PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Proprotein Convertase 2”

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 307 records · Page 17Linked to original sources
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↗
PubMed2003

The proprotein convertase PC2 is involved in the maturation of prosomatostatin to somatostatin-14 but not in the somatostatin deficit in Alzheimer's disease.

A somatostatin deficit occurs in the cerebral cortex of Alzheimer's disease patients without a major loss in somatostatin-containing neurons. This deficit could be related to a reduction in the rate of proteolytic processing of peptide precursors. Since the two proprotein convertases (PC)1 and PC2 are responsible for the processing of neuropeptide precursors directed to the regulated secretory pathway, we examined whether they are involved first in the proteolytic processing of prosomatostatin in mouse and human brain and secondly in somatostatin defect associated with Alzheimer's disease. By size exclusion chromatography, the cleavage of prosomatostatin to somatostatin-14 is almost totally abolished in the cortex of PC2 null mice, while the proportions of prosomatostatin and somatostatin-28 are increased. By immunohistochemistry, PC1 and PC2 were localized in many neuronal elements in human frontal and temporal cortex. The convertases levels were quantified by Western blot, as well as the protein 7B2 which is required for the production of active PC2. No significant change in PC1 levels was observed in Alzheimer's disease. In contrast, a marked decrease in the ratio of the PC2 precursor to the total enzymatic pool was observed in the frontal cortex of Alzheimer patients. This decrease coincides with an increase in the binding protein 7B2. However, the content and enzymatic activity of the PC2 mature form were similar in Alzheimer patients and controls. Therefore, the cortical somatostatin defect is not due to convertase alteration occuring during Alzheimer's disease. Further studies will be needed to assess the mechanisms involved in somatostatin deficiency in Alzheimer's disease.

Adult↗
PubMed2001

Neuroendocrine secretory protein 7B2: structure, expression and functions.

7B2 is an acidic protein residing in the secretory granules of neuroendocrine cells. Its sequence has been elucidated in many phyla and species. It shows high similarity among mammals. A Pro-Pro-Asn-Pro-Cys-Pro polyproline motif is its most conserved feature, being carried by both vertebrate and invertebrate sequences. It is biosynthesized as a precursor protein that is cleaved into an N-terminal fragment and a C-terminal peptide. In neuroendocrine cells, 7B2 functions as a specific chaperone for the proprotein convertase (PC) 2. Through the sequence around its Pro-Pro-Asn-Pro-Cys-Pro motif, it binds to an inactive proPC2 and facilitates its transport from the endoplasmic reticulum to later compartments of the secretory pathway where the zymogen is proteolytically matured and activated. Its C-terminal peptide can inhibit PC2 in vitro and may contribute to keep the enzyme transiently inactive in vivo. The PC2-7B2 model defines a new neuroendocrine paradigm whereby proteolytic activation of prohormones and proneuropeptides in the secretory pathway is spatially and temporally regulated by the dynamics of interactions between converting enzymes and their binding proteins. Interestingly, unlike PC2-null mice, which are viable, 7B2-null mutants die early in life from Cushing's disease due to corticotropin ('ACTH') hypersecretion by the neurointermediate lobe, suggesting a possible involvement of 7B2 in secretory granule formation and in secretion regulation. The mechanism of this regulation is yet to be elucidated. 7B2 has been shown to be a good marker of several neuroendocrine cell dysfunctions in humans. The possibility that anomalies in its structure and expression could be aetiological causes of some of these dysfunctions warrants investigation.

Amino Acid Sequence↗
PubMed1994

The post-translational processing of chromogranin A in the pancreatic islet: involvement of the eukaryote subtilisin PC2.

