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PubMed1990

The helminth Schistosoma mansoni expresses a peptide similar to human beta-endorphin and possesses a proopiomelanocortin-related gene.

Opioid peptides, a group of transmitter substances with a high degree of phylogenic conservation, have many different functions, including a role in modulation of cells of the immune system. We have postulated the existence of such peptides in the parasite Schistosoma mansoni in view of their possible role in host-parasite interactions. In this report we show that beta-endorphin, which is a member of the opiate family and is derived from the proopiomelanocortin (POMC) precursor, is present in S. mansoni. Southern blots of cercarial genomic DNA, hybridized with two oligonucleotide probes complementary to highly conserved POMC sequences, showed a POMC-related gene in this trematode. Northern blot analysis of adult worm RNA indicated that this gene was actively transcribed. Significant amounts of beta-endorphin, adrenocorticotropin (ACTH), and alpha-melanocyte stimulating hormone (alpha-MSH) were detected in all developmental stages of the parasite by radioimmunoassays with the use of antisera to human peptides. By means of reverse-phase high-performance liquid chromatography (HPLC), we found that the parasite beta-endorphin-like material and the human opiate have a high degree of homology. These results appear to constitute the first demonstration of a POMC-related gene transcribed in an invertebrate.

Adrenocorticotropic Hormone↗
PubMed1991

Expression of pre-opiomelanocortin (POMC) mRNA in undifferentiated and in vitro differentiated human neuroblastoma cell lines.

Several human neuroblastoma cell lines express transcripts of the proopiomelanocortin (POMC) gene of 0.8 kb and 9.5 kb, thus different from the characteristic pituitary 1.2 kb POMC mRNA. The expression of POMC mRNA decreases when the cell lines are differentiated with retinoic acid or alpha-difluoromethylornithine. Since both POMC and MYCN genes are present on the same chromosome segment (2p23), it is possible that the 2 genes are coamplified and coexpressed. Alternatively the expression of opiate peptides by neuroblastoma cells might act as an autocrine factor or it may modulate the action of tumour infiltrating lymphocytes.

Cell Differentiation↗
PubMed1990

An in vivo characterization of the cleavage site specificity of the insulin cell prohormone processing enzymes.

Most peptide hormones and neurotransmitters are synthesized as larger precursor proteins, which are post-translationally processed to mature bioactive products. An early event in prohormone maturation is endoproteolytic cleavage, occurring usually at pairs of basic amino acids (e.g. Lys-Arg). Since many of the characteristics of a prohormone endoprotease are unknown, distinguishing these enzymes from other cellular proteases in vitro has been difficult. In this report, the substrate specificity of a model prohormone processing system, the insulinoma cell line Rin m5F, was characterized in vivo to establish a set of criteria by which putative proinsulin endoproteases may be assessed. To determine the role of composition of the paired basic amino acid site in directing cleavage, a series of mutant prohormones containing altered cleavage sites was constructed and expressed in Rin m5F cells. Proopiomelanocortin (POMC) was used as a substrate since this prohormone was previously shown to be processed by these cells. To control for positional effects, all four permutations of lysine and arginine (Lys-Arg, Arg-Arg, Arg-Lys, and Lys-Lys) were introduced at both the efficiently processed cleavage site separating the ACTH and beta-lipotropin (beta-LPH) domains of POMC and at the inefficiently processed site in the beta-endorphin sequence near the COOH-terminus of the precursor. His-Arg and Met-Arg sites were also introduced at the ACTH/beta-LPH junction to assess the requirement for paired lysines and arginines. Identification of POMC-derived peptides demonstrated efficient processing of Lys-Arg and inefficient processing of Lys-Lys and Arg-Lys sites at both positions in the prohormone. The Arg-Arg sequence, however, was processed in a position-dependent manner, being efficiently cleaved between ACTH and beta-LPH but only about 50% processed within beta-endorphin. His-Arg was not cleaved in Rin m5F cells, although surprisingly Met-Arg was partially processed. These results indicate a strict preference of the insulinoma prohormone endoprotease(s) for paired basic amino acids ending in arginine, but that processing efficiency of some sequences may be modulated by location within the precursor molecule.

Amino Acid Sequence↗
PubMedDate not supplied

[Expression in Escherichia coli of hybrid genes containing sequences coding the bovine adrenocorticotropic hormone].

