Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P01189 (beta-endorphin)
 21,003 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Specific antisera against mammalian prohormone convertases PC1 and PC2 have been used to examine, light-immunocytochemically, the distribution of these enzymes in the pituitary gland of five different species of anuran amphibians (Rana catesbeiana, Bufo japonicus formosus, Xenopus laevis, Rana brevipoda porosa, and Buergeria japonica). A differential pattern of immunoreactivity of PC1 and PC2 was found among these species. Only PC1 was found in the corticotrope cells of the pars distalis in R. catesbeiana, B. japonicus formosus, and X. laevis. Only PC2 was observed in these cells in B. japonica, whereas both PC1 and PC2 were present in these cells in R. brevipoda porosa. PC2 immunoreactivity was always observed in melanotrope cells in the pars intermedia of all of the species, but it coexisted with PC1 immunoreactivity only in R. catesbeiana and X. laevis. The nerve fibers and terminals in the pars nervosa in all of the species were intensely immunopositive with both PC1 and PC2 antibodies. Immunoelectron microscopy on B. japonicus formosus and B. japonica, by means of double-labeling with gold particles of different sizes, revealed that almost all the adrenocorticotropin-positive secretory granules in the corticotrope cells and alpha-melanophore-stimulating-hormone-positive secretory granules in the melanotrope cells were also labeled with either PC1 or PC2 antibodies. This study suggests that PC1 and PC2 are involved in the intracellular proteolytic cleavage of proopiomelanocortin in amphibian pituitary glands, a situation similar to that proposed for mammals.
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PMID:Immunocytochemical localization of prohormone convertases PC1 and PC2 in the anuran pituitary gland: subcellular localization in corticotrope and melanotrope cells. 913 61

Prohormone substrates are required for investigation of the proteolytic processing of prohormones and proproteins into active peptide hormones and neurotransmitters. However, the lack of prohormone proteins has been a limiting factor in elucidating proteolytic mechanisms for conversion of prohormones into active peptides. Therefore, in this study, cloned cDNAs encoding the prohormones proenkephalin (PE), pro-neuropeptide Y (pro-NPY), pro-opiomelanocortin (POMC), and beta-protachykinin (beta-PT) were utilized to express recombinant prohormones in Escherichia coli. High-level expression of milligrams of prohormones was achieved with the pET3c expression vector utilizing the T7 promoter for production of PE, pro-NPY, and POMC, as demonstrated by SDS-PAGE gel electrophoresis, Western blots, and 35S-methionine labeling. In addition, beta-PT was expressed at high levels as fusion proteins with the maltose-binding protein and glutathione S-transferase by the pMAL-c and pGEX-2T expression vectors, respectively. Relative rates of processing by the established processing proteases "prohormone thiol protease" (PTP), 70-kDa aspartyl protease, and PC1/ 3 and PC2 (PC, prohormone convertase) were examined with purified PE, pro-NPY, and POMC. Distinct preferences of processing enzymes for different prohormones was demonstrated. PTP preferred PE and pro-NPY substrates, whereas little processing of POMC was detected. In contrast, the 70-kDa aspartyl protease cleaved POMC more readily than pro-NPY or PE. However, PC1/3 and PC2 prefer POMC as substrate. Demonstration of selectivity of processing enzymes for prohormone substrates illustrates the importance of expressing recombinant prohormones for in vitro processing studies.
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PMID:High-level expression of the prohormones proenkephalin, pro-neuropeptide Y, proopiomelanocortin, and beta-protachykinin for in vitro prohormone processing. 917 94