The post-translational processing of chromogranin A (CGA) and the nature of the enzyme(s) involved were investigated in rat pancreatic islet and insulinoma tissue. Pulse-chase radiolabelling experiments using sequence-specific antisera showed that the 98 kDa (determined by SDS/PAGE) precursor was processed to an N-terminal 21 kDa peptide, a C-terminal 14 kDa peptide and a 45 kDa centrally located peptide with a rapid time course (t1/2 approx. 30 min) after an initial delay of 30-60 min. The 45 kDa peptide was, in turn, converted partially into a 5 kDa peptide with pancreastatin immunoreactivity and a 3 kDa peptide with WE-14 immunoreactivity over a longer time period. Incubation of bovine CGA with rat insulinoma secretory-granule lysate produced peptides of 18, 16 and 40 kDa via intermediates of 65 and 55 kDa. N-terminal sequence analysis indicated that cleavage occurred at the conserved paired basic sites Lys114-Arg115 and Lys330-Arg331, suggesting that cleavage of the equivalent sites (Lys129-Arg130 and Lys357-Arg358) in the rat molecule produced the initial post-translational products observed in intact pancreatic beta-cells. The enzyme activity responsible for the cleavage of bovine CGA co-chromatographed on DEAE-cellulose with the type-2 proinsulin endopeptidase and with PC2 immunoreactivity. The type-1 enzyme (PC1/3) appeared inactive towards CGA. The requirement for Ca2+ ions and an acidic pH for conversion was consistent with the involvement of a member of the eukaryote subtilisin family, and the composition of the released peptides in pulse-chase and secretion studies suggested that conversion occurred in the secretory-granule compartment. The overall catalytic rate as well as the relative susceptibilities of the Lys114-Arg115 and Lys330-Arg331 sites to cleavage were affected by pH, suggesting that the ionic environment of the processing compartment may play a role in the differential processing of CGA which is evident in various neuroendocrine cells.

Adrenal Glands↗
PubMed1995

Differences between the catalytic properties of recombinant human PC2 and endogenous rat PC2.

Human prohormone convertase PC2 was expressed in Xenopus oocytes and its properties were compared with those of the Type-2 endopeptidase of rat insulin secretory granules, previously identified as PC2 [Bennett, Bailyes, Nielson, Guest, Rutherford, Arden and Hutton (1992) J. Biol. Chem. 267, 15229-15236]. Recombinant PC2 had the same substrate specificity as the Type-2 endopeptidase, cleaving at the CA-junction (Lys64, Arg65) of human des-31,32-proinsulin to generate insulin; little activity was found toward human des-64,65-proinsulin or proinsulin itself. Recombinant PC2 was maximally active in 5-7 mM Ca2+ (K0.5 = 1.6 mM) whereas the Type-2 endopeptidase was maximally active in 0.5-1 mM Ca2+ (K0.5 = 40 microM). Both enzymes had a pH optimum of 5.0-5.5 but the Type-2 endopeptidase was active over a wider pH range. Two molecular forms of recombinant PC2 (71 kDa and 68 kDa) were found, both had an intact C-terminus but differed by the presence of the propeptide. The endogenous PC2 comprised several overlapping forms (size range 64-68 kDa), approximately two-thirds of which lacked C-terminal immunoreactivity. Part of the size difference between recombinant and endogenous PC2 was attributable to differences in N-glycosylation. The different post-translational proteolytic modifications of recombinant and endogenous PC2 did not account for the different pH and Ca2+ sensitivities shown by the enzymes. A modulating effect of carbohydrate on enzyme activity could not be excluded.

Animals↗
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↗
PubMed1997

pH-dependent processing of secretogranin II by the endopeptidase PC2 in isolated immature secretory granules.

