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Sample records for adenylate cyclase inhibitor

  1. Protein kinase C sensitizes olfactory adenylate cyclase.

    PubMed

    Frings, S

    1993-02-01

    Effects of neurotransmitters on cAMP-mediated signal transduction in frog olfactory receptor cells (ORCs) were studied using in situ spike recordings and radioimmunoassays. Carbachol, applied to the mucosal side of olfactory epithelium, amplified the electrical response of ORCs to cAMP-generating odorants, but did not affect unstimulated cells. A similar augmentation of odorant response was observed in the presence of phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC). The electrical response to forskolin, an activator of adenylate cyclase (AC), was also enhanced by PDBu, and it was attenuated by the PKC inhibitor Goe 6983. Forskolin-induced accumulation of cAMP in olfactory tissue was potentiated by carbachol, serotonin, and PDBu to a similar extent. Potentiation was completely suppressed by the PKC inhibitors Goe 6983, staurosporine, and polymyxin B, suggesting that the sensitivity of olfactory AC to stimulation by odorants and forskolin was increased by PKC. Experiments with deciliated olfactory tissue indicated that sensitization of AC was restricted to sensory cilia of ORCs. To study the effects of cell Ca2+ on these mechanisms, the intracellular Ca2+ concentration of olfactory tissue was either increased by ionomycin or decreased by BAPTA/AM. Increasing cell Ca2+ had two effects on cAMP production: (a) the basal cAMP production was enhanced by a mechanism sensitive to inhibitors of calmodulin; and (b) similar to phorbol ester, cell Ca2+ caused sensitization of AC to stimulation by forskolin, an effect sensitive to Goe 6983. Decreasing cell Ca2+ below basal levels rendered AC unresponsive to stimulation by forskolin. These data suggest that a crosstalk mechanism is functional in frog ORCs, linking the sensitivity of AC to the activity of PKC. At increased activity of PKC, olfactory AC becomes more responsive to stimulation by odorants, forskolin, and cell Ca2+. Neurotransmitters appear to use this crosstalk mechanism to regulate olfactory

  2. Adenylate cyclases involvement in pathogenicity, a minireview.

    PubMed

    Costache, Adriana; Bucurenci, Nadia; Onu, Adrian

    2013-01-01

    Cyclic AMP (cAMP), one of the most important secondary messengers, is produced by adenylate cyclase (AC) from adenosine triphosphate (ATP). AC is a widespread enzyme, being present both in prokaryotes and eukaryotes. Although they have the same enzymatic activity (ATP cyclization), the structure of these proteins varies, depending on their function and the producing organism. Some pathogenic bacteria utilize these enzymes as toxins which interact with calmodulin (or another eukaryote activator), causing intense cAMP synthesis and disruption of infected cell functions. In contrast, other pathogenic bacteria benefit of augmentation of AC activity for their own function. Based on sequence analysis ofAC catalytic domain from two pathogenic bacteria (Bacillus anthracis and Bordetellapertussis) with known three-dimensional structures, a possible secondary structure for 1-255 amino acid fragment from Pseudomonas aeruginosa AC (with 80TKGFSVKGKSS90 as the ATP binding site) is proposed. PMID:23947014

  3. Guanylate cyclase in Dictyostelium discoideum with the topology of mammalian adenylate cyclase.

    PubMed Central

    Roelofs, J; Snippe, H; Kleineidam, R G; Van Haastert, P J

    2001-01-01

    The core of adenylate and guanylate cyclases is formed by an intramolecular or intermolecular dimer of two cyclase domains arranged in an antiparallel fashion. Metazoan membrane-bound adenylate cyclases are composed of 12 transmembrane spanning regions, and two cyclase domains which function as a heterodimer and are activated by G-proteins. In contrast, membrane-bound guanylate cyclases have only one transmembrane spanning region and one cyclase domain, and are activated by extracellular ligands to form a homodimer. In the cellular slime mould, Dictyostelium discoideum, membrane-bound guanylate cyclase activity is induced after cAMP stimulation; a G-protein-coupled cAMP receptor and G-proteins are essential for this activation. We have cloned a Dictyostelium gene, DdGCA, encoding a protein with 12 transmembrane spanning regions and two cyclase domains. Sequence alignment demonstrates that the two cyclase domains are transposed, relative to these domains in adenylate cyclases. DdGCA expressed in Dictyostelium exhibits high guanylate cyclase activity and no detectable adenylate cyclase activity. Deletion of the gene indicates that DdGCA is not essential for chemotaxis or osmo-regulation. The knock-out strain still exhibits substantial guanylate cyclase activity, demonstrating that Dictyostelium contains at least one other guanylate cyclase. PMID:11237875

  4. Adenylate cyclase activity in a higher plant, alfalfa (Medicago sativa).

    PubMed Central

    Carricarte, V C; Bianchini, G M; Muschietti, J P; Téllez-Iñón, M T; Perticari, A; Torres, N; Flawiá, M M

    1988-01-01

    An adenylate cyclase activity in Medicago sativa L. (alfalfa) roots was partially characterized. The enzyme activity remains in the supernatant fluid after centrifugation at 105,000 g and shows in crude extracts an apparent Mr of about 84,000. The enzyme is active with Mg2+ and Ca2+ as bivalent cations, and is inhibited by EGTA and by chlorpromazine. Calmodulin from bovine brain or spinach leaves activates this adenylate cyclase. PMID:3128270

  5. Glucagon and adenylate cyclase: binding studies and requirements for activation.

    PubMed

    Levey, G S; Fletcher, M A; Klein, I

    1975-01-01

    Solubilization of myocardial adenylate cyclase abolished responsiveness to glucagon and catecholamines, two of the hormones which activate the membrane-bound enzyme. Adenylate cyclase freed of detergent by DEAE-cellulose chromatography continues to remain unresponsive to hormone stimulation. However, adding purified bovine brain phospholipids--phosphotidylserine and monophosphatidylinositol--restored responsiveness to glucagon and catecholamines, respectively. 125-i-glucagon binding appeared to be independent of phospholipid, since equal binding was observed in the presence or absence of detergent and in the presence or absence of phospholipids. Chromatography of the solubilized preparation on Sephadex G-100 WAS CHARACTERIZED BY 125-I-glucagon binding and fluoride-stimulatable adenylate cyclase activity appearing in the fractions consistent with the void volume, suggesting a molecular weight greater than 100,000 for the receptor-adenylate cyclase complex. Prior incubation of the binding peak with 125-I-glucagon and rechromatography of the bound glucagon on Sephadex G-100 shifted its elution to a later fraction consistent with a smaller-molecular-weight peak. The molecular weight of this material was 24,000 to 28,000, as determined by SDS polyacrylamide gel electrophoresis. The latter findings are consistent with a dissociable receptor site for glucagon on myocardial adenylate cyclase. PMID:165684

  6. Identification of sea urchin sperm adenylate cyclase

    PubMed Central

    1990-01-01

    Calmodulin (CaM) affinity chromatography of a detergent extract of sea urchin sperm yielded approximately 20 major proteins. One of these proteins, of Mr 190,000, was purified and used to immunize rabbits. After absorption with living sperm, the serum reacted monospecifically on one- and two-dimensional Western immunoblots with the Mr 190,000 protein. The anti-190-kD serum inhibited 94% of the adenylate cyclase (AC) activity of the CaM eluate. An immunoaffinity column removed 95% of the AC activity, and the purified (but inactive) Mr 190,000 protein was eluted from the column. The antiserum also inhibited 23% of the activity of bovine brain CaM-sensitive AC and 90% of the activity of horse sperm CaM-sensitive AC. These data support the hypothesis that the Mr 190,000 protein is sea urchin sperm AC. Although this AC bound to CaM, it was not possible to demonstrate directly a Ca2+ or CaM sensitivity. However, two CaM antagonists, calmidazolium and chlorpromazine, both inhibited AC activity, and the inhibition was released by added CaM, suggesting the possibility of regulation of this AC by CaM. Indirect immunofluorescence showed the Mr 190,000 protein to be highly concentrated on only the proximal half of the sea urchin sperm flagellum. This asymmetric localization of AC may be important to its function in flagellar motility. This is the first report of the identification of an AC from animal spermatozoa. PMID:2121742

  7. Neurohypophyseal Hormone-Responsive Adenylate Cyclase from Mammalian Kidney

    PubMed Central

    Douša, Thomas; Hechter, Oscar; Schwartz, Irving L.; Walter, Roderich

    1971-01-01

    The investigation was undertaken to evaluate the direct stimulatory effects of neurohypophyseal hormones upon adenylate cyclase activity in a cell-free, particulate fraction derived from the kidney medulla of various mammalian species. The relative affinity of neurohypophyseal hormones for the receptor component of the adenylate cyclase system (as defined by the concentration of hormone required for half-maximal stimulation) had the order [8-arginine]-vasopressin > [8-lysine]-vasopressin ≫ oxytocin (AVP > LVP ≫ OT) for rat, mouse, rabbit, and ox; in the pig, the order was LVP > AVP ≫ OT. The relative affinities of the three hormones in rat and pig cyclase systems were found to correspond with the relative antidiuretic potencies of these hormones in the intact rat and pig. These findings show that the renal receptor for neurohypophyseal hormones in a particular species exhibits the highest affinity for the specific antidiuretic hormone that occurs naturally in that species. Some of the molecular requirements for the stimulation of rabbit adenylate cyclase were defined by studies of several neurohypophyseal analogs possessing structural changes in positions 1, 2, 3, 4, 5, 8, and 9. This investigation introduces the particulate preparation of renal medullary adenylate cyclase as a tool for the analysis of neurohypophyseal hormone-receptor interactions and indicates that this preparation can be adapted to serve as an in vitro bioassay system for antidiuretic hormonal activity. PMID:4331557

  8. Modification of adenylate cyclase by photoaffinity analogs of forskolin

    SciTech Connect

    Ho, L.T.; Nie, Z.M.; Mende, T.J.; Richardson, S.; Chavan, A.; Kolaczkowska, E.; Watt, D.S.; Haley, B.E.; Ho, R.J. )

    1989-01-01

    Photoaffinity labeling analogs of the adenylate cyclase activator forskolin (PF) have been synthesized, purified and tested for their effect on preparations of membrane-bound, Lubrol solubilized and forskolin affinity-purified adenylate cyclase (AC). All analogs of forskolin significantly activated AC. However, in the presence of 0.1 to 0.3 microM forskolin, the less active forskolin photoaffinity probes at 100 microM caused inhibition. This inhibition was dose-dependent for PF, suggesting that PF may complete with F for the same binding site(s). After cross-linking (125I)PF-M to either membrane or Lubrol-solubilized AC preparations by photolysis, a radiolabeled 100-110 kDa protein band was observed after autoradiography following SDS-PAGE. F at 100 microM blocked the photoradiolabeling of this protein. Radioiodination of forskolin-affinity purified AC showed several protein bands on autoradiogram, however, only one band (Mr = 100-110 kDa) was specifically labeled by (125I)PF-M following photolysis. The photoaffinity-labeled protein of 100-110 kDa of AC preparation of rat adipocyte may be the catalytic unit of adenylate cyclase of rat adipocyte itself as supported by the facts that (a) no other AC-regulatory proteins are known to be of this size, (b) the catalytic unit of bovine brain enzyme is in the same range and (c) this PF specifically stimulates AC activity when assayed alone, and weekly inhibits forskolin-activation of cyclase. These studies indicate that radiolabeled PF probes may be useful for photolabeling and detecting the catalytic unit of adenylate cyclase.

