Yeast AMP pathway genes respond to adenine through regulated synthesis of a metabolic intermediate.

In Saccharomyces cerevisiae, AMP biosynthesis genes (ADE genes) are transcriptionally activated in the absence of extracellular purines by the Bas1p and Bas2p (Pho2p) transcription factors. We now show that expression of the ADE genes is low in mutant strains affected in the first seven steps of the pathway, while it is constitutively derepressed in mutant strains affected in later steps. Combined with epistasy studies, these results show that 5'-phosphoribosyl-4-succinocarboxamide-5-aminoimidazole (SAICAR), an intermediate metabolite of the pathway, is needed for optimal activation of the ADE genes. Two-hybrid studies establish that SAICAR is required to promote interaction between Bas1p and Bas2p in vivo, while in vitro experiments suggest that the effect of SAICAR on Bas1p-Bas2p interaction could be indirect. Importantly, feedback inhibition by ATP of Ade4p, catalyzing the first step of the pathway, appears to regulate SAICAR synthesis in response to adenine availability. Consistently, both ADE4 dominant mutations and overexpression of wild-type ADE4 lead to deregulation of ADE gene expression. We conclude that efficient transcription of yeast AMP biosynthesis genes requires interaction between Bas1p and Bas2p which is promoted in the presence of a metabolic intermediate whose synthesis is controlled by feedback inhibition of Ade4p acting as the purine nucleotide sensor within the cell.

Synergistic action of upstream elements and a promoter-proximal CRE is required for neuroendocrine cell-specific expression and second-messenger regulation of the gene encoding the human secretory protein secretogranin II.

Secretogranin II (SgII) is a secretory polypeptide stored in large dense core vesicles of neuroendocrine and neuronal cells. In order to characterize the molecular mechanisms underlying the tissue-specific expression of the SgII gene and its regulation by second-messenger pathways in endocrine and neuronal cells, we have cloned and characterized the human SgII gene. Sequence analysis revealed the existence of numerous putative cis-regulatory elements in the SgII gene promoter, including a consensus cyclic AMP-responsive element (CRE). Constructs containing different portions of the human SgII promoter fused to the luciferase reporter were transfected in AtT-20, SH-SY5Y, LLC-PK1 or COS-7 cells. Northern blot analysis showed that the endogenous SgII gene is more highly expressed in AtT-20 cells than in SH-SY5Y cells, and not expressed at all in LLC-PK1 cells. Treatment by forskolin or 12-O-tetradecanoylphorbol-13-acetate (TPA) caused a 1.5- and 10-fold increase, respectively, in SgII mRNA levels in SH-SY5Y cells but not in AtT-20 cells. Transfection experiments revealed that 4 kb of the human SgII promoter is sufficient to impart cell-specific expression of the reporter gene in the four cell lines studied. Specifically, in AtT-20 cells, a positive element located between -1.38 and -4 kb, in addition to the CRE, is responsible for the high expression of the SgII gene. In SH-SY5Y cells, a negative element located between -0.66 and -1.4 kb represses the activating effect of the CRE leading to an overall lower activity of fusion genes in these cells compared to the activity in AtT-20 cells. Finally, the promoter activity was very low in LLC-PK1 and COS-7 cells. Forskolin and TPA stimulated the activity of a SgII-luciferase fusion gene in SH-SY5Y but not in AtT-20 cells. Disruption of the CRE abolished the stimulatory effect of forskolin and TPA. These data suggest that the basal activity of the human SgII gene relies on cell-specific trans-acting factors in addition to factors that bind to the CRE and show that the regulation of this gene by second messengers is cell-specific and requires an intact CRE.

Identification of a novel secretogranin II-derived peptide (SgII(187-252)) in adult and fetal human adrenal glands using antibodies raised against the human recombinant peptide.