We studied the extent of cellular inhibitory activity of alpha1-antitrypsin Portland (alpha1-PDX), a potent inhibitor of proprotein convertases of the subtilisin/kexin type. We compared the inhibitory effects of alpha1-PDX on the intracellular processing of two model precursors (pro-7B2 and POMC) mediated by six of the seven known mammalian convertases, namely furin, PC1, PC2, PACE4, PC5-A, PC5-B, and PC7. The substrates selected were pro7B2, a precursor cleaved within the trans-Golgi network (TGN), and pro-opiomelanocortin, which is processed in the TGN and secretory granules. Biosynthetic analyses were performed using either vaccinia virus expression in BSC40, GH4C1, and AtT20 cells, or stable transfectants of alpha1-PDX in AtT20 cells. Results revealed that alpha1-PDX inhibits processing of these precursors primarily within the constitutive secretory pathway and that alpha1-PDX is cleaved into a shorter form by some convertases. Evidence is presented demonstrating that in contrast to the full-length alpha1-PDX (64 kDa), the cleaved (56 kDa) secreted product does not significantly inhibit furin activity in vitro. Cellular expression of alpha1-PDX results in modified contents of mature secretory granules with increased levels of partially processed products. Biosynthetic and immunocytochemical analyses of AtT20/alpha1-PDX cells demonstrated that alpha1-PDX is primarily localized within the TGN, and that a small proportion enters secretory granules where it is mostly stored as the cleaved product.
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PMID:Alpha1-antitrypsin Portland inhibits processing of precursors mediated by proprotein convertases primarily within the constitutive secretory pathway. 933 89

A variety of proteins and peptides are produced through limited proteolysis of precursors at paired basic residues. This proteolytic bioactivation is carried out by subtilisin-like proteases, called convertases. The mRNAs of several convertases are expressed during prenatal life as well as in P19 embryonal carcinoma cells, which are a model of the totipotent cells of the embryo before and at the time of implantation. To determine whether converting activities accompany convertase mRNA expression in the early embryo, we transferred the gene of pro-opiomelanocortin (POMC) into P19 cells, by lipofection, and searched for the presence of mature peptides by high-performance liquid chromatography and radioimmunoassay techniques. In P19 cells, POMC, a precursor of several endocrine peptides, is mainly processed to beta-lipotropin rather than to beta-endorphin, both peptides having been identified by their immunoreactivity, polarity, and molecular size. These results indicate that converting capacities appear early in the embryo and that they are more similar to the activity of furin and of convertase PC1 than that of convertase PC2 in their cleavage selectivity of POMC sites. Efficiency of POMC processing can reach 50%, suggesting that convertases, with other proteases, can have an important role in ontogenesis. As for other peptide precursors in endocrine cells, the conversion of POMC in P19 cells was inhibited by the biosynthetic replacement of its arginine residues by the analog canavanine. However, the incorporation of canavanine into P19 cells also inhibited peptide secretion, suggesting that inhibition of conversion in these cells as well as in endocrine cells could indirectly result from the impairment of intracellular traffic and not only from a direct inhibition of the converting activity.
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PMID:Proteolytic profile of recombinant pro-opiomelanocortin in embryonal carcinoma P19 cells: conversion to beta-lipotropin and secretion are inhibited following incubation with canavanine. 940 43

Prohormone convertase (PC) 1/3 and PC2 are involved in post-translational processing of endocrine tissues, including the pancreatic islets and pituitary glands. Our immunohistochemical studies disclosed the presence of PC1/3 and PC2 in non-neoplastic pituitary glands, especially in corticotrophs, gonadotrophs, and thyrotrophs. Among 58 pituitary adenomas obtained by trans-sphenoidal surgery, adrenocorticotropin (ACTH)-secreting adenomas showed a high incidence of the presence of PC1/3 and PC2, i.e., nine of nine cases were positive for ACTH. Five of nine cases showed consistency between PC2 localization and alpha-melanocyte stimulating hormone immunoreactivity, which suggests the functional correlation between PC2 and the processing of ACTH. In four cases, we observed inconsistency in immunolocalization, which suggested the possibility of inactive PC2 and abnormal processing of alpha-melanocyte stimulating hormone. The high incidence of PC1/3 and PC2 in nonfunctioning adenomas might be related to the processing of chromogranin A.
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PMID:Localization of prohormone convertases 1/3 and 2 in the human pituitary gland and pituitary adenomas: analysis by immunohistochemistry, immunoelectron microscopy, and laser scanning microscopy. 952 68