  9. Bordetella pertussis adenylate cyclase inactivation by the host cell.

    PubMed Central

    Gilboa-Ron, A; Rogel, A; Hanski, E

    1989-01-01

    Bordetella pertussis produces a calmodulin-dependent adenylate cyclase (AC) which acts as a toxin capable of penetrating eukaryotic cells and generating high levels of intracellular cyclic AMP. Transfer of target cells into B. pertussis AC-free medium leads to a rapid decay in the intracellular AC activity, implying that the invasive enzyme is unstable in the host cytoplasm. We report here that treatment of human lymphocytes with a glycolysis inhibitor and an uncoupler of oxidative phosphorylation completely blocked the intracellular inactivation of B. pertussis AC. Lymphocyte lysates inactivated all forms of B. pertussis AC in the presence of exogenous ATP. This inactivation was associated with degradation of an 125I-labelled 200 kDa form of B. pertussis AC. It appears that ATP is required for the proteolytic pathway, but not as an energy source, since non-hydrolysable ATP analogues supported inactivation and complete degradation of the enzyme. The possibility that binding of ATP to B. pertussis AC renders it susceptible to degradation by the host cell protease is discussed. Images Fig. 2. Fig. 4. PMID:2554887

  10. Mechanism of activation of adenylate cyclase by Vibrio cholerae enterotoxin.

    PubMed

    Bennett, V; Cuatrecasas, P

    1975-06-01

    The kinetics and properties of the activation of adenylate cyclase by cholera enterotoxin have been examined primarily in toad erythrocytes, but also in avian erythrocytes, rat fat cells and cultured melanoma cells. When cholera toxin is incubated with intact cells it stimulates adenylate cyclase activity, as measured in the subsequently isolated plasma membranes, according to a triphasic time course. This consists of a true lag period of about 30 min, followed by a stage of exponentially increasing adenylate cyclase activity which continues for 110 to 130 min, and finally a period of slow activation which may extend as long as 30 hr in cultured melanoma cells. The progressive activation of adenylate cyclase activity by cholera toxin is interrupted by cell lysis; continued incubation of the isolated membranes under nearly identical conditions does not lead to further activation of the enzyme. The delay in the action of the toxin is not grossly dependent of the number of toxin-receptor (GM1 ganglioside) complexes, and is still seen upon adding a second dose of toxin to partially stimulated cells. Direct measurements indicate negligible intracellular levels of biologically active radioiodinated toxin in either a soluble or a nuclear-bound form. The effects are not prevented by Actinomycin D (20 mug/ml), uromycin (30 mug/ml), cycloheximide (30 mug/ml), sodium fluoride (10 mM) or sodium azide (1 mM); KCN, however, almost completely prevents the action of cholera toxin. The action of the toxin is temperature dependent, occurring at very slow or negligible rates below certain critical temperatures, the values of which depend on the specific animal species. Thetransition for toad erythrocytes occurs at 15 to 17 degrees C, while rat adipocytes and turkey erythrocytes demonstrate a discontinuity at 26 to 30 degrees C. The temperature effects are evident during the lag period as well as during the exponential phase of activation. The rate of decay of the stimulated adenylate

  11. Interaction of Trypanosoma cruzi adenylate cyclase with liver regulatory factors.

    PubMed Central

    Eisenschlos, C; Flawiá, M M; Torruella, M; Torres, H N

    1986-01-01

    Trypanosoma cruzi adenylate cyclase catalytic subunits may interact with regulatory factors from rat liver membranes, reconstituting heterologous systems which are catalytically active in assay mixtures containing MgATP. The systems show stimulatory responses to glucagon and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) or fluoride. Reconstitution was obtained by three different methods: fusion of rat liver membranes (pretreated with N-ethylmaleimide) to T. cruzi membranes; interaction of detergent extracts of rat liver membranes with T. cruzi membranes; or interaction of purified preparations of T. cruzi adenylate cyclase and of liver membrane factors in phospholipid vesicles. The liver factors responsible for the guanine nucleotide effect were characterized as the NS protein. Data also indicate that reconstitution requires the presence of a membrane substrate. PMID:2947568

  12. High skeletal muscle adenylate cyclase in malignant hyperthermia.

    PubMed Central

    Willner, J H; Cerri, C G; Wood, D S

    1981-01-01

    Malignant hyperthermia occurs in humans with several congenital myopathies, usually in response to general anesthesia. Commonly, individuals who develop this syndrome lack symptoms of muscle disease, and their muscle lacks specific pathological changes. A biochemical marker for this myopathy has not previously been available; we found activity of adenylate cyclase and content of cyclic AMP to be abnormally high in skeletal muscle. Secondary modification of protein phosphorylation could explain observed abnormalities of phosphorylase activation and sarcoplasmic reticulum function. PMID:6271806

  13. Yeast mating pheromone alpha factor inhibits adenylate cyclase.

    PubMed Central

    Liao, H; Thorner, J

    1980-01-01

    The pheromone alpha factor, secreted by Saccharomyces cerevisiae cells of the alpha mating type, serves to synchronize the opposite mating type (a cells) at G1 as a prelude to fusion of the two cell types. We found that, in vitro, alpha factor inhibited the membrane-bound adenylate cyclase of these cells in a dose-dependent manner. Moreover, one class (ste5) of a cell mutants that grow normally at either 23 degrees or 34 degrees C but that are unable to respond to alpha factor or to mate at the higher temperature possessed an adenylate cyclase activity that was not inhibited by alpha factor at 34 degrees C but was fully sensitive to inhibition at 23 degrees C. Furthermore, addition of cyclic AMP to a cell culture medium shortened the period of pheromone-induced G1 arrest. We conclude that inhibition of adenylate cyclase activity by alpha factor may constitute, at least in part, the biochemical mode of action of the pheromone in vivo. PMID:6246513

  14. Adenylate cyclase mediates olfactory transduction for a wide variety of odorants.

    PubMed Central

    Lowe, G; Nakamura, T; Gold, G H

    1989-01-01

    An odor-stimulated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] is thought to mediate olfactory transduction in vertebrates. However, it is not known whether the adenylate cyclase serves this function for all odorants or for only certain classes of odorants. To investigate this question, we have compared the abilities of 35 odorants to stimulate the adenylate cyclase and to elicit an electrophysiological response. We report a strong positive correlation between the magnitude of adenylate cyclase stimulation and the summated electrical response of the olfactory epithelium (electro-olfactogram) evoked by individual odorants. We also show that the adenylate cyclase stimulator forskolin equally attenuates the electro-olfactogram response for all odorants tested. These data provide evidence that the adenylate cyclase mediates transduction for a wide variety of odorants. PMID:2787513

  15. Adenylate cyclase regulates elongation of mammalian primary cilia

    SciTech Connect

    Ou, Young; Ruan, Yibing; Cheng, Min; Moser, Joanna J.; Rattner, Jerome B.; Hoorn, Frans A. van der

    2009-10-01

    The primary cilium is a non-motile microtubule-based structure that shares many similarities with the structures of flagella and motile cilia. It is well known that the length of flagella is under stringent control, but it is not known whether this is true for primary cilia. In this study, we found that the length of primary cilia in fibroblast-like synoviocytes, either in log phase culture or in quiescent state, was confined within a range. However, when lithium was added to the culture to a final concentration of 100 mM, primary cilia of synoviocytes grew beyond this range, elongating to a length that was on average approximately 3 times the length of untreated cilia. Lithium is a drug approved for treating bipolar disorder. We dissected the molecular targets of this drug, and observed that inhibition of adenylate cyclase III (ACIII) by specific inhibitors mimicked the effects of lithium on primary cilium elongation. Inhibition of GSK-3{beta} by four different inhibitors did not induce primary cilia elongation. ACIII was found in primary cilia of a variety of cell types, and lithium treatment of these cell types led to their cilium elongation. Further, we demonstrate that different cell types displayed distinct sensitivities to the lithium treatment. However, in all cases examined primary cilia elongated as a result of lithium treatment. In particular, two neuronal cell types, rat PC-12 adrenal medulla cells and human astrocytes, developed long primary cilia when lithium was used at or close to the therapeutic relevant concentration (1-2 mM). These results suggest that the length of primary cilia is controlled, at least in part, by the ACIII-cAMP signaling pathway.

  16. ADENYLATE CYCLASE REGULATES ELONGATION OF MAMMALIAN PRIMARY CILIA

    PubMed Central

    Ou, Young; Ruan, Yibing; Cheng, Min; Moser, Joanna J.; Rattner, Jerome B.; van der Hoorn, Frans A.

    2011-01-01

    The primary cilium is a non-motile microtubule-based structure that shares many similarities with the structures of flagella and motile cilia. It is well known that the length of flagella is under stringent control, but it is not known whether this is true for primary cilia. In this study, we found that the length of primary cilia in fibroblast-like synoviocytes, either in log phase culture or in quiescent state, was confined within a range. However, when lithium was added to the culture to a final concentration of 100 mM, primary cilia of synoviocytes grew beyond this range, elongating to a length that was on average approximately 3 times the length of untreated cilia. Lithium is a drug approved for treating bipolar disorder. We dissected the molecular targets of this drug, and observed that inhibition of adenylate cyclase III (ACIII) by specific inhibitors mimicked the effects of lithium on primary cilium elongation. Inhibition of GSK-3β by four different inhibitors did not induce primary cilia elongation. ACIII was found in primary cilia of a variety of cell types, and lithium treatment of these cell types led to their cilium elongation. Further, we demonstrate that different cell types displayed distinct sensitivities to the lithium treatment. However, in all cases examined primary cilia elongated as a result of lithium treatment. In particular, two neuronal cell types, rat PC-12 adrenal medulla cells and human astrocytes, developed long primary cilia when lithium was used at or close to the therapeutic relevant concentration (1–2 mM). These results suggest that the length of primary cilia is controlled, at least in part, by the ACIII-cAMP signaling pathway. PMID:19576885

  17. Adenylate cyclase in Arthrospira platensis responds to light through transcription.

    PubMed

    Kashith, M; Keerthana, B; Sriram, S; Ramamurthy, V

    2016-08-19

    Cyclic 3',5' adenosine monophosphate (cAMP) is a ubiquitous signaling molecule, but its role in higher plants was in doubt due to its very low concentration. In this study we wanted to look at the flux of cAMP in response to light in algae, considered to be the more primitive form of photosynthetic organisms. While it did not fluctuate very much in the tested green algae, in the cyanobacterium Arthrospira platensis its level was closely linked to exposure to light. The expression from cyaC, the major isoform of adenylate cyclase was strongly influenced by exposure of the cells to light. There was about 300 fold enhancement of cyaC transcripts in cells exposed to light compared to the transcripts in cells in the dark. Although post-translational regulation of adenylate cyclase activity has been widely known, our studies suggest that transcriptional control could also be an important aspect of its regulation in A. platensis. PMID:27311855

  18. Cellular levels of feedback regulator of adenylate cyclase and the effect of epinephrine and insulin.

    PubMed Central

    Ho, R j; Russell, T R; Asakawa, T; Sutherland, E W

    1975-01-01

    We have obtained direct evidence that shows the cellular formation and subsequent release of a potent inhibitor (feedback regulator) of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by adipocytes, upon stimulation with epinephrine. The appearance of such a feedback regulator in adipocytes preceded its release into the medium. During a 30 min incubation, intracellular regulator levels rose rapidly and reached 39-61 units/g of adipocyte at 10 min. Release of inhibitor into the medium increased slowly and was 11-16 units/g of adipocyte at 10 min. Upon continued incubation, the cells at 30 min contained 30-41 units/g of ingibitor, slightly less than the content at 30 min; meanwhile, the medium content rose more than 3-fold. The inhibitor from both locations appeared to have the same characteristics, judging from the purification procedures and the biological activities on hormone-stimulated adenylate cyclase. Adenylate cyclase was inhibited by the feedback regulator in vitro when either epinephrine, corticotropin (ACTH), or glucagon was used as activator. The site of action of this inhibitor is therefore most likely beyond the specific hormone receptors. A new in vitro action of insulin has been found. Insulin, 50-500 microunits/ml, inhibited the formation and release of this factor from isolated rat or hamster adipocytes by 29-81% after these cells were stimulated by hormones that raise intracellular adenosine 3':5'-cyclic monophosphate. This factor enhaced the effect of insulin in lowering the adenosine 3':5'-cyclic monophosphate levels in fresh rat adipocytes. A reduced formation of such a factor may modify the metabolic events in adipocytes, and some as yet unexplained effects of insulin could therefore be linked to the metabolic effects of this factor. PMID:174073

  19. Prokaryotic adenylate cyclase toxin stimulates anterior pituitary cells in culture

    SciTech Connect

    Cronin, M.J.; Evans, W.S.; Rogol, A.D.; Weiss, A.A.; Thorner, M.O.; Orth, D.N.; Nicholson, W.E.; Yasumoto, T.; Hewlett, E.L.