Molecular cloning of secretogranin II (SgII) in phylogenetically distant species has recently revealed the existence of a highly conserved 66-amino acid peptide flanked by preserved pairs of basic residues. This observation suggested that this peptide, named EM66, which had not been described to date, could be an important processing product of SgII. The aim of the present study was to investigate the possible occurrence of EM66 in the human adrenal gland. The EM66 peptide was generated in Escherichia coli, which was programmed to make a fusion protein containing the human EM66 sequence. The affinity-purified fusion protein was used to raise polyclonal antibodies in rabbits. The free EM66 peptide was obtained by cleavage of the fusion protein followed by high performance liquid chromatography purification. Immunohistochemical analysis using the EM66 antibodies revealed intense labeling of adrenochromaffin cells in the adult adrenal medulla and the fetal adrenal gland. A sensitive and specific RIA was developed and applied to the detection of EM66-like immunoreactivity in extracts of adult adrenal medulla and whole fetal adrenal gland after high performance liquid chromatographic analysis. A major immunoreactive species exhibiting the same retention time as recombinant EM66 was detected in both adult and fetal adrenal extracts. Taken together, these data demonstrate that posttranslational processing of SgII actually generates EM66 in the adrenal gland. The strong conservation of the amino acid sequence of EM66 in the vertebrate phylum and the occurrence of the mature peptide in both fetal and adult chromaffin cells suggest that EM66 could play an important physiological role in the human adrenal gland.

Identification of a novel secretogranin II-derived peptide in the adult and fetal human adrenal gland.

Molecular cloning of secretogranin II (SgII) in different species has revealed the existence of a highly conserved 66-amino acid peptide (EM66) flanked by preserved pairs of basic residues. In the present study we have localized and characterized EM66 in the human adrenal gland. A fusion protein containing the human EM66 peptide was produced in E. coli and used to raise polyclonal antibodies in rabbits. Immunohistochemical staining of human adrenal slices revealed intense labeling of adrenochromaffin cells in the adult and fetal gland. HPLC analysis of adrenal extracts showed the presence of an immunoreactive peak exhibiting the same retention time as recombinant EM66 in both adult and fetus. These data demonstrate that post-translational processing of SgII actually generates a novel peptide in the human adrenal gland. The conservation of the sequence of EM66 in vertebrates and the occurrence of the mature peptide during early ontogenesis of the human adrenal gland strongly suggest that EM66 could exert physiological activities.

Neuropeptides and related nucleic acid sequences detected in peneid shrimps by immunohistochemistry and molecular hybridizations.

The neurosecretory cells in the eyestalks of Penaeus indicus and P. vannamei were studied by immunocytochemistry using polyclonal antisera raised against purified Homarus americanus neuropeptides. Cross-reactions between two H. americanus anti-crustacean hyperglycemic hormone antisera and Penaeus neurosecretory material were observed. The specific anti-vitellogenesis inhibiting hormone antiserum only showed an immunological reaction in the nervous tract and the sinus gland of Penaeus, suggesting a progressive exposure of a characteristic epitope which was amenable to immunological detection. Molecular hybridizations were performed in P. indicus and P. vannamei with a digoxigenin tailed 23mer oligonucleotide probe deduced from two partial sequences of uncharacterized, purified P. duorarum neuropeptides. Two distinct clusters of positive cells were observed by in situ hybridization experiments in the medulla terminalis ganglionic X-organ. Classical control tests gave negative results. Northern and Southern blot analyses were performed with the same tailed probed and allowed the determination of molecular weights for the mRNA and for a DNA restriction fragment (Pst 1), 1.7 kb and 200 bp, respectively. These observations show the existence of a strong homology between the P. duorarum sequence (selected for synthesizing the probe), and some P. indicus and P. vannamei neuropeptide sequence(s). Heterologous antisera were tested in other Arthropods to complete our analyses. In the centipede Lithobius forficatus and the scorpion Euscorpius carpathicus, the anti-crustacean hyperglycemic hormone antiserum induced a strong cross-reaction. A monoclonal anti-bombyxin-1 antiserum showed an immunoreaction in the neurosecretory system of the insects Tenebrio molitor and Labidura riparia. In contrast, the anti-bombyxin-1 antiserum did not react either in Penaeus or in Lithobius, and the Homarus hyperglycemic hormone antiserum did not react in the insects that were tested. A comprehensive view of these observations is discussed in relation to a divergence in Arthropod evolution.