The prohormone convertase PC2 is one of the major subtilisin/kexin-like enzymes responsible for the formation of small bioactive peptides in neural and endocrine cells. This convertase is unique among the members of the subtilisin/kexin-like mammalian serine proteinase family in that it undergoes zymogen processing of its inactive precursor proPC2 late along the secretory pathway and requires the help of a PC2-specific binding protein known as 7B2. We hypothesized that some of these unique properties of PC2 are dictated by the presence of PC2-specific amino acids, which in the six other known mammalian convertases are otherwise conserved but distinct. Accordingly, six sites were identified within the catalytic segment of PC2. Herein we report on the site-directed mutagenesis of Tyr194 and of the oxyanion hole Asp309 and the consequences of such mutations on the cellular expression and enzyme activity of PC2. The data show that the Y194D mutation markedly increases the ex vivo ability of PC2 to process proopiomelanocortin (POMC) into beta-endorphin in cells devoid of 7B2, e.g. BSC40 cells. In these cells, expression of native PC2 does not result in the secretion of measurable in vitro activity against a pentapeptide fluorogenic substrate. In contrast, secreted Y194D-PC2 exhibited significant enzymatic activity, even in the absence of 7B2. Based on co-immunoprecipitations and Western blots, binding assays indicate that Tyr194 participates in the interaction of PC2 with 7B2, and that the oxyanion hole Asp309 is critical for the binding of proPC2 with pro7B2.
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PMID:Residues unique to the pro-hormone convertase PC2 modulate its autoactivation, binding to 7B2 and enzymatic activity. 964 70

In the central and peripheral nervous systems, the neuropeptide precursor proenkephalin must be endoproteolytically cleaved by enzymes known as prohormone convertases 1 and 2 (PC1 and PC2) to generate opioid-active enkephalins. In this study, we have investigated the specificity of recombinant mouse PC2 for proenkephalin-related internally quenched (IQ) peptides, for methylcoumarin amide-based fluorogenic peptides, and for recombinant rat proenkephalin. IQ peptides exhibited specificity constants (kcat/Km) between 9.4 x 10(4) M-1 s-1 (Abz-Val-Pro-Arg-Met-Glu-Lys-Arg-Tyr-Gly-Gly-Phe-Met-Gln-EDDnp+ ++; where Abz is ortho-aminobenzoic acid and EDDnp is N-(2, 4-dinitrophenyl)ethylenediamine)) and 0.24 x 10(4) M-1 s-1 (Abz-Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg-Pro-Glu-EDDnp), with the peptide B to Met-enk-Arg-Phe cleavage preferred (Met-enk is met-enkephalin). Fluorogenic substrates with P1, P2, and P4 basic amino acids were hydrolyzed with specificity constants ranging between 2.0 x 10(3) M-1 s-1 (Ac-Orn-Ser-Lys-Arg-MCA; where MCA is methylcoumarin amide) and 1.8 x 10(4) M-1 s-1 (<Glu-Arg-Thr-Lys-Arg-MCA; where <Glu is pyroglutamic acid). Substrates containing only a single basic residue were not appreciably hydrolyzed, and substrates lacking a P4 Arg exhibited kcat of less than 0.05 s-1. Substitution of ornithine for Lys at the P4 position did not significantly affect the kcat but increased the Km 2-fold. Data from both sets of fluorogenic substrates supported the contribution of a P4 Arg to PC2 preference. Analysis of proenkephalin reaction products using immunoblotting and gel permeation chromatography demonstrated that PC2 can directly cleave proenkephalin and that the generation of small opioid peptides from intermediates is mediated almost entirely by PC2 rather than by PC1. These results are in accord with the analysis of PC2 knock-out brains, in which the amounts of three mature enkephalins were depleted by more than three-quarters.
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PMID:Specificity of prohormone convertase 2 on proenkephalin and proenkephalin-related substrates. 971 97

