis of an active enzyme and requires a C-terminal basic residue for its function.

The Saccharomyces cerevisiae prohormone-processing enzyme Kex2p is biosynthesized as an inactive precursor extended by its N-terminal proregion. Here we show that deletion of the proregion renders Kex2p inactive both in vivo and in vitro. Absence of the proregion impaired glycosylation and stability and resulted in the retention of the enzyme in the endoplasmic reticulum. These phenotypes were partially complemented by expression of the proregion in trans. Trans complementation was specific to Kex2p proregion because expression of any of the seven mammalian prohormone convertase propeptides had no effect. These data are consistent with a model whereby Kex2p proregion functions as an intramolecular chaperone and indicate that covalent linkage to the protein is not an absolute requirement for proregion function. Furthermore, extensive mutagenesis revealed that, in addition to their function as proteolytic recognition sites, C-terminal basic residues play an active role in proregion-dependent Kex2p activation.

Amino Acid Sequence↗
PubMed2006

RT-PCR analysis of the expression of POMC and its processing enzyme PC1 in amphibian melanotropes.

The frog intermediate lobe comprises two functionally distinct cell subtypes, referred to as secretory and storage melanotropes, which differ in their ultrastructure, secretory, and synthetic rates, and display dissimilar responses to hypothalamic regulatory factors. All these differences make melanotrope subtypes an excellent model to analyze the expression and regulation of genes involved in the control and maintenance of the secretory state of endocrine cells. However, quantification of the expression levels of genes involved in the secretory process requires the characterization of a gene whose expression remains constant irrespective of the secretory state of the cells. In this study, we have cloned the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene from frog pituitary and have evaluated its suitability as internal standard in gene expression studies in melanotropes. A semiquantitative RT-PCR system developed to this end revealed that secretory melanotropes and storage melanotropes possess similar expression levels of GAPDH, whereas, as expected, secretory melanotropes showed higher levels of POMC transcripts than storage cells. Furthermore, we found that the expression of the convertase PC1, an intracellular protease involved in POMC processing, parallels that of POMC, thus suggesting that the higher secretory rate of the POMC-derived peptide alpha-MSH exhibited by secretory melanotropes is supported by their higher PC1 expression levels. In addition, we have shown that both POMC and PC1 mRNAs are up-regulated by the hypothalamic factor TRH in melanotrope cell cultures. In contrast, the inhibitory factor NPY reduced the expression level of the convertase but did not modify that of POMC. Taken together, these results demonstrate that PC1 expression is regulated in melanotropes by both stimulatory (TRH) and inhibitory (NPY) hypothalamic signals, in a manner which essentially parallels that observed for the precursor POMC.

Amino Acid Sequence↗
PubMed2002

Gastrin biosynthesis in canine G cells.

Gastrin requires extensive posttranslational processing for full biological activity. It is presumed that progastrin is cleaved at pairs of basic amino acids by a prohormone convertase to form a glycine-extended intermediate (G-Gly) that serves as a substrate for peptidyl-glycine alpha-amidating monooxygenase (PAM), resulting in COOH-terminally amidated gastrin. To confirm the nature of progastrin processing in a primary cell line, we performed [(35)S]methionine-labeled pulse-chase biosynthetic experiments in canine antral G cells. Radiolabeled progastrin reached a peak earlier than observed for G-Gly or amidated gastrin. G-Gly radioactivity accumulated in G cells and preceded the appearance of radioactivity in amidated gastrin. The conversion of G-Gly to amidated gastrin was enhanced by the PAM cofactor ascorbic acid. To determine whether one member of the prohormone convertase family (PC2) was responsible for progastrin cleavage, G cells were incubated with PC2 antisense oligonucleotide probes. Cells treated with antisense probes had reduced PC2 expression, an accumulation of radiolabeled progastrin, and a delay in the formation of amidated gastrin. Progastrin in antral G cells is cleaved via PC2 to form G-Gly that is converted to amidated gastrin via the actions of PAM.

Animals↗
PubMed1995

Downregulation of prohormone convertase-1 by a phorbol ester.