E. coli strains producing a hybrid protein, containing adrenocorticotropic hormone (ACTH) and protein A of S. aureus was obtained. The sequence coding for ACTH was obtained from the bovine proopiomelanocortin cDNA and, after the modification of the 5'- and 3'-terminal parts, was linked with the protein A gene and its derivatives due to synthetic adaptors. Three forms of ACTH gene, coding this hormone with differing N-terminal amino acid were used to construct the fusion gene. The hybrid proteins contain Asp-Pro or (Asp)4-Lys sequences for obtaining ACTH by acid or enterokinase treatment, respectively. It is shown that each of the constructed plasmids direct the synthesis of hybrid protein in E. coli. This protein was purified by the use of IgG-sepharose. The level of the expression of the hybrid protein is 4 mg/l of the bacterial culture. Most of the synthesized protein is secreted into the periplasmic space.

Adrenocorticotropic Hormone↗
PubMed1990

Yeast KEX1 protease cleaves a prohormone processing intermediate in mammalian cells.

A vaccinia virus vector was used to express the yeast KEX1 gene, which encodes a prohormone carboxypeptidase specific for the removal of basic amino acids from prohormone processing intermediates, in mammalian cells. When produced in BSC-40 cells, Kex1p was localized to the perinuclear region and conferred a large increase in enzymatic activity characteristic of this carboxypeptidase. Expression of the KEX1 gene together with the yeast KEX2 gene, which encodes a prohormone endopeptidase specific for cleavage at pairs of basic amino acids, and the mouse proopiomelanocortin (mPOMC) cDNA in BSC-40 cells resulted in the full conversion of mPOMC to mature peptides including gamma-lipotropin. This in vivo processing of mPOMC to mature peptides by the KEX2/KEX1 gene products demonstrates a significant functional homology of the basic prohormone processing machinery in yeast and neuroendocrine cells.

Animals↗
PubMed1990

Variants of the carboxyl-terminal KDEL sequence direct intracellular retention.

Soluble proteins which reside in the lumen of the endoplasmic reticulum share a common carboxyl-terminal tetrapeptide Lys-Asp-Glu-Leu (KDEL). Addition of the tetrapeptide to a normally secreted protein is both necessary and sufficient to cause retention in the endoplasmic reticulum. In order to characterize the critical residues in the KDEL signal, cDNAs encoding proneuropeptide Y (pro-NPY) with the 4-amino acid carboxyl-terminal extension KDEL or a series of KDEL variants were expressed in the AtT-20 cell line. AtT-20 cells, a mouse anterior pituitary corticotrope cell line, synthesize, process, and secrete the pro-ACTH/endorphin precursor. Since post-translational processing in AtT-20 cells has been extensively characterized, it provides a model system in which the processing of a foreign peptide precursor (pro-NPY) and the endogenous precursor (pro-ACTH/endorphin) can be compared. Altered cDNAs encoding pro-NPY with KDEL, DKEL, RDEL, KNEL, KDQL, or KDEA at the COOH terminus were used to generate stable AtT-20 cell lines. The processing of pro-NPY to neuropeptide Y and the carboxyl-terminal peptide was studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, tryptic peptide mapping, and radiosequencing. Addition of the tetrapeptides KDEL, DKEL, RDEL, or KNEL to the COOH terminus of the neuropeptide Y precursor, a peptide hormone normally processed and secreted from neuronal cells, caused complete intracellular retention of the unprocessed prohormone in AtT-20 cells. However, KDQL and KDEA-extended pro-NPY molecules were processed and secreted like wild-type pro-NPY when expressed in AtT-20 cells. The secretion of proNPY-derived peptides in these cell lines paralleled secretion of endogenous pro-ACTH/endorphin-derived products under both basal and stimulated conditions. These mutagenesis studies demonstrate that variants of the KDEL retention signal can direct intracellular retention.

Amino Acid Sequence↗
PubMed1990

Participation of the POMC-system in the effects of morphine on the avoidance reaction in albino rats.

The influence of the POMC-system on the effects of morphine during learning are studied using physical (adrenalectomy, stress) and drug impacts (morphine, methamizol, acetysal) on the functional activity of the system. Adrenalectomized rats are found to manifest a sharp rise in the plasma and adenopituitary levels of beta-endorphin and inhibition of learning. The facilitating effect of morphine on learning (in a dose of 5 mg/kg) is inverted against the background of adrenalectomy. Dexamethasone prevents this effect, which coincides with the decrease in the beta-endorphin level. Morphine in a dose of 10 mg/kg body mass enhances learning against the background of stress applied in advance. In unstressed animals the same dose of morphine results in sharp deterioration of learning. Naloxone prevents the positive effect of morphine under stress. The dose-dependent opposite effects of morphine on the processes of learning are assumed to be modulated by the changes taking place in the ratio of the opioid/antiopioid POMC-derivatives under functional and drug influences.