    1986-08-01

    Bordetella pertussis synthesis a variety of virulence factors including a calmodulin-dependent adenylate cyclase (AC) toxin. Treatment of anterior pituitary cells with this AC toxin resulted in an increase in cellular cAMP levels that was associated with accelerated exocytosis of growth hormone (GH), prolactin, adrenocorticotropic hormone (ACTH), and luteinizing hormone (LH). The kinetics of release of these hormones, however, were markedly different; GH and prolactin were rapidly released, while LH and ACTH secretion was more gradually elevated. Neither dopamine agonists nor somatostatin changes the ability of AC toxin to generate cAMP (up to 2 h). Low concentrations of AC toxin amplified the secretory response to hypophysiotrophic hormones. The authors conclude that bacterial AC toxin can rapidly elevate cAMP levels in anterior pituitary cells and that it is the response that explains the subsequent acceleration of hormone release.

  20. 3',5'-cyclic adenosine monophosphate and adenylate cyclase in phototransduction by limulus ventral photoreceptors.

    PubMed Central

    Brown, J E; Kaupp, U B; Malbon, C C

    1984-01-01

    Biochemical and electrophysiological measurements were made on photoreceptor cells from Limulus ventral eyes to investigate the possible role of cyclic AMP and adenylate cyclase in the visual transduction mechanism. Cyclic AMP content in a photoreceptor-enriched fraction (the end organs) of Limulus ventral eyes was approximately 15 pmol/mg protein. The cyclic AMP content was increased by bathing eyes in 1-methyl-3-isobutyl xanthine or forskolin and was increased almost 100-fold when bathed in both. Illumination did not change cyclic AMP content significantly in any of these conditions. Discrete events that can be recorded electrophysiologically occur spontaneously in darkness. An increase in the frequency of discrete events is evoked by dim illumination. The discrete events are a sign of excitation of Limulus photoreceptor cells. Drug-induced changes in the rate of occurrence of discrete events recorded electrophysiologically in darkness were not correlated with changes in cyclic AMP content. Adenylate cyclase activity measured from a small number of pooled photoreceptor clusters was stimulated by fluoride and vanadate ions, hydrolysis-resistant analogues of GTP, cholera toxin and forskolin. The Limulus enzyme is similar pharmacologically to mammalian and avian adenylate cyclases. Activation of adenylate cyclase by drugs was not correlated with changes in the rate of occurrence of discrete events recorded electrophysiologically in darkness. A heat-treated Lubrol extract of membranes from Limulus ventral eyes reconstituted the adenylate cyclase activity of membranes from S49 mouse lymphoma cyc- mutant cells which lack a functional regulatory protein. These findings suggest that Limulus ventral eye photoreceptors contain a regulatory protein that mediates the activation of adenylate cyclase by guanine nucleotides, fluoride or cholera toxin. This regulatory protein is homologous with that found in mammalian and avian adenylate cyclases. Our findings suggest that

  1. Crystal Structure of Human Soluble Adenylate Cyclase Reveals a Distinct, Highly Flexible Allosteric Bicarbonate Binding Pocket

    PubMed Central

    Saalau-Bethell, Susanne M; Berdini, Valerio; Cleasby, Anne; Congreve, Miles; Coyle, Joseph E; Lock, Victoria; Murray, Christopher W; O'Brien, M Alistair; Rich, Sharna J; Sambrook, Tracey; Vinkovic, Mladen; Yon, Jeff R; Jhoti, Harren

    2014-01-01

    Soluble adenylate cyclases catalyse the synthesis of the second messenger cAMP through the cyclisation of ATP and are the only known enzymes to be directly activated by bicarbonate. Here, we report the first crystal structure of the human enzyme that reveals a pseudosymmetrical arrangement of two catalytic domains to produce a single competent active site and a novel discrete bicarbonate binding pocket. Crystal structures of the apo protein, the protein in complex with α,β-methylene adenosine 5′-triphosphate (AMPCPP) and calcium, with the allosteric activator bicarbonate, and also with a number of inhibitors identified using fragment screening, all show a flexible active site that undergoes significant conformational changes on binding of ligands. The resulting nanomolar-potent inhibitors that were developed bind at both the substrate binding pocket and the allosteric site, and can be used as chemical probes to further elucidate the function of this protein. PMID:24616449

  2. Adenylate cyclase in prothoracic glands during the last larval instar of the silkworm, Bombyx mori.

    PubMed

    Chen, C H; Gu, S H; Chow, Y S

    2001-04-27

    We have previously reported that the absence of prothoracicotropic hormone (PTTH) signal transduction during the early last larval instar of Bombyx mori plays a role in leading to very low ecdysteroid levels in the hemolymph, inactivation of the corpora allata, as well as larval-pupal transformation. In the present study, adenylate cyclase was characterized in crude preparations of prothoracic gland cell membranes in an effort to localize the cause of refractoriness to PTTH. It was found that cyclase activity of the prothoracic glands from the day 6 last instar showed activation responses to fluoride, a guanine nucleotide analogue, as well as calmodulin (CaM) in dose-dependent fashions. The additive effects of day 5 prothoracic gland adenylate cyclase stimulation by fluoride and CaM imply that there may exist Gs protein-dependent and CaM-dependent forms of adenylate cyclase. For day 1 last instar prothoracic glands, which showed no response to stimulation by PTTH in either cAMP generation or ecdysteroidogenesis, adenylate cyclase activity exhibited far less responsiveness to Ca(2+)/CaM than did that from day 5 glands. These findings suggest that day 1 prothoracic glands may possess some lesions in the receptor-Ca(2+) influx-adenylate cyclase signal transduction pathway and these impairments in PTTH signal transduction may be, at least in part, responsible for decreased ecdysteroidogenesis. PMID:11267904

  3. Adenylate Cyclase Toxin promotes bacterial internalisation into non phagocytic cells

    PubMed Central

    Martín, César; Etxaniz, Asier; Uribe, Kepa B.; Etxebarria, Aitor; González-Bullón, David; Arlucea, Jon; Goñi, Félix M.; Aréchaga, Juan; Ostolaza, Helena

    2015-01-01

    Bordetella pertussis causes whooping cough, a respiratory infectious disease that is the fifth largest cause of vaccine-preventable death in infants. Though historically considered an extracellular pathogen, this bacterium has been detected both in vitro and in vivo inside phagocytic and non-phagocytic cells. However the precise mechanism used by B. pertussis for cell entry, or the putative bacterial factors involved, are not fully elucidated. Here we find that adenylate cyclase toxin (ACT), one of the important toxins of B. pertussis, is sufficient to promote bacterial internalisation into non-phagocytic cells. After characterization of the entry route we show that uptake of “toxin-coated bacteria” proceeds via a clathrin-independent, caveolae-dependent entry pathway, allowing the internalised bacteria to survive within the cells. Intracellular bacteria were found inside non-acidic endosomes with high sphingomyelin and cholesterol content, or “free” in the cytosol of the invaded cells, suggesting that the ACT-induced bacterial uptake may not proceed through formation of late endolysosomes. Activation of Tyr kinases and toxin-induced Ca2+-influx are essential for the entry process. We hypothesize that B. pertussis might use ACT to activate the endocytic machinery of non-phagocytic cells and gain entry into these cells, in this way evading the host immune system. PMID:26346097

  4. The adenylate cyclase receptor complex and aqueous humor formation.

    PubMed Central

    Caprioli, J.; Sears, M.

    1984-01-01

    The secretory tissue of the eye, the ciliary processes, contains an enzyme receptor complex, composed of membrane proteins, the catalytic moiety of the enzyme adenylate cyclase, a guanyl nucleotide regulatory protein (or N protein), and other features. The enzyme can be activated by well-known neurohumoral or humoral agents, catecholamines, glycoprotein hormones produced by the hypothalamic pituitary axis, and other related compounds, including placental gonadotropin, organic fluorides, and forskolin, a diterpene. These compounds cause the ciliary epithelia to produce cyclic AMP at an accelerated rate. Cyclic AMP, as a second messenger, causes, either directly or indirectly, a decrease in the net rate of aqueous humor inflow that may be modulated by cofactors. Clinical syndromes fit the experimental data so that an integrated explanation can be given for the reduced intraocular pressure witnessed under certain central nervous system and adrenergic influences. The molecular biology of this concept provides important leads for future investigations that bear directly both upon the regulation of intraocular pressure and upon glaucoma. Images FIG. 11 PMID:6093393

  5. Stimulation of hormone-responsive adenylate cyclase activity by a factor present in the cell cytosol.

    PubMed Central

    MacNeil, S; Crawford, A; Amirrasooli, H; Johnson, S; Pollock, A; Ollis, C; Tomlinson, S

    1980-01-01

    1. Homogenates of whole tissues were shown to contain both intracellular and extracellular factors that affected particulate adenylate cyclase activity in vitro. Factors present in the extracellular fluids produced an inhibition of basal, hormone- and fluoride-stimulated enzyme activity but factors present in the cell cytosol increased hormone-stimulated activity with relatively little effect on basal or fluoride-stimulated enzyme activity. 2. The existence of this cytosol factor or factors was investigated using freshly isolated human platelets, freshly isolated rat hepatocytes, and cultured cells derived from rat osteogenic sarcoma, rat calvaria, mouse melanoma, pig aortic endothelium, human articular cartilage chondrocytes and human bronchial carcinoma (BEN) cells. 3. The stimulation of the hormone response by the cytosol factor ranged from 60 to 890% depending on the tissue of origin of the adenylate cyclase. 4. In each case the behaviour of the factor was similar to the action of GTP on that particular adenylate cyclase preparation. 5. No evidence of tissue or species specificity was found, as cytosols stimulated adenylate cyclase from their own and unrelated tissues to the same degree. 6. In the human platelet, the inclusion of the cytosol in the assay of adenylate cyclase increased the rate of enzyme activity in response to stimulation by prostaglandin E1 without affecting the amount of prostaglandin E1 required for half-maximal stimulation or the characteristics of enzyme activation by prostaglandin E. PMID:7396869

  6. Localization of nigrostriatal dopamine receptor subtypes and adenylate cyclase

    SciTech Connect

    Filloux, F.; Dawson, T.M.; Wamsley, J.K.