We have previously characterized the processing of secretogranin II (SgII) in PC12 cells that were stably transfected with the endopeptidase PC2. Here we show that processing of SgII can be observed in isolated immature secretory granules (ISGs) derived from this cell line in a temperature- and ATP-dependent manner. The stimulatory effect of ATP on processing can be attributed to the activation of the vacuolar H(+)-ATPase and a concomitant decrease in intragranular pH. The immature secretory granule therefore provides an adequate environment for correct processing of SgII by PC2. The rate of SgII processing was strongly dependent on the intragranular pH, suggesting that processing of SgII can be used as a pH indicator for the granule interior. A standard curve was prepared using SgII processing in ISGs equilibrated at a range of pH values. The extent of processing in ISGs incubated in the presence of ATP at physiological pH was compared with the standard curve, and the intragranular pH was determined. From these observations, we propose an intragranular pH of 6.3 +/- 0.1 for ISGs in a physiological buffer in the presence of ATP. Hence, the pH of ISGs seems to be similar to the pH of the trans-Golgi network (TGN) and is clearly higher than the pH of mature secretory granules (pH 5.0-5.5). Interestingly, no processing of SgII could be observed in a membrane fraction that is highly enriched in TGN under conditions for which processing was readily obtained in isolated ISGs.

Animals↗
PubMed1997

Mutations within the propeptide, the primary cleavage site or the catalytic site, or deletion of C-terminal sequences, prevents secretion of proPC2 from transfected COS-7 cells.

PC2 is a neuroendocrine endoprotease involved in the processing of prohormones and proneuropeptides. PC2 is synthesized as a proenzyme which undergoes proteolytic maturation within the cellular secretory apparatus. Cleavage occurs at specific sites to remove the N-terminal propeptide. The aim of the present study was to investigate structural requirements for the transfer of proPC2 through the secretory pathway. A series of mutant proPC2 constructs were transfected into COS-7 cells and the fate of the expressed proteins followed by pulse-chase analysis and immunocytochemistry. Human PC2 was secreted relatively slowly, and appeared in the medium primarily as proPC2 (75 kDa), together with much lower amounts of a processed intermediate (71 kDa) and mature PC2 (68 kDa). Mutations within the primary processing site or the catalytic triad caused the protein to accumulate intracellularly, whereas deletion of part of the propeptide, the P-domain or the C-terminal regions also prevented secretion. Immunocytochemistry showed that wild-type hPC2 was localized mainly in the Golgi, whereas two representative mutants showed a distribution typical of proteins resident in the endoplasmic reticulum. The results suggest that proenzyme processing is not essential for secretion of PC2, but peptides containing mutations that affect the ability of the propeptide (and cleavage sites) to fold within the catalytic pocket are not transferred beyond the early stages of the secretory pathway. C-terminal sequences may be involved in stabilizing such conformations.

Amino Acid Sequence↗
PubMed1996

Role of PC2 in proenkephalin processing: antisense and overexpression studies.

The contribution of the prohormone-processing enzyme PC2 to the proteolytic maturation of proenkephalin was examined in three sets of studies. In the first, the processing of this precursor was compared in PC2-rich (Rin5f) and PC2-lacking (AtT-20) cell lines expressing proenkephalin by virtue of stable transfection. These studies showed that the time frame for processing of this precursor is cell line specific, with AtT-20 cells processing proenkephalin to peptide B much more rapidly than Rin cells. However, the latter cell line processed proenkephalin much more extensively, i.e., produced a greater proportion of the penta- to octapeptide enkephalins. The involvement of PC2 in these later processing events was analyzed by examining the processing of proenkephalin in PC2-overexpressing AtT-20 cell lines. These experiments yielded a processing profile similar to that observed for Rin cells, although the time frame of initial processing was similar to that found in AtT-20 cells. To confirm the physiological involvement of proenkephalin in the production of the small opioid peptides, we generated a Rin cell line in which the production of PC2 was impaired due to stable expression of antisense mRNA to this enzyme. These experiments provided conclusive evidence that the generation of Met-enkephalin-Arg-Phe and Met-enkephalin-Arg-Gly-Leu, but not the larger enkephalin-containing peptides, is mediated by PC2. Taken together, our data support the idea that PC2 is physiologically capable of mediating only the later processing steps of neuropeptide precursors. PC2 thus appears to be the primary enzyme responsible for the generation of bioactive opioid peptide species from proenkephalin.