Acute TPA treatment (1h, 100nM) of a human pancreatic carcinoid cell line (BON) depletes cell contents of chromogranin A (CGA) and pancreastatin (PST), a peptide derived posttranslationally from CGA. Despite removal of TPA, BON cells continue to release CGA in an unregulated fashion whereas PST secretion is reduced substantially. TPA treatment also reduced prohormone convertase-1 (PC-1) protein and increased PC-1 mRNA levels. Together, these findings indicate that the TPA-induced switch from a regulated to unregulated pattern of CGA secretion is accompanied by a decrease in the processing of CGA to PST and a decrease in the active form of a processing enzyme potentially involved in processing CGA to a smaller peptide, PST.

Aspartic Acid Endopeptidases↗
PubMed2006

Further EST analysis of endocrine genes that are preferentially expressed in the neural complex of Ciona intestinalis: receptor and enzyme genes associated with endocrine system in the neural complex.

Identification of orthologs of vertebrate neuropeptides and hypothalamic hormones in the neural complex of ascidians suggests integral roles of the ascidian neural complex in the endocrine system. In the present study, we investigated endocrine-related genes expressed in the neural complex of Ciona intestinalis. Comprehensive analyses of 3'-end sequences of the neural complex cDNAs placed 10,029 clones into 4051 independent clusters or genes, 1524 of them being expressed preferentially in this organ. Comparison of the 1524 genes with the human proteome databank demonstrated that 476 matched previously identified human proteins with distinct functions. Further analyses of sequence similarity of the 476 genes demonstrated that 21 genes are candidates for those involved in the endocrine system. Although we cannot detect hormone or peptide candidates, we found 21 genes such as receptors for peptide ligands, receptor-modulating proteins, and processing enzymes. We then characterized the Ciona prohormone convertase 2 (Ci-PC2) and carboxypeptidase E (Ci-CPE), which are associated with endoproteolytic processing of peptide hormone precursors. Furthermore, genes encoding these transcripts are expressed specifically in the neural complex of young adult ascidians. These data provide the molecular basis for further functional studies of the endocrine role of the neural complex of ascidians.

Amino Acid Sequence↗
PubMed1998

Mapping of the domain in the neuroendocrine protein 7B2 important for its helper function towards prohormone convertase PC2.

In the secretory pathway, the neuroendocrine polypeptide 7B2 transiently associates with its physiological target, the prohormone convertase PC2. The 7B2 protein carries two functional domains involved in the control of proPC2 maturation and activation, an inhibitory C-terminal region and an N-terminal domain displaying facilitative activity. Here, we show that a short segment in 7B2 (His120-Pro131) is important for its facilitative activity. Site-directed mutagenesis indicates that within this segment. Tyr130 is particularly important for the ability of 7B2 to act as a helper protein for PC2.

Animals↗
PubMed1998

Increased expression of prohormone convertase-2 in the irradiated rat brain.

Changes in gene expression have been suggested to play a role in radiotherapy-induced central nervous system (CNS) injury. To begin to identify radiation-inducible genes in the CNS, we have applied the differential display of reverse transcription-polymerase chain reaction products to RNA extracted from the brain of adult rats. RNA was isolated from a rat brain 6 h after whole-body exposure to 10 Gy and compared with RNA from unirradiated brain. A cDNA band was consistently observed at about 600 bp in samples from the irradiated rat but not from unirradiated (control) rat. Amplification and sequencing of the cDNA revealed that it corresponded to the prohormone convertase-2 (PC2) gene, which is involved in the processing of inert prohormones and neuropeptides to their bioactive forms. Enhanced PC2 expression was detected after irradiation of neuronal cultures but not in cultures of astrocytes, suggesting that the cell type in the CNS responsible for the PC2 induction after in vivo irradiation is the neuron. These results indicate that radiation induces the expression of a neuronal enzyme that is critical to the activation of a number of prohormones and neuropeptides, which may influence the radioresponse of the CNS.

Animals↗
PubMed1999

Exposure of human islets to cytokines can result in disproportionately elevated proinsulin release.