    1988-04-01

    Quantitative autoradiography using (/sup 3/H)-SCH 23390, (/sup 3/H)-sulpiride and (/sup 3/H)-forskolin was used to assess the effects of single and combined neurotoxin lesions of the nigrostriatal pathway in the rat brain on dopamine (DA) receptor subtypes and adenylate cyclase (AC), respectively. Ibotenic acid (IA) lesions of the caudate-putamen (CPu) resulted in near total loss of both (/sup 3/H)-SCH 23390 and of (/sup 3/H)-forskolin binding in the ipsilateral CPu and substantia nigra reticulata (SNR). (/sup 3/H)-sulpiride binding in the CPu was only partially removed by this same lesion, and nigral (/sup 3/H)-sulpiride binding was virtually unchanged. 6-Hydroxydopamine (6-OHDA) and IA lesions of the substantia nigra compacta (SNC) did not affect (/sup 3/H)-SCH 23390 or (/sup 3/H)-forskolin binding, but largely removed (/sup 3/H)-sulpiride binding in the SNC. A 6-OHDA lesion of the nigrostriatal pathway followed by an ipsilateral IA injection of the CPu failed to further reduce (/sup 3/H)-sulpiride binding in the CPu. These results demonstrate that postsynaptic DA receptors in the CPu are of both the D1 and D2 variety; however, a portion of D2 receptors in the CPu may be presynaptic on afferent nerve terminals to this structure. D1 receptors in the SNR are presynaptic on striatonigral terminals, whereas the D2 receptors of the SNC are autoreceptors on nigral DA neurons. The existence of presynaptic D2 receptors on nigrostriatal DA-ergic terminals could not be confirmed by this study. Co-localization of D1 receptors and AC occurs in both the CPu and SNR.

  7. Role of Adenylate Cyclase 1 in Retinofugal Map Development

    PubMed Central

    Dhande, Onkar S.; Bhatt, Shivani; Anishchenko, Anastacia; Elstrott, Justin; Iwasato, Takuji; Swindell, Eric C.; Xu, Hong-Ping; Jamrich, Milan; Itohara, Shigeyoshi; Feller, Marla B.; Crair, Michael C.

    2013-01-01

    The development of topographic maps of the sensory periphery is sensitive to the disruption of adenylate cyclase 1 (AC1) signaling. AC1 catalyzes the production of cAMP in a Ca2+/calmodulin-dependent manner, and AC1 mutant mice (AC1−/−) have disordered visual and somatotopic maps. However, the broad expression of AC1 in the brain and the promiscuous nature of cAMP signaling have frustrated attempts to determine the underlying mechanism of AC1-dependent map development. In the mammalian visual system, the initial coarse targeting of retinal ganglion cell (RGC) projections to the superior colliculus (SC) and lateral geniculate nucleus (LGN) is guided by molecular cues, and the subsequent refinement of these crude projections occurs via an activity-dependent process that depends on spontaneous retinal waves. Here, we show that AC1−/− mice have normal retinal waves but disrupted map refinement. We demonstrate that AC1 is required for the emergence of dense and focused termination zones and elimination of inaccurately targeted collaterals at the level of individual retinofugal arbors. Conditional deletion of AC1 in the retina recapitulates map defects, indicating that the locus of map disruptions in the SC and dorsal LGN of AC1−/− mice is presynaptic. Finally, map defects in mice without AC1 and disrupted retinal waves (AC1−/−;β2−/− double KO mice) are no worse than those in mice lacking only β2−/−, but loss of AC1 occludes map recovery in β2−/− mice during the second postnatal week. These results suggest that AC1 in RGC axons mediates the development of retinotopy and eye-specific segregation in the SC and dorsal LGN. PMID:22102330

  8. Dynamics of adenylate cyclase regulation via heterotrimeric G-proteins.

    PubMed

    Milde, Markus; Werthmann, Ruth C; von Hayn, Kathrin; Bünemann, Moritz

    2014-04-01

    A wide variety of G-protein-coupled receptors either activate or inhibit ACs (adenylate cyclases), thereby regulating cellular cAMP levels and consequently inducing proper physiological responses. Stimulatory and inhibitory G-proteins interact directly with ACs, whereas G(q)-coupled receptors exert their effects primarily via Ca2+. Using the FRET-based cAMP sensor Epac1 (exchange protein directly activated by cAMP 1)-cAMPS (adenosine 3',5'-cyclic monophosphorothioate), we studied cAMP levels in single living VSMCs (vascular smooth muscle cells) or HUVECs (human umbilical vein endothelial cells) with subsecond temporal resolution. Stimulation of purinergic (VSMCs) or thrombin (HUVECs) receptors rapidly decreased cAMP levels in the presence of the β-adrenergic agonist isoprenaline via a rise in Ca2+ and subsequent inhibition of AC5 and AC6. Specifically in HUVECs, we observed that, in the continuous presence of thrombin, cAMP levels climbed slowly after the initial decline with a delay of a little less than 1 min. The underlying mechanism includes phospholipase A2 activity and cyclo-oxygenase-mediated synthesis of prostaglandins. We studied further the dynamics of the inhibition of ACs via G(i)-proteins utilizing FRET imaging to resolve interactions between fluorescently labelled G(i)-proteins and AC5. FRET between Gα(i1) and AC5 developed at much lower concentration of agonist compared with the overall G(i)-protein activity. We found the dissociation of Gα(i1) subunits and AC5 to occur slower than the G(i)-protein deactivation. This led us to the conclusion that AC5, by binding active Gα(i1), interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation. PMID:24646224

  9. Evidence for a dissociable protein subunit required for calmodulin stimulation of brain adenylate cyclase.

    PubMed Central

    Toscano, W A; Westcott, K R; LaPorte, D C; Storm, D R

    1979-01-01

    An adenylate cyclase [ATP pyrophosphatelyase (cyclizing), EC 4.6.1.1] preparation that is not stimulated by NaF,5'-guanylyl imidodiphosphate, or Ca2+.calmodulin has been isolated from bovine cerebral cortex by Affi-Gel Blue chromatography and calmodulin-Sepharose chromatography. Sensitivity to these effectors was restored by incubation of the adenylate cyclase preparation with detergent-solubilized protein from bovine cerebral cortex. Reconstitution of of Ca2+.calmodulin activation required the presence of 5'-guanylyl imidodiphosphate. The factor required for restoration of Ca2+.calmodulin stimulation was sensitive to heat, trypsin digestion, and N-ethylmaleimide. These observations suggest that this adenylate cyclase activity requires the presence of one or more guanyl nucleotide binding subunits for calmodulin sensitivity. PMID:293663

  10. Cooperative phenomena in binding and activation of Bordetella pertussis adenylate cyclase by calmodulin.

    PubMed

    Bouhss, A; Krin, E; Munier, H; Gilles, A M; Danchin, A; Glaser, P; Bârzu, O

    1993-01-25

    The catalytic domain of Bordetella pertussis adenylate cyclase located within the first 400 amino acids of the protein can be cleaved by trypsin in two subdomains (T25 and T18) corresponding to ATP-(T25) and calmodulin (CaM)-(T18) binding sites. Reassociation of subdomains by CaM is a cooperative process, which is a unique case among CaM-activated enzymes. To understand better the molecular basis of this phenomenon, we used several approaches such as partial deletions of the adenylate cyclase gene, isolation of peptides of various size, and site-directed mutagenesis experiments. We found that a stretch of 72 amino acid residues overlapping the carboxyl terminus of T25 and the amino terminus of T18 accounts for 90% of the binding energy of adenylate cyclase-CaM complex. The hydrophobic "side" of the helical region situated around Trp242 plays a major role in the interaction of adenylate cyclase with CaM, whereas basic residues that alternate with acidic residues in bacterial enzyme play a much less important role. The amino-terminal half of the catalytic domain of adenylate cyclase contributes only 10% to the binding energy of CaM, whereas the last 130 amino acid residues are not at all involved in binding. However, these segments of adenylate cyclase might affect protein/protein interaction and catalysis by propagating conformational changes to the CaM-binding sequence which is located in the middle of the catalytic domain of bacterial enzyme. PMID:8420945

  11. Tachyphylaxis to PACAP-27 after inhibition of NO synthesis: a loss of adenylate cyclase activation

    NASA Technical Reports Server (NTRS)

    Whalen, E. J.; Johnson, A. K.; Lewis, S. J.

    1999-01-01

    The vasodilator effects of pituitary adenylate cyclase activating polypeptide (PACAP-27) are subject to tachyphylaxis in rats treated with the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). This study examined whether this tachyphylaxis is due to the loss of vasodilator potency of cAMP generated by activation of the G(s) protein-coupled PACAP receptors. Five successive treatments with PACAP-27 (2 nmol/kg iv) produced pronounced vasodilator responses in saline-treated rats that were not subject to tachyphylaxis. The first injection of PACAP-27 (2 nmol/kg iv) in L-NAME (50 micromol/kg iv)-treated rats produced vasodilator responses of similar magnitude to those in saline-treated rats, whereas four subsequent injections produced progressively and markedly smaller responses. The hemodynamic effects of the membrane-permeable cAMP analog 8-(4-chlorophenylthiol)-cAMP (8-CPT-cAMP; 5-15 micromol/kg iv) were similar in L-NAME-treated rats and in L-NAME-treated rats that had received the five injections of PACAP-27. In addition, five injections of 8-CPT-cAMP (10 micromol/kg iv) produced pronounced vasodilator responses in saline- and L-NAME-treated rats that were not subject to the development of tachyphylaxis. These results suggest that a loss of biological potency of cAMP is not responsible for tachyphylaxis to PACAP-27 in L-NAME-treated rats. This tachyphylaxis may be due to the inability of the G(s) protein-coupled PACAP receptor to activate adenylate cyclase.

  12. Cytochemical localization of adenylate cyclase in the various tissues of Locusta migratoria (migratorioides R.F.).

    PubMed

    Benedeczky, I; Rózsa, K S

    1981-01-01

    The ultrastructural cytochemical procedure to demonstrate adenyl cyclase in mammalian organs was used in insects. After several modifications, an utilizable method was applied for the detection of the enzyme in the various tissues. Adenylate cyclase which can be stimulated with octopamine was localized on the membrane of the glial cells and the axolemma of certain large axons in the insect brain. Adenylate cyclase which could be activated by NaF and isoproterenol was also demonstrated in the lipid droplets of glial cells of the brain. With the simultaneous application of NaF and isoproterenol, rather strong adenylate cyclase activity could be detected on the surface of the corpora allata cells both in the cells situated on the glandular surface and the central part of the gland. In contrast in the corpus cardiacum enzyme activity was only observable on the basal lamina of the glandular surface. An appreciable amount of reaction product, indicating the presence of the enzyme, could be found on the surface of the lipid droplets in the fat body situated near the glandular tissues. In the heart muscle, reaction product referring to enzyme activation could not be demonstrated with the help of the methods applied. PMID:7216835

  13. Modulation of receptors and adenylate cyclase activity during sucrose feeding, food deprivation, and cold exposure

    SciTech Connect

    Scarpace, P.J.; Baresi, L.A.; Morley, J.E. Univ. of California, Los Angeles )

    1987-12-01

    Thermogenesis in brown adipose tissue (BAT) serves as a regulator of body temperature and weight maintenance. Thermogenesis can be stimulated by catecholamine activation of adenylate cyclase through the {beta}-adrenergic receptor. To investigate the effects of sucrose feeding, food deprivation, and cold exposure on the {beta}-adrenergic pathway, adenylate cyclase activity and {beta}-adrenergic receptors were assessed in rat BAT after 2 wk of sucrose feeding, 2 days of food deprivation, or 2 days of cold exposure. {beta}-Adrenergic receptors were identified in BAT using ({sup 125}I)iodocyanopindolol. Binding sites had the characteristics of mixed {beta}{sub 1}- and {beta}{sub 2}-type adrenergic receptors at a ratio of 60/40. After sucrose feeding or cold exposure, there was the expected increase in BAT mitochondrial mass as measured by total cytochrome-c oxidase activity but a decrease in {beta}-adrenergic receptor density due to a loss of the {beta}{sub 1}-adrenergic subtype. This BAT {beta}-adrenergic receptor downregulation was tissue specific, since myocardial {beta}-adrenergic receptors were unchanged with either sucrose feeding or cold exposure. Forskolin-stimulated adenylate cyclase activity increased in BAT after sucrose feeding or cold exposure but not after food deprivation. These data suggest that in BAT, sucrose feeding or cold exposure result in downregulation of {beta}-adrenergic receptors and that isoproterenol-stimulated adenylate cyclase activity was limited by receptor availability.