Animals↗
PubMed1998

Manipulation of disulfide bonds differentially affects the intracellular transport, sorting, and processing of neuroendocrine secretory proteins.

To investigate if the prevention of disulfide bond formation affects the intracellular transport, sorting, and processing of a distinct set of neuroendocrine proteins in the regulated secretory pathway, we have treated Xenopus intermediate pituitaries with the thiol-reducing agent dithiothreitol. Pulse-chase incubations in combination with immunoprecipitation analysis were used to monitor the fates of the prohormone proopiomelanocortin (POMC), prohormone convertase PC2 and its helper protein 7B2, as well as secretogranin III. Manipulation of the disulfide bonds in POMC and proPC2 blocked their transport to the trans-Golgi network and strongly inhibited their processing. Reduction of the single disulfide bond in 7B2 did not disturb its transport and cleavage, but caused its missorting to the constitutive secretory pathway. Moreover, the liaison between proPC2 and 7B2 was prevented. Dithiothreitol did not affect transport, sorting, and cleavage of secretogranin III, which lacks disulfide bonds. When the reducing agent was washed away, POMC processing, proPC2 maturation, and the association between proPC2 and 7B2 were reestablished. Collectively, our findings indicate that manipulation of disulfide bonds differentially affects the fates of neuroendocrine proteins during their transit through the secretory pathway.

Animals↗
PubMed2006

Improving function and survival of pancreatic islets by endogenous production of glucagon-like peptide 1 (GLP-1).

Glucagon-like peptide 1 (GLP-1) is a hormone that has received significant attention as a therapy for diabetes because of its ability to stimulate insulin biosynthesis and release and to promote growth and survival of insulin-producing beta cells. While GLP-1 is produced from the proglucagon precursor by means of prohormone convertase (PC) 1/3 activity in enteroendocrine L cells, the same precursor is differentially processed by PC2 in pancreatic islet alpha cells to release glucagon, leaving GLP-1 trapped within a larger fragment with no known function. We hypothesized that we could induce GLP-1 production directly within pancreatic islets by means of delivery of PC1/3 and, further, that this intervention would improve the viability and function of islets. Here, we show that adenovirus-mediated expression of PC1/3 in alpha cells increases islet GLP-1 secretion, resulting in improved glucose-stimulated insulin secretion and enhanced survival in response to cytokine treatment. PC1/3 expression in alpha cells also improved performance after islet transplantation in a mouse model of type 1 diabetes, possibly by enhancing nuclear Pdx1 and insulin content of islet beta cells. These results demonstrate a unique strategy for liberating GLP-1 from directly within the target organ and highlight the potential for up-regulating islet GLP-1 production as a means of treating diabetes.

Adenoviridae↗
PubMed2006

Mu opioid receptor A118G polymorphism in association with striatal opioid neuropeptide gene expression in heroin abusers.

Mu opioid receptors are critical for heroin dependence, and A118G SNP of the mu opioid receptor gene (OPRM1) has been linked with heroin abuse. In our population of European Caucasians (n = 118), approximately 90% of 118G allelic carriers were heroin users. Postmortem brain analyses showed the OPRM1 genotype associated with transcription, translation, and processing of the human striatal opioid neuropeptide system. Whereas down-regulation of preproenkephalin and preprodynorphin genes was evident in all heroin users, the effects were exaggerated in 118G subjects and were most prominent for preproenkephalin in the nucleus accumbens shell. Reduced opioid neuropeptide transcription was accompanied by increased dynorphin and enkephalin peptide concentrations exclusively in 118G heroin subjects, suggesting that the peptide processing is associated with the OPRM1 genotype. Abnormal gene expression related to peptide convertase and ubiquitin/proteosome regulation was also evident in heroin users. Taken together, alterations in opioid neuropeptide systems might underlie enhanced opiate abuse vulnerability apparent in 118G individuals.