Infiltration of immunocytes into pancreatic islets precedes loss of beta cells in type 1 diabetes. It is conceivable that local release of cytokines affects the function of beta cells before their apoptosis. This study examines whether the elevated proinsulin levels that have been described in prediabetes can result from exposure of beta cells to cytokines. Human beta-cell preparations were cultured for 48 or 72 hours with or without IL-1beta, TNF-alpha, or IFN-gamma, alone or in combination. None of these conditions were cytotoxic, nor did they reduce insulin biosynthetic activity. Single cytokines did not alter medium or cellular content in insulin or proinsulin. Cytokine combinations, in particular IL-1beta plus IFN-gamma, disproportionately elevated medium proinsulin levels. This effect expresses an altered functional state of the beta cells characterized by preserved proinsulin synthesis, a slower hormone conversion, and an increased ratio of cellular proinsulin over insulin content. The delay in proinsulin conversion can be attributed to lower expression of PC1 and PC2 convertases. It is concluded that disproportionately elevated proinsulin levels in pre-type 1 diabetic patients might result from exposure of their beta cells to cytokines released from infiltrating immunocytes. This hormonal alteration expresses an altered functional state of the beta cells that can occur independently of beta-cell death.

Adolescent↗
PubMed2000

Localization of new, microdissection- generated, anonymous markers and of the genes Pcsk1, Dhfr, Ndub13, and Ccnb1 to rat chromosome region 2q1.

The centromeric region of rat chromosome 2 (2q1) harbors unidentified quantitative trait loci of genes that control tumor growth or development. To improve the mapping of this chromosome region, we microdissected it and generated 10 new microsatellite markers, which we included in the linkage map and/or radiation hybrid map of 2q1, together with other known markers, including four genes: Pcsk1 (protein convertase 1), Dhfr (dihydrofolate reductase), Ndub13 (NADH ubiquinone oxidoreductase subunit b13), and Ccnb1 (cyclin B1). To generate anchor points between the different maps, the gene Ndub13 and the microsatellite markers D2Ulb25 and D2Mit1 were also localized cytogenetically. The radiation map generated in region 2q1 extends its centromeric end of about 150 cR.

Animals↗
PubMed1997

PACE4: a subtilisin-like endoprotease with unique properties.

PACE4 is one of the neuroendocrine-specific mammalian subtilisin-related endoproteases believed to function in the secretory pathway. The biosynthesis and secretion of PACE4 have been studied using transfected neuroendocrine and fibroblast cell lines. as well as primary pituitary cultures. ProPACE4 (approx. 106 kDa) is cleaved intracellularly before secretion of PACE4 (approx. 97 kDa); the N-terminal propeptide cleavage is accelerated in a truncated form of PACE4 lacking the Cys-rich C-terminal region (PACE4s). Neither PACE4 nor PACE4s is stored in regulated neuroendocrine secretory granules, whereas pro-opiomelanocortin-derived peptides and prohormone convertase I enter the regulated secretory pathway efficiently. The relatively slow cleavage of the proregion of proPACE4 in primary anterior pituitary cells, followed by rapid secretion of PACE4, is similar to the results for proPACE4 in transfected cell lines. The enzyme activity of PACE4 is distinct from furin and prohormone convertases, both in the marked sensitivity of PACE4 to inhibition by leupeptin and the relative insensitivity of PACE4 to inhibition by Ca2+ chelators and dithiothreitol; PACE4 is not inhibited by the alpha1-antitrypsin Portland variant that is very potent at inhibiting furin. The unique biosynthetic and enzymic patterns seen for PACE4 suggest a role for this neuroendocrine-specific subtilisin-like endoprotease outside the pathway for peptide biosynthesis.

Blotting, Western↗
PubMed2003

Trafficking of mutant carboxypeptidase E to secretory granules in a beta-cell line derived from Cpe(fat)/Cpe(fat) mice.

We have reinvestigated the stability and intracellular routing of mutant carboxypeptidase E in NIT3 cells, a pancreatic beta-cell line derived from the Cpe(fat)/Cpe(fat) mouse. Pulse-chase experiments demonstrated that this protein has a half-life of approximately 3 h in these cells and that up to 45% of the proCPE(202) can escape degradation by the proteosome. In double-label immunofluorescence microscopy, a portion of the mutant CPE did not colocalize with calnexin, an endoplasmic reticulum marker, but was found in prohormone convertase 2-containing secretory granules, demonstrating that it had escaped degradation and arrived at a post-Golgi compartment. The mutant CPE as well as prohormone convertase 2 were secreted into the medium in a stimulated manner by treatment with the physiological secretagogue, glucagon-like peptide-1, consistent with its presence in granules of the regulated secretory pathway. The presence of mutant carboxypeptidase E in granules supports a potential role for its involvement as a sorting/retention receptor in the trafficking of proinsulin to the regulated secretory pathway.

Animals↗