  14. Mechanisms of nonhormonal activation of adenylate cyclase based on target analysis

    SciTech Connect

    Verkman, A.S.; Ausiello, D.A.; Jung, C.Y.; Skorecki, K.L.

    1986-08-12

    Radiation inactivation was used to examine the mechanism of activation of adenylate cyclase in the cultured renal epithelial cell line LLC-PK1 with hormonal (vasopressin) and nonhormonal (GTP, forskolin, fluoride, and chloride) activating ligands. Intact cells were frozen, irradiated at -70 degrees C (0-14 Mrad), thawed, and assayed for adenylate cyclase activity in the presence of activating ligands. The ln (adenylate cyclase activity) vs. radiation dose relation was linear (target size 162 kDa) for vasopressin- (2 microM) stimulated activity and concave downward for unstimulated (10 mM Mn/sup 2 +/), NaF- (10 mM) stimulated, and NaCl- (100 mM) stimulated activities. Addition of 2 microM vasopressin did not alter the ln activity vs. dose relation for NaF- (10 mM) stimulated activity. The dose-response relations for adenylate cyclase activation and for transition in the ln activity vs. dose curve shape were measured for vasopressin and NaF. On the basis of our model for adenylate cyclase subunit interactions reported previously (Verkman, A. S., Skorecki, K. L., and Ausiello, D. A. (1986) Am. J. Physiol. 260, C103-C123) and of new mathematical analyses, activation mechanisms for each ligand are proposed. In the unstimulated state, equilibrium between alpha beta and alpha + beta favors alpha beta; dissociated alpha binds to GTP (rate-limiting step), which then combines with the catalytic (C) subunit to form active enzyme. Vasopressin binding to receptor provides a rapid pathway for GTP binding to alpha. GTP and its analogues accelerate the rate of alpha GTP formation. Forskolin inhibits the spontaneous deactivation of activated C. Activation by fluoride may occur without alpha beta dissociation or GTP addition through activation of C by an alpha beta-F complex.

  15. Hyperexpression and purification of Escherichia coli adenylate cyclase using a vector designed for expression of lethal gene products.

    PubMed

    Reddy, P; Peterkofsky, A; McKenney, K

    1989-12-25

    We describe the construction of a new generation of vectors (pRE) for the hyperexpression of lethal gene products such as adenylate cyclase in Escherichia coli. The pRE vectors are based on the lambda PL promoter and lambda cII ribosome binding site described by Shimatake and Rosenberg (Nature, 292, 128-132, 1981). They have a unique NdeI restriction endonuclease site 3' of the lambda cII ribosome binding site that includes the ATG initiation codon, multilinker cloning sites 3' to the NdeI site, and two lambda transcription terminators 5' and 3' of the lambda PL promoter to eliminate nonspecific transcription and reduce leaky PL transcription, respectively. For hyperexpression of adenylate cyclase, tight control of transcription was necessary since elevation of cAMP levels above the physiological range is lethal to E. coli. Lethality associated with the overproduction of adenylate cyclase was shown to be mediated through the cAMP receptor protein. We used this expression system to overproduce adenylate cyclase 7500 fold, corresponding to 30% of the total cellular protein. Under these conditions the enzyme precipitated with significant loss of activity. Reducing the rate and amount of adenylate cyclase expression to 16% of the total cell protein produced one fourth of the enzyme in a soluble form with high specific activity. The soluble adenylate cyclase was purified to near homogeneity. PMID:2557591

  16. Alkaline phosphatase relieves desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocyte membranes

    SciTech Connect

    Stadel, J.M.; Rebar, R.; Crooke, S.T.

    1987-05-01

    Desensitization of adenylate cyclase-coupled ..beta..-adrenergic receptors in avian erythrocytes results in 40-65% decrease in agonist-stimulated adenylate cyclase activity and correlates with increased phosphorylation of ..beta..-adrenergic receptors. To assess the role of phosphorylation in desensitization, membranes from isoproterenol- and cAMP-desensitized turkey erythrocytes were incubated with alkaline phosphatase for 30 min at 37/sup 0/C, pH = 8.0. In both cases alkaline phosphatase treatment significantly reduced desensitization of agonist-stimulated adenylate cyclase activity by 40-60%. Similar results were obtained following alkaline phosphatase treatment of membranes from isoproterenol- and cAMP-desensitized duck erythrocytes. In addition, alkaline phosphatase treatment of membranes from duck erythrocytes desensitized with phorbol 12-mystrate 13-acetate returned adenylate cyclase activity to near control values. In all experiments inclusion of 20 mM NaPO/sub 4/ to inhibit alkaline phosphatase during treatment of membranes blocked the enzyme's effect on agonist-stimulated adenylate cyclase activity. These results demonstrate a role for phosphorylation in desensitization of adenylate cyclase-coupled ..beta..-adrenergic receptors in avian erythrocytes.

  17. Homology modeling and molecular docking of human pituitary adenylate cyclase-activating polypeptide I receptor

    PubMed Central

    WU, LUSHENG; GUANG, WENHUA; CHEN, XIAOJIA; HONG, AN

    2014-01-01

    Pituitary adenylate cyclase-activating peptide I receptor (PAC1R) is member of the B class of G protein-coupled seven-transmembrane receptors, with molecular functions associated with neural cell differentiation, regeneration and the inhibition of apoptosis. However, the integrity of the protein structure is difficult to be determined in vitro. In the present study, the physicochemical properties of PAC1R were analyzed, the extracellular, transmembrane and intracellular regions were constructed and a three-dimensional structure model of PAC1R was produced using extracellular loop region optimization and the energy minimization homology modeling method. Preliminary studies on the PAC1R protein and ligand interactions used a molecular docking method. The results indicated that the interaction sites of PAC1R were at Ile63, Ser100 and Gln105. These were the sites where the PAC1R combined with a hydrazide small molecule inhibitor. This study provides a theoretical basis for further studies on the model for the development of PAC1R target drugs. PMID:25069645

  18. Adenylate cyclase responsiveness to hormones in various portions of the human nephron.

    PubMed Central

    Chabardès, D; Gagnan-Brunette, M; Imbert-Teboul, M; Gontcharevskaia, O; Montégut, M; Clique, A; Morel, F

    1980-01-01

    The action sites for parathyroid hormone (PTH), salmon calcitonin (SCT), and arginine-vasopressin (AVP) were investigated along the human nephron by measuring adenylate cyclase activity, using a single tubule in vitro microassay. Well-localized segments of tubule were isolated by microdissection from five human kidneys unsuitable for transplantation. PTH (10 IU/ml) increased adenylate cyclase activity in the convoluted and the straight proximal tubule, in the medullary and cortical portions of the thick ascending limb, and in the early portion of the distal convoluted tubule (corresponding stimulated:basal activity ratios were 64, 19, 10, 18, and 22, respectively). SCT (10 ng/ml) increased adenylate cyclase activity in the medullary and cortical portions of the thick ascending limb, in the early portion of the distal convoluted tubule, and, to a lesser extent, in the cortical and the medullay collecting tubule (activity ratios were 7, 14, 15, 3, and 3, respectively). AVP (1 microM) stimulated adenylate cyclase activity in the terminal nephron segments only, i.e., the late portion of the distal convoluted tubule, the cortical and medullary portions of the collecting tubule (activity ratios 81, 51, and 97, respectively). As measured in one experiment, nearly one-half maximal responses were obtained with 0.1 IU/ml PTH or 0.3 ng/ml SCT in thick ascending limbs and with 1 nM AVP in collecting tubules, suggesting that enzyme sensitivity to hormones as well preserved under the conditions used in this study. PMID:7356689

  19. Non-co-ordinate development of beta-adrenergic receptors and adenylate cyclase in chick heart.

    PubMed Central

    Alexander, R W; Galper, J B; Neer, E J; Smith, T W

    1982-01-01

    We have studied the properties of beta-adrenergic receptors and of their interaction with adenylate cyclase in the chick myocardium during embryogenesis. Between 4.5 and 7.5 days in ovo the number of receptors determined by (-)-[3H]dihydroalprenolol ([3H]DHA) binding is constant at approx. 0.36 pmol of receptor/mg of protein. By day 9 the density decreases significantly to 0.22 pmol of receptor/mg of protein. At day 12.5--13.5 the number was 0.14--0.18 pmol of receptor/mg of protein. This number did not change further up to day 16. The same results were obtained with guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) added to the assay mixtures. There was no significant change in receptor affinity for the antagonist [3H]DHA between days 5.5 and 13. Despite the decrease in numbers of beta-adrenergic receptors, there was no change in basal, p[NH]ppG-, isoprenaline- or isoprenaline-plus-p[NH]ppG-stimulated adenylate cyclase activity between days 3 and 12 of development. We conclude that beta-adrenergic receptors and adenylate cyclase are not co-ordinately regulated during early embryonic development of the chick heart. Some of the beta-adrenergic receptors present very early in the ontogeny of cardiac tissue appear not to be coupled to adenylate cyclase since their loss is not reflected in decreased activation of the enzyme. PMID:6289805

  20. Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in Syrian hamster hypothalamus

    SciTech Connect

    Niles, L.P.; Hashemi, F. )

    1990-12-01

    1. The effect of melatonin on forskolin-stimulated adenylate cyclase activity was measured in homogenates of Syrian hamster hypothalamus. In addition, the saturation binding characteristics of the melatonin receptor ligand, ({sup 125}I)iodomelatonin, was examined using an incubation temperature (30{degree}C) similar to that used in enzyme assays. 2. At concentrations ranging from 10 pM to 1 nM, melatonin caused a significant decrease in stimulated adenylate cyclase activity with a maximum inhibition of approximately 22%. 3. Binding experiments utilizing ({sup 125}I)iodomelatonin in a range of approximately 5-80 pM indicated a single class of high-affinity sites: Kd = 55 +/- 9 pM, Bmax = 1.1 +/- 0.3 fmol/mg protein. 4. The ability of picomolar concentrations of melatonin to inhibit forskolin-stimulated adenylate cyclase activity suggests that this affect is mediated by picomolar-affinity receptor binding sites for this hormone in the hypothalamus.