The hypothesis proposed here presents a mechanism of melatonin action, which may explain the role of this neurohormone in the genesis of various human pathologies, including fetal abnormalities. It assumes that monomeric or dimeric forms of indoloderived compounds such as melatonin and precursors of melanin have the ability to selectively stimulate the synthesis of prohormone 1 convertase (PC1) or prohormone 2 convertase (PC2), in proportion to their concentrations in the body. Thus, the mean circadian level of melatonin, by determining the manner and rapidity of proopiomelanocortin (POMC) cleavage, would also determine the mean proopiomelanocortin (POMC) level, maintained in dynamic equilibrium as a result of the simultaneous influence of testosterone, estradiol and cortisol on the intensity of POMC mRNA synthesis. The correlative proportions between the activity of PC1 and PC2 would therefore shape the character of hormonal balance in the organism, and in particular the mean ACTH concentration that determines the level of cyclic adenosine monophosphate (cAMP) concentration in its cells. The hypothesis also suggests that melatonin, by influencing the concentration of ACTH and beta-endorphin and their relative proportion could determine the stimulation or suppression of the immune system, thereby confirming its role as an immunomodulator. A disturbance in the above model of immunohormonal equilibrium, resulting from, for example, decreased pineal efficiency, would lead to stimulation of an alternative mode of achieving homeostasis, i.e. increase in concentration of melanin monomers and dimers, with concomitant high activity of tyrosine kinase and high cyclic guanosine monophosphate (cGMP) concentration in the cells. According to the proposed hypothesis, the risk of bearing a developmentally handicapped child would be highest in a woman with a high circadian secretion of melatonin, i.e. with domination of melatonin dimers and high PC1 activity, a condition which may be additionally aggravated by the exposure of the mother to adverse environmental factors or by immunohormonal disturbances. The hypothetical break-up of maternal melatonin dimers when crossing placenta would be the cause of excessive concentration of melatonin monomers and high PC2 activity in the fetus, and thus it should be the reason for very low levels of vimentin filaments and cAMP concentration in embryonal cells, the latter being directly responsible for inducing fetal pathologies.
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PMID:Can high maternal melatonin concentrations be responsible for inducing fetal pathologies, and can melatonin participate in immunohormonal homeostasis by determining prohormone convertase activity?--Hypothesis and facts. 982 29

We have carried out an investigation into the processing of the enkephalin-like immunoreactivity reported in breast tissue using two human breast tumour cell lines and a mouse tumour cell line. A 46 kDa form of proenkephalin (PE) has been observed in the cell lysates of two human breast tumour cell lines (MCF-7, ZR-75-1) and the mouse androgen-responsive Shionogi breast carcinoma cell line (SC115). PE processing in the cell lysates of these cells was assessed by a specific met-enkephalin RIA. The basal levels of processed PE in the MCF-7, ZR-75-1 and SC115 cell lysates were 30, 30 and 76% respectively. The processing enzymes PC1 and PC2, which have been implicated in the differential processing of PE, were detected by immunoblot analysis in these cells. PC1 was found within the cell extracts of all three cell lines. PC2 was only observed in the SC115 cell line, which may account for the higher percentage of processed PE measured. The cDNA of PC2 has been transfected into ZR-75-1 cells and this was accompanied by an increase in the level of processed PE from 30 to 76%. These breast tumour cell lines may provide a useful insight into the function of enkephalin-containing peptides in breast cancer.
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PMID:The differential processing of proenkephalin A in mouse and human breast tumour cell lines. 1033 50

Many peptide hormones and neuropeptides are processed by members of the subtilisin-like family of prohormone convertases (PCs), which are either soluble or integral membrane proteins. PC1 and PC2 are soluble PCs that are primarily localized to large dense core vesicles in neurons and endocrine cells. We examined whether PC1 and PC2 were active when expressed as membrane-tethered proteins, and how tethering to membranes alters the biosynthesis, enzymatic activity, and intracellular routing of these PCs. PC1 and PC2 chimeras were constructed using the transmembrane domain and cytoplasmic domain of the amidating enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The membrane-tethered PCs were rerouted from large dense core vesicles to the Golgi region. In addition, the chimeras were transiently expressed at the cell surface and rapidly internalized to the Golgi region in a fashion similar to PAM. Membrane-tethered PC1 and PC2 exhibited changes in pro-domain maturation rates, N-glycosylation, and in the pH and calcium optima required for maximal enzymatic activity against a fluorogenic substrate. In addition, the PC chimeras efficiently cleaved endogenous pro-opiomelanocortin to the correct bioactive peptides. The PAM transmembrane domain/cytoplasmic domain also prevented stimulated secretion of pro-opiomelanocortin products in AtT-20 cells.
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PMID:Activation and routing of membrane-tethered prohormone convertases 1 and 2. 1045 38


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