Adrenalectomy↗
PubMed1990

The neuroendocrine polypeptide 7B2 is a precursor protein.

The neuroendocrine protein 7B2 is highly conserved and widely present in neurons and endocrine cells. It is coexpressed with the prohormone proopiomelanocortin (POMC) in the intermediate lobe of the pituitary gland of Xenopus laevis. To study the biosynthesis of 7B2 in this amphibian, an anti-7B2 monoclonal antibody was used in immunoprecipitation analysis of newly synthesized radiolabeled proteins, produced by pulse and pulse-chase-incubated neurointermediate lobes. Following a 15-min pulse incubation, a single immunoprecipitable protein of 25 kDa was synthesized. During subsequent chase incubation, this newly synthesized 7B2 protein was processed to an 18-kDa immunoprecipitable form. Analysis of the chase incubation medium revealed that only the 18-kDa processed product of 7B2, and not 7B2 itself, had been secreted. This secretion is a regulated process because it was blocked completely by the dopamine receptor agonist apomorphine. A study of protein biosynthesis in lobes treated with tunicamycin to prevent N-linked glycosylation showed that in contrast to POMC and an 18-kDa derivative of POMC, neither 7B2 nor its 18-kDa derivative was glycosylated. Chemical and enzymatic peptide mapping showed that processing of 7B2 occurs in the carboxyl-terminal region. The function of the 7B2 protein is unknown; the present results show that 7B2 itself is a precursor molecule and can only have an intracellular function whereas an extracellular function can only be attributed to 7B2-derived peptides.

Amino Acid Sequence↗
PubMed1987

Pharmacological modulation of CRH-stimulated ACTH secretion by ketoconazole.

The effects of ketoconazole (KC) on secretion and biosynthesis of ACTH in rat anterior pituitary cells were investigated in vitro. KC inhibits the corticotropin releasing hormone (CRH) stimulated ACTH release from rat anterior pituitary fragments in a dose dependent fashion between 1.5 and 100 microM. The effect of CRH to release ACTH was fully restored after KC was removed from the medium. Similar effects were observed in primary cultures of rat anterior pituitary cells: KC decreased dose dependently the basal and CRH stimulated ACTH release. In addition, basal and CRH-stimulated mRNA coding for the ACTH precursor was reduced after preincubation with CK. The effects of KC on ACTH release and biosynthesis seems to be mediated by cyclic AMP (cAMP) since KC inhibits basal and CRH stimulated cAMP release and content within the same dose range. Since the stimulatory effect of cholera toxin, sodium fluoride and forskolin were dose dependently inhibited by KC and since the addition of dibutyryl cAMP abolished the inhibiting effect of KC, it is concluded that KC acts by inhibition of the catalytic component of the adenylate cyclase holoenzyme.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Expression of mouse proopiomelanocortin in an insulinoma cell line. Requirements for beta-endorphin processing.

Proopiomelanocortin (POMC) is a neuroendocrine precursor protein which is processed at paired basic amino acids in a tissue-specific manner. To study this phenomenon, a vaccinia virus recombinant, which directs the synthesis of mouse POMC (VV:mPOMC) was constructed and used to infect epithelial (BSC-40) and endocrine (Rin m5F) cell lines. Bona fide mPOMC was produced in both cell types and beta-endorphin immunoreactivity was secreted in a nonregulated manner from BSC-40 cells and in a regulated manner from Rin m5F cells. Although the precursor was not cleaved to smaller beta-MSH or beta-endorphin immunoreactive peptides in BSC-40 cell extracts, Rin m5F cells produced primarily authentic gamma-lipotropin and des-acetyl beta-endorphin. Furthermore, production of these peptides was restricted to the regulated secretory pathway in Rin m5F cells. Site-directed mutagenesis was then used to change the inefficiently recognized Lys-Lys potential cleavage site near the carboxyl terminus of beta-endorphin to Lys-Arg. Expression of the mutant precursor in Rin m5F cells resulted in the synthesis of both des-acetyl beta-endorphin and beta-endorphin.

Adenoma, Islet Cell↗