  1. Modulation of ischemic-induced damage to cerebral adenylate cyclase in gerbils by calcium channel blockers.

    PubMed

    Christie-Pope, B C; Palmer, G C

    1986-12-01

    It has been previously established that prolonged bilateral carotid occlusion followed by recirculation produces damage to the synaptic enzyme adenylate cyclase in the frontal cortex of the gerbil. Since calcium entrance into the brain may account in part for the deleterious consequences of stroke, the present study examined whether pretreatment with calcium channel blockers would modify the effects of 60 min of bilateral ischemia plus 40 min of reflow on various parameters of cortical adenylate cyclase activation. In this context activation of cerebral homogenates by norepinephrine with or without 5'-guanylyl imidodiphosphate was preserved by pretreatment of ischemic gerbils with verapamil but worsened by flunarizine. In contrast, in particulate fractions (treated with EGTA to reduce metallic ion levels) the damage to the Mn2+-sensitive catalytic site of adenylate cyclase was prevented only by flunarizine. Pretreatment with the two calcium channel blockers resulted in an elevated basal activity of the enzyme, thereby reducing the response in the homogenate preparation to forskolin. Gerbils pretreated with verapamil tended to have an increased ability for survival resulting from the ischemic episode. Under in vitro conditions the enzyme preparations were not markedly influenced by either drug. PMID:3508245

  2. Persistent stimulation of adenylate cyclase and urea transport by an AVP photolabel

    SciTech Connect

    Eggena, P.; Ma, C.L.; Fahrenholz, F.; Schwartz, I.L.

    1985-07-01

    The effects of a photoaffinity label for arginine vasopressin receptors, (Phe2, Phe(p-N3)3)AVP (N3-AVP), on urea permeability and adenylate cyclase activity have been investigated in the toad urinary bladder. This compound, when activated by ultraviolet light, induced a maximal and persistent increase in the urea permeability of the intact bladder and a persistent increase in the adenylate cyclase activity of toad bladder epithelial cell homogenates. Covalent attachment of the analogue to target tissue during photolysis was equivalent at 4 and 20 degrees C. Bladders exposed to N3-AVP in the presence of AVP during photolysis were substantially less permeable to urea than controls that had been exposed to N3-AVP alone. These findings constitute further evidence in support of the previous suggestion that N3-AVP binds covalently to AVP receptors and, in addition, demonstrates that N3-AVP evokes a persistent increase in adenylate cyclase activity which, in turn, triggers a persistent increase in bladder permeability to urea.

  3. Opioid inhibition of adenylate cyclase in the striatum and vas deferens of the rat.

    PubMed Central

    Bhoola, K. D.; Pay, S.

    1986-01-01

    The activity of adenylate cyclase in striatal membrane-enriched fractions (25,000 g) was inhibited by morphine, beta-endorphin, [D-Ala2-D-Leu5] enkephalin (DADLenk), fentanyl and bremazocine. Whereas guanosine triphosphate (GTP) appeared essential for the expression of this effect, sodium chloride seemed to enhance the degree of inhibition. Dopamine stimulation and sodium fluoride activation of the enzyme was also suppressed by morphine, beta-endorphin and DADLenk. beta-Endorphin and DADLenk inhibited adenylate cyclase activity in vasa deferentia membrane-enriched fractions (25,000 g); both opioids required GTP and NaCl and were inhibited by a delta-opioid receptor antagonist and by naloxone. Morphine, bremazocine and tifluadom did not significantly alter the activity of the vas deferens enzyme. Basal cyclic AMP values of striatal slices were not significantly altered by morphine, beta-endorphin or DADLenk. However, dopamine-induced elevation of cyclic AMP was reduced by morphine and this effect of the opiate was suppressed by naloxone. Only beta-endorphin lowered the basal cyclic AMP values in the vas deferens. The physiological relevance of adenylate cyclase coupling to opioid receptor subtypes is considered. PMID:3026542

  4. A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity

    PubMed Central

    Israeli, Ma’ayan; Rotem, Shahar; Elia, Uri; Bar-Haim, Erez; Cohen, Ofer; Chitlaru, Theodor

    2016-01-01

    Edema Factor (EF), the toxic sub-unit of the Bacillus anthracis Edema Toxin (ET) is a calmodulin-dependent adenylate cyclase whose detrimental activity in the infected host results in severe edema. EF is therefore a major virulence factor of B. anthracis. We describe a simple, rapid and reliable functional adenylate-cyclase assay based on inhibition of a luciferase-mediated luminescence reaction. The assay exploits the efficient adenylate cyclase-mediated depletion of adenosine tri-phosphate (ATP), and the strict dependence on ATP of the light-emitting luciferase-catalyzed luciferin-conversion to oxyluciferin, which can be easily visualized. The assay exhibits a robust EF-dose response decrease in luminescence, which may be specifically reverted by anti-EF antibodies. The application of the assay is exemplified in: (a) determining the presence of EF in B. anthracis cultures, or its absence in cultures of EF-defective strains; (b) evaluating the anti-EF humoral response in experimental animals infected/vaccinated with B. anthracis; and (c) rapid discrimination between EF producing and non-producing bacterial colonies. Furthermore, the assay may be amenable with high-throughput screening for EF inhibitory molecules. PMID:27548219

  5. Isolation and characterization of an Escherichia coli mutant affected in the regulation of adenylate cyclase.

    PubMed Central

    Guidi-Rontani, C; Danchin, A; Ullmann, A

    1981-01-01

    A mutant, cyaR1, affecting regulation of adenylate cyclase expression or activity is described. It was obtained as a thermoresistant revertant of a strain harboring a thermosensitive transcription termination factor, rho (rho-15). This mutant failed to synthesize adenosine 3',5'-phosphate and exhibited a carbohydrate-negative phenotype. A secondary mutation at the crp locus (crpC) restored the ability of the mutant to synthesize adenosine 3',5'-phosphate, enabled the expression of catabolite-sensitive operons, and conferred on the strain an extreme sensitivity to catabolite repression. In addition, we showed that the crpC mutation restored the pleiotropic carbohydrate-positive phenotype even in a delta cya background. We interpret this to mean that the adenosine 3',5'-phosphate receptor protein regulates negatively either the activity or synthesis of adenylate cyclase and that the cyaR1 mutation is either in a regulatory protein or a regulatory site of adenylate cyclase. Images PMID:6273380

  6. A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity.

    PubMed

    Israeli, Ma'ayan; Rotem, Shahar; Elia, Uri; Bar-Haim, Erez; Cohen, Ofer; Chitlaru, Theodor

    2016-01-01

    Edema Factor (EF), the toxic sub-unit of the Bacillus anthracis Edema Toxin (ET) is a calmodulin-dependent adenylate cyclase whose detrimental activity in the infected host results in severe edema. EF is therefore a major virulence factor of B. anthracis. We describe a simple, rapid and reliable functional adenylate-cyclase assay based on inhibition of a luciferase-mediated luminescence reaction. The assay exploits the efficient adenylate cyclase-mediated depletion of adenosine tri-phosphate (ATP), and the strict dependence on ATP of the light-emitting luciferase-catalyzed luciferin-conversion to oxyluciferin, which can be easily visualized. The assay exhibits a robust EF-dose response decrease in luminescence, which may be specifically reverted by anti-EF antibodies. The application of the assay is exemplified in: (a) determining the presence of EF in B. anthracis cultures, or its absence in cultures of EF-defective strains; (b) evaluating the anti-EF humoral response in experimental animals infected/vaccinated with B. anthracis; and (c) rapid discrimination between EF producing and non-producing bacterial colonies. Furthermore, the assay may be amenable with high-throughput screening for EF inhibitory molecules. PMID:27548219

  7. Hemodynamic actions of systemically injected pituitary adenylate cyclase activating polypeptide-27 in the rat

    NASA Technical Reports Server (NTRS)

    Whalen, E. J.; Johnson, A. K.; Lewis, S. J.

    1999-01-01

    The aims of this study were (1) to characterize the hemodynamic mechanisms underlying the hypotensive effects of pituitary adenylate cyclase activating polypeptide-27 (PACAP-27 0.1-2.0 nmol/kg, i.v.) in pentobarbital-anesthetized rats, and (2) to determine the roles of the autonomic nervous system, adrenal catecholamines and endothelium-derived nitric oxide (NO) in the expression of PACAP-27-mediated effects on hemodynamic function. PACAP-27 produced dose-dependent decreases in mean arterial blood pressure and hindquarter and mesenteric vascular resistances in saline-treated rats. PACAP-27 also produced pronounced falls in mean arterial blood pressure in rats treated with the ganglion blocker, chlorisondamine (5 mg/kg, i.v.). The hypotensive and vasodilator actions of PACAP-27 were not attenuated by the beta-adrenoceptor antagonist, propranolol (1 mg/kg, i.v.), or the NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME 50 micromol/kg, i.v.). PACAP-27 produced dose-dependent increases in heart rate whereas the hypotensive response produced by the nitrovasodilator, sodium nitroprusside (10 microg/kg, i.v.), was associated with a minimal tachycardia. The PACAP-27-induced tachycardia was unaffected by chlorisondamine, but was virtually abolished by propranolol. These results suggest that the vasodilator effects of PACAP-27 are due to actions in the microcirculation rather than to the release of adrenal catecholamines and that this vasodilation may not involve the release of endothelium-derived NO. These results also suggest that PACAP-27 produces tachycardia by directly releasing norepinephrine from cardiac sympathetic nerve terminals rather than by direct or baroreceptor reflex-mediated increases in sympathetic nerve activity.

  8. Reconstitution of beta 1-adrenoceptor-dependent adenylate cyclase from purified components.

    PubMed Central

    Feder, D; Im, M J; Klein, H W; Hekman, M; Holzhöfer, A; Dees, C; Levitzki, A; Helmreich, E J; Pfeuffer, T

    1986-01-01

    In continuation of our efforts to reconstitute from purified components into lipid vesicles the signal transmission chain from beta 1-adrenoceptors to adenylate cyclase, we now report on the total reconstitution of the hormone-dependent adenylate cyclase. In these reconstitution experiments we have employed the purified adenylate cyclase (C) from bovine brain and rabbit heart, the stimulatory GTP-binding protein (GS) purified from turkey erythrocytes and rabbit liver and the beta 1-adrenoceptor (R) from turkey erythrocytes. Several detergents were compared with respect to their suitability to allow reconstitution of subunits into phospholipid vesicles. While octyl-polyoxyethylene (octyl-POE) was almost as potent as lauroyl-sucrose for preparation of vesicles containing GS.C, the latter detergent was clearly superior for vesicles enabling productive R.GS and R.GS.C coupling. The catalytic subunit from either bovine brain or rabbit heart was equally efficient in reconstitution. However, GS from turkey erythrocytes and rabbit liver revealed significant differences in RGS and RGS.C containing vesicles. While isoproterenol-induced activation of GS by GTP gamma S was first order in both instances, kon with turkey GS was 0.12 min-1, whereas kon with rabbit liver GS was 0.6 min-1. Moreover, GTP gamma S activation of erythrocyte GS was significantly more dependent on the presence of hormone than that of liver GS, confirming observations made on the native membrane-bound system. Compared with stimulation by isoproterenol (GTP gamma S) (4-fold), stimulation by isoproterenol/GTP was modest (1.3- to 1.6-fold).(ABSTRACT TRUNCATED AT 250 WORDS) Images Fig. 1. PMID:3017696

  9. Insect Stage-Specific Adenylate Cyclases Regulate Social Motility in African Trypanosomes

    PubMed Central

    Lopez, Miguel A.; Saada, Edwin A.