Adolescent↗
PubMed1992

Identification and analysis of the gene encoding human PC2, a prohormone convertase expressed in neuroendocrine tissues.

In recent studies we have identified PC2 and PC3, members of a family of serine proteases that are related structurally to subtilisin, and have provided evidence that these are involved in the tissue-specific processing of prohormones and neuropeptides. PC2 is expressed at high levels in the islets of Langerhans, where it participates in the processing of proinsulin to insulin (S.P.S. and D.F.S., unpublished data). To evaluate the regulated expression of the human PC2 (hPC2) gene we have analyzed its structure and characterized its promoter. A map of the gene was constructed by using 11 clones isolated from two human genomic DNA libraries. The gene spans greater than 130 kilobase pairs and consists of 12 exons. Comparison with the structure of the gene encoding human furin, another member of this superfamily, revealed a high degree of conservation of exon-intron junctions. The hPC2 gene was localized to chromosome 20, band p11.2. The 5' flanking region of the hPC2 gene is very G+C-rich and contains six potential Sp1 binding sites but no TATA or CAAT box. Expression of chloramphenicol acetyltransferase reporter fusions containing the putative promoter region was observed to occur in beta TC-3 mouse insulinoma cells but not in HepG2 human hepatoma cells, consistent with the known tissue-specific pattern of expression of the hPC2 gene. Analysis of the level of chloramphenicol acetyltransferase activity with several deletion mutants identified the region from -1100 to -539 from the translation start site as essential for hPC2 promoter activity.

Amino Acid Sequence↗
PubMed1994

The neuroendocrine polypeptide 7B2 is an endogenous inhibitor of prohormone convertase PC2.

The subtilisin-like prohormone convertase PC2 and the polypeptide 7B2 (an intracellularly cleaved protein of unknown function) are both selectively present in the regulated secretory pathway of neurons and endocrine cells. Here we demonstrate that intact recombinant 7B2 is a potent inhibitor of PC2 and prevents proPC2 cleavage in vitro, whereas the 7B2 cleavage product is virtually inactive. The PC2-related proteinase PC1/PC3 is not inhibited by 7B2. Furthermore, the carboxyl-terminal half of the 7B2 protein sequence is distantly related to the so-called potato inhibitor I family (which includes subtilisin inhibitors). Our findings indicate that 7B2 is a physiological inhibitor of PC2 and may provide alternative avenues for the manipulation of peptide hormone levels.

Amino Acid Sequence↗
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↗
PubMed1997

Defective prohormone processing and altered pancreatic islet morphology in mice lacking active SPC2.

The prohormone convertase SPC2 (PC2) participates in the processing of proinsulin, proglucagon, and a variety of other neuroendocrine precursors, acting either alone or in conjunction with the structurally related dense-core granule convertase SPC3 (PC3/PC1). We have generated a strain of mice lacking active SPC2 by introducing the neomycin resistance gene (Neor) into the third exon of the mSPC2 gene. This gene insertion results in the synthesis of an exon 3-deleted form of SPC2 that does not undergo autoactivation and is not secreted. The homozygous mutant mice appear to be normal at birth. However, they exhibit a small decrease in rate of growth. They also have chronic fasting hypoglycemia and a reduced rise in blood glucose levels during an intraperitoneal glucose tolerance test, which is consistent with a deficiency of circulating glucagon. The processing of proglucagon, prosomatostatin, and proinsulin in the alpha, delta, and beta cells, respectively, of the pancreatic islets is severely impaired. The islets in mutant mice at 3 months of age show marked hyperplasia of alpha and delta cells and a relative diminution of beta cells. SPC2-defective mice offer many possibilities for further delineating neuroendocrine precursor processing mechanisms and for exploring more fully the physiological roles of many neuropeptides and peptide hormones.

Animals↗