    2014-01-01

    Sophisticated systems for cell-cell communication enable unicellular microbes to act as multicellular entities capable of group-level behaviors that are not evident in individuals. These group behaviors influence microbe physiology, and the underlying signaling pathways are considered potential drug targets in microbial pathogens. Trypanosoma brucei is a protozoan parasite that causes substantial human suffering and economic hardship in some of the most impoverished regions of the world. T. brucei lives on host tissue surfaces during transmission through its tsetse fly vector, and cultivation on surfaces causes the parasites to assemble into multicellular communities in which individual cells coordinate their movements in response to external signals. This behavior is termed “social motility,” based on its similarities with surface-induced social motility in bacteria, and it demonstrates that trypanosomes are capable of group-level behavior. Mechanisms governing T. brucei social motility are unknown. Here we report that a subset of receptor-type adenylate cyclases (ACs) in the trypanosome flagellum regulate social motility. RNA interference-mediated knockdown of adenylate cyclase 6 (AC6), or dual knockdown of AC1 and AC2, causes a hypersocial phenotype but has no discernible effect on individual cells in suspension culture. Mutation of the AC6 catalytic domain phenocopies AC6 knockdown, demonstrating that loss of adenylate cyclase activity is responsible for the phenotype. Notably, knockdown of other ACs did not affect social motility, indicating segregation of AC functions. These studies reveal interesting parallels in systems that control social behavior in trypanosomes and bacteria and provide insight into a feature of parasite biology that may be exploited for novel intervention strategies. PMID:25416239

  10. Aluminum: a requirement for activation of the regulatory component of adenylate cyclase by fluoride.

    PubMed Central

    Sternweis, P C; Gilman, A G

    1982-01-01

    Activation of the purified guanine nucleotide-binding regulatory component (G/F) of adenylate cyclase by F- requires the presence of Mg2+ and another factor. This factor, which contaminates commercial preparations of various nucleotides and disposable glass test tubes, has been identified as Al3+. In the presence of 10 mM Mg2+ and 5 mM F-, AlCl3 causes activation of G/F with an apparent activation constant of approximately 1-5 muM. The requirement for Al3+ is highly specific; of 28 other metals tested, only Be2+ promoted activation of G/F by F-. PMID:6289322

  11. Effect of serum lipoproteins on the adenylate cyclase activity of rat liver plasma membranes.

    PubMed Central

    Ghiselli, G; Sirtori, C R; Nicosia, S

    1981-01-01

    Four rat lipoprotein classes [lymph chylomicrons, VLD (very-low-density), LD (low-density) and HD (high-density) lipoproteins] were tested for their ability to affect basal adenylate cyclase (EC 4.6.1.1) activity of rat liver plasma membranes. All the lipoproteins, with the exception of lymph chylomicrons, effectively increase the enzyme activity. VLD lipoproteins are the most active class (67% maximal increase), followed by HD lipoproteins (33%) and LD lipoproteins (23%). The effect of VLD lipoproteins is additive to that elicited by GTP or GTP plus glucagon (at least within a certain concentration range). VLD lipoproteins affect only the Vmax. of the enzyme, not the Km. PMID:7317023

  12. Binding of (/sup 3/H)forskolin to solubilized preparations of adenylate cyclase

    SciTech Connect

    Nelson, C.A.; Seamon, K.B.

    1988-01-01

    The binding of (/sup 3/H)forskolin to proteins solubilized from bovine brain membranes was studied by precipitating proteins with polyethylene glycol and separating (/sup 3/H)forskolin bound to protein from free (/sup 3/H)forskolin by rapid filtration. The K/sub d/ for (/sup 3/H)forskolin binding to solubilized proteins was 14 nM which was similar to that for (/sup 3/H)forskolin binding sites in membranes from rat brain and human platelets. Forskolin analogs competed for (/sup 3/H)forskolin binding sites with the same rank potency in both brain membranes and in proteins solubilized from brain membranes. (/sup 3/H)forskolin bound to proteins solubilized from membranes with a Bmax of 38 fmolmg protein which increased to 94 fmolmg protein when GppNHp was included in the binding assay. In contrast, GppNHp had no effect on (/sup 3/H)forskolin binding to proteins solubilized from membranes preactivated with GppNHp. Solubilized adenylate cyclase from non-preactivated membranes had a basal activity of 130 pmolmgmin which was increased 7-fold by GppNHp. In contrast, adenylate cyclase from preactivated membranes had a basal activity of 850 pmolmgmin which was not stimulated by GppNHp or forskolin

  13. Influence of the beta-adrenergic receptor concentration on functional coupling to the adenylate cyclase system.

    PubMed Central

    Severne, Y; Coppens, D; Bottari, S; Riviere, M; Kram, R; Vauquelin, G

    1984-01-01

    Only part of the beta-adrenergic receptors can undergo functional coupling to the adenylate cyclase regulatory unit. This receptor subpopulation shows an increased affinity for agonists in the presence of Mg2+ and undergoes rapid "inactivation" (locking-in of the agonist) by the alkylating reagent N-ethylmaleimide in the presence of agonists. Several experimental conditions, known to modify the total receptor concentration without alteration of the other components of the adenylate cyclase system, do not affect the percentage of receptors that can undergo functional coupling: (i) homologous regulation of beta 1 receptors in rat brain by noradrenaline (through antidepressive drug or reserpine injections); (ii) up- and down-regulation of the beta 2 receptors in Friend erythroleukemia cells by, respectively, sodium butyrate and cinnarizine treatment; and (iii) dithiothreitol-mediated inactivation of receptors in turkey erythrocytes, Friend erythroleukemia cells, and rat brain. Our findings argue against a stoichiometric limitation in the number of regulatory components, genetically different receptor subpopulations, bound guanine nucleotides, or reduced accessibility of part of the receptors to the agonists as the cause for functional receptor heterogeneity. Differences in either the receptor conformation or its membrane microenvironment are more plausible explanations. PMID:6087337

  14. Adenylate cyclase regulation in the spermatogenic cell plasma membrane: Modulating effects of TPA and TCDD

    SciTech Connect

    Beebe, L.E.

    1989-01-01

    This research was designed to compare the effects of TPA, a phorbol ester, and TCDD in a spermatogenic cell population, a target of TCDD toxicity. Membrane-bound adenylate cyclase activity was used an index of membrane function, and was quantified by the amount of {sup 32}P-cAMP formed from {sup 32}P-ATP following chromatographic separation. Exposure to male germ cells in-vitro to TPA and TCDD followed by direct measurement of enzyme activity was used to investigate the potential of each agent to perturb membrane function. TPA and TCDD consistently inhibited adenylate cyclase activity at the levels of G{sub s}-catalytic unit coupling and hormone-receptor activation, as measured by the stimulation of enzyme activity by concomitant addition of forskolin and GTP and FSH and GTP, respectively. The effect on coupling required at least 60 minutes of exposure to TPA or TCDD. Concentration-response curves demonstrated a progressive desensitization with increasing TPA concentration, while TCDD exhibited consistent inhibition over the same concentration range.

  15. Pituitary Adenylate Cyclase-Activating Polypeptide Receptors Signal via Phospholipase C Pathway to Block Apoptosis in Newborn Rat Retina.

    PubMed

    Lakk, Monika; Denes, Viktoria; Gabriel, Robert

    2015-07-01

    Glutamate induced cell death mechanisms gained considerable attention lately as excessive release of extracellular glutamate was reported to cause neurodegeneration in brain areas including the retina. Conversely, pituitary adenylate cyclase-activating polypeptide (PACAP) was shown to provide neuroprotection through anti-apoptotic effects in the glutamate-model and also in other degeneration assays. Although PACAP is known to orchestrate complex intracellular signaling primarily through cAMP production, the mechanism that mediates the anti-apoptotic effect in glutamate excitotoxicity remains to be clarified. To study this mechanism we induced retinal neurodegeneration in newborn Wistar rats by subcutaneous monosodium-glutamate injection. 100 pmol PACAP and enzyme inhibitors were administered intravitreally. Levels of caspase 3, 9, and phospho-protein kinase A were assessed by Western blots. Changes in cAMP levels were detected employing a competitive immunoassay. We found that cAMP blockade by an adenylyl-cyclase inhibitor (2',4'-dideoxy-adenosine) did not abrogate the neuroprotective effect of PACAP1-38. We show that following intravitreal PACAP1-38 treatment cAMP was unaltered, consistent with the inhibitor results and phospho-protein kinase A, an effector of the cAMP pathway was also unaffected. On the other hand, blockade of the alternative phosphatidylcholine-specific PLC pathway using an inhibitor (D609CAS) abrogated the neuroprotective effects of PACAP1-38. Our results highlight PACAP1-38 ability in protecting retinal cells against apoptosis through diverse signaling cascades. It seems that at picomolar concentrations, PACAP does not trigger cAMP production, but nonetheless, exerts a significant anti-apoptotic effect through PLC activation. In conclusion, PACAP1-38 may signal via both AC and PLC activation producing the same protective outcome. PMID:25975365

  16. Fetal nicotine exposure produces postnatal up-regulation of adenylate cyclase activity in peripheral tissues

    SciTech Connect

    Slotkin, T.A.; Navarro, H.A.; McCook, E.C.; Seidler, F.J. )

    1990-01-01

    Gestational exposure to nicotine has been shown to affect development of noradrenergic activity in both the central and peripheral nervous systems. In the current study, pregnant rats received nicotine infusions of 6 mg/kg/day throughout gestation, administered by osmotic minipump implants. After birth, offspring of the nicotine-infused dams exhibited marked increases in basal adenylate cyclase activity in membranes prepared from kidney and heart, as well as supersensitivity to stimulation by either a {beta}-adrenergic agonist, isoproterenol, or by forskolin. The altered responses were not accompanied by up-regulation of {beta}-adrenergic receptors: in fact, ({sup 125}I)pindolol binding was significantly decreased in the nicotine group. These results indicate that fetal nicotine exposure affects enzymes involved in membrane receptor signal transduction, leading to altered responsiveness independently of changes at the receptor level.

  17. Characterization of beta-adrenergic receptors and adenylate cyclase activity in rat brown fat

    SciTech Connect

    Baresi, L.A.; Morley, J.E.; Scarpace, P.J.

    1986-03-01

    Catecholamines stimulate thermogenesis in rat brown fat through a mechanism which involves binding to the beta-adrenergic receptor (BAR), stimulation of adenylate cyclase (AC) and culminating with uncoupling of mitochondrial respiration from ATP synthesis. The authors characterized BAR, AC and cytochrome (cyt) c oxidase in CDF (F-344) interscapular brown fat. Scatchard analysis of (/sup 125/)Iodopindolol binding yields a straight line consistent with a single class of antagonist binding sites with 41.8 +/- 12.0 fmol BAR/mg protein and a K/sub d/ of 118 +/- 15 pM. Binding was both specific and stereospecific. Competition with 1-propranolol (K/sub d/ = 6.7 nM) was 15 times more potent than d-propranolol (K/sub d/ = 103 nM). Competition with isoproterenol (K/sub d/ = 79 nM) was 10 times more potent than epinephrine (K/sub d/ = 820 nM) which was 35 times more potent than norepinephrine (K/sub d/ = 2.9 x 10/sup -5/ M) suggesting predominate beta/sub 2/-type BAR. Cyt c oxidase activity was assessed in brown fat mitochrondrial preparations. The ratio of BAR to cyt c activity was 959 +/- 275 nmol BAR/mol cyc c/min. Isoproterenol (0.1 mM) stimulated AC activity was 24 times GTP (0.1 mM) stimulated AC (98.5 vs 40.7 pmol cAMP/min/mg). NaF-stimulated AC was nine times basal activity (90.5 vs 11.3 pmol cAMP/min/mg). These data demonstrate the presence of a beta-/sub 2/-type BAR coupled to adenylate cyclase in rat brown fat.

  18. Subtyping of Salmonella enterica Subspecies I Using Single-Nucleotide Polymorphisms in Adenylate Cyclase.

    PubMed

    Guard, Jean; Abdo, Zaid; Byers, Sara Overstreet; Kriebel, Patrick; Rothrock, Michael J

    2016-07-01

    Methods to rapidly identify serotypes of Salmonella enterica subspecies I are of vital importance for protecting the safety of food. To supplement the serotyping method dkgB-linked intergenic sequence ribotyping (ISR), single-nucleotide polymorphisms were characterized within adenylate cyclase (cyaA). The National Center for Biotechnology Information (NCBI) database had 378 cyaA sequences from S. enterica subspecies I, which included 42 unique DNA sequences and 19 different amino acid sequences. Five representative isolates, namely serotypes Typhimurium, Kentucky, Enteritidis phage type PT4, and two variants of Enteritidis phage type PT13a, were differentiated within a microsphere-based fluidics system in cyaA by allele-specific primer extension. Validation against 25 poultry-related environmental Salmonella isolates representing 11 serotypes yielded a ∼89% success rate at identifying the serotype of the isolate, and a different region could be targeted to achieve 100%. When coupled with ISR, all serotypes were differentiated. Phage lineages of serotype Enteritidis 13a and 4 were identified, and a biofilm-forming strain of PT13a was differentiated from a smooth phenotype within phage type. Comparative ranking of mutation indices to genes such as the tRNA transferases, the diguanylate cyclases, and genes used for multilocus sequence typing indicated that cyaA is an appropriate gene for assessing epidemiological trends of Salmonella because of its relative stability in nucleotide composition. PMID:27035032

  19. Adenylate cyclase 3: a new target for anti-obesity drug development.

    PubMed

    Wu, L; Shen, C; Seed Ahmed, M; Östenson, C-G; Gu, H F

    2016-09-01

    Obesity has become epidemic worldwide, and abdominal obesity has a negative impact on health. Current treatment options on obesity, however, still remain limited. It is then of importance to find a new target for anti-obesity drug development based upon recent molecular studies in obesity. Adenylate cyclase 3 (ADCY3) is the third member of adenylyl cyclase family and catalyses the synthesis of cAMP from ATP. Genetic studies with candidate gene and genome-wide association study approaches have demonstrated that ADCY3 genetic polymorphisms are associated with obesity in European and Chinese populations. Epigenetic studies have indicated that increased DNA methylation levels in the ADCY3 gene are involved in the pathogenesis of obesity. Furthermore, biological analyses with animal models have implicated that ADCY3 dysfunction resulted in increased body weight and fat mass, while reduction of body weight is partially explained by ADCY3 activation. In this review, we describe genomic and biological features of ADCY3, summarize genetic and epigenetic association studies of the ADCY3 gene with obesity and discuss dysfunction and activation of ADCY3. Based upon all data, we suggest that ADCY3 is a new target for anti-obesity drug development. Further investigation on the effectiveness of ADCY3 activator and its delivery approach to treat abdominal obesity has been taken into our consideration. PMID:27256589

  20. Subtyping of Salmonella enterica Subspecies I Using Single-Nucleotide Polymorphisms in Adenylate Cyclase

    PubMed Central

    Abdo, Zaid; Byers, Sara Overstreet; Kriebel, Patrick; Rothrock, Michael J.

    2016-01-01

    Abstract Methods to rapidly identify serotypes of Salmonella enterica subspecies I are of vital importance for protecting the safety of food. To supplement the serotyping method dkgB-linked intergenic sequence ribotyping (ISR), single-nucleotide polymorphisms were characterized within adenylate cyclase (cyaA). The National Center for Biotechnology Information (NCBI) database had 378 cyaA sequences from S. enterica subspecies I, which included 42 unique DNA sequences and 19 different amino acid sequences. Five representative isolates, namely serotypes Typhimurium, Kentucky, Enteritidis phage type PT4, and two variants of Enteritidis phage type PT13a, were differentiated within a microsphere-based fluidics system in cyaA by allele-specific primer extension. Validation against 25 poultry-related environmental Salmonella isolates representing 11 serotypes yielded a ∼89% success rate at identifying the serotype of the isolate, and a different region could be targeted to achieve 100%. When coupled with ISR, all serotypes were differentiated. Phage lineages of serotype Enteritidis 13a and 4 were identified, and a biofilm-forming strain of PT13a was differentiated from a smooth phenotype within phage type. Comparative ranking of mutation indices to genes such as the tRNA transferases, the diguanylate cyclases, and genes used for multilocus sequence typing indicated that cyaA is an appropriate gene for assessing epidemiological trends of Salmonella because of its relative stability in nucleotide composition. PMID:27035032

  1. The Bordetella Adenylate Cyclase Repeat-in-Toxin (RTX) Domain Is Immunodominant and Elicits Neutralizing Antibodies*

    PubMed Central

    Wang, Xianzhe; Maynard, Jennifer A.

    2015-01-01

    The adenylate cyclase toxin (ACT) is a multifunctional virulence factor secreted by Bordetella species. Upon interaction of its C-terminal hemolysin moiety with the cell surface receptor αMβ2 integrin, the N-terminal cyclase domain translocates into the host cell cytosol where it rapidly generates supraphysiological cAMP concentrations, which inhibit host cell anti-bacterial activities. Although ACT has been shown to induce protective immunity in mice, it is not included in any current acellular pertussis vaccines due to protein stability issues and a poor understanding of its role as a protective antigen. Here, we aimed to determine whether any single domain could recapitulate the antibody responses induced by the holo-toxin and to characterize the dominant neutralizing antibody response. We first immunized mice with ACT and screened antibody phage display libraries for binding to purified ACT. The vast majority of unique antibodies identified bound the C-terminal repeat-in-toxin (RTX) domain. Representative antibodies binding two nonoverlapping, neutralizing epitopes in the RTX domain prevented ACT association with J774A.1 macrophages and soluble αMβ2 integrin, suggesting that these antibodies inhibit the ACT-receptor interaction. Sera from mice immunized with the RTX domain showed similar neutralizing activity as ACT-immunized mice, indicating that this domain induced an antibody response similar to that induced by ACT. These data demonstrate that RTX can elicit neutralizing antibodies and suggest it may present an alternative to ACT. PMID:25505186

  2. Mechanism of activation of light-activated phosphodiesterase and evidence for homology with hormone-activated adenylate cyclase

    SciTech Connect

    Bitensky, M.W.; Yamazaki, A.; Wheeler, M.A.; George, J.S.; Rasenick, M.M.

    1983-01-01

    Light-activated cGMP phosphodiesterase (PDE) is one of the effector proteins in the rod outer segments in vertebrate retina. The hydrolysis of cGMP in rod occurs with a speed and light sensitivity which suggests a role for this hydrolysis in visual transduction. In fact, there is electrophysiological data which supports the possibility that cGMP could regulate rod membrane voltage. PDE shows very rapid activation in the presence of photons and GTP. We have called attention to the intriguing analogy between light activated rod phosphodiesterase and hormone activated adenylate cyclase. A number of studies have implicated the binding of GTP to a GTP binding protein as a factor in the hormone dependent activation of adenylate cyclase. Moreover, Cassel and Selinger have shown that hydrolysis of GTP is a component in the inactivation of the hormone dependent adenylate cyclase. We review here recent additional data which provide specific molecular details of the mechanism of light activation of rod PDE as well as demonstrate the exchange of components between light activated PDE and hormone activated cyclase.

  3. Functional non-nucleoside adenylyl cyclase inhibitors.

    PubMed

    Lelle, Marco; Hameed, Abdul; Ackermann, Lisa-Maria; Kaloyanova, Stefka; Wagner, Manfred; Berisha, Filip; Nikolaev, Viacheslav O; Peneva, Kalina

    2015-05-01

    In this study, we describe the synthesis of novel functional non-nucleoside adenylyl cyclase inhibitors, which can be easily modified with thiol containing biomolecules such as tumour targeting structures. The linkage between inhibitor and biomolecule contains cleavable bonds to enable efficient intracellular delivery in the reductive milieu of the cytosol as well as in the acidic environment within endosomes and lysosomes. The suitability of this synthetic approach was shown by the successful bioconjugation of a poor cell-permeable inhibitor with a cell-penetrating peptide. Additionally, we have demonstrated the excellent inhibitory effect of the compounds presented here in a live-cell Förster resonance energy transfer-based assay in human embryonic kidney cells. PMID:25319071

  4. Cell-cycle arrest induced by the bacterial adenylate cyclase toxins from Bacillus anthracis and Bordetella pertussis

    PubMed Central

    Gray, Mary C.; Hewlett, Erik L.

    2014-01-01

    Summary Bacillus anthracis Edema Toxin (ET) and Bordetella pertussis Adenylate Cyclase Toxin (ACT) enter host cells and produce cAMP. To understand the cellular consequences, we exposed J774 cells to these toxins at ng/ml (pM) concentrations, then followed cell number and changes in cell signaling pathways. Under these conditions, both toxins produce a concentration-dependent inhibition of cell proliferation without cytotoxicity. ET and ACT increase the proportion of cells in G1/G0 and reduce S-phase, such that a single addition of ET or ACT inhibits cell division for 3 to 6 days. Treatment with ET or ACT produces striking changes in proteins controlling cell cycle, including virtual elimination of phosphorylated ERK 1/2 and Cyclin D1 and increases in phospho-CREB and p27Kip1. Importantly, PD98059, a MEK inhibitor, elicits a comparable reduction in Cyclin D1 to that produced by the toxins and blocks proliferation. These data show that non-lethal concentrations of ET and ACT impose a prolonged block on the proliferation of J774 cells by impairment of the progression from G1/G0 to S-phase in a process involving cAMP-mediated increases in phospho-CREB and p27Kip1 and reductions in phospho-ERK 1/2 and Cyclin D1. This phenomenon represents a new mechanism by which these toxins affect host cells. PMID:20946259

  5. Studies of the cAMP mediated aggregation in Dictyostelium discoideum: receptor mediated activation of the adenylate cyclase

    SciTech Connect

    Theibert, W.E.A.B.

    1985-01-01

    Dictyostelium discoideum, a eukaryotic amoeba of the cellular slime mold family, provides an interesting paradigm in developmental biology. During development, hundreds of thousands of cells aggregate to form a multicellular aggregate. Aggregation is mediated by chemotaxis and chemical signaling. Waves of adenosine 3'-5' cyclic monophosphate (cAMP) propagate through the monolayer and provide transient gradients for chemotaxis. The author has used a reversible inhibitor of the cAMP signaling response to demonstrate that adaptation to cAMP is independent of the activation of the adenylate cyclase and therefore is not caused by the rise in intracellular cAMP. Next, it is shown that adenosine inhibits the cAMP signaling response. Inhibition is rapid, reversible, and depends on the cAMP stimulus concentration. Then the specificity of the cAMP receptors which mediates signaling is determined and compared with the receptors which mediate chemotaxis, the cGMP response, and cAMP binding antagonism. The cAMP surface receptor has been identified by photoaffinity labeling intact cells with (/sup 32/P)-8-N/sub 3/-cAMP using an ammonium sulfate binding stabilization technique. The photoactivated ligand specifically labels a polypeptide, localized to the membrane fraction, which migrates as a closely spaced doublet on SDS Page.

  6. Characterization of a novel serotonin receptor coupled to adenylate cyclase in the hybrid neuroblastoma cell line NCB. 20

    SciTech Connect

    Conner, D.A.

    1988-01-01

    Pharmacological characterization of the serotonin activation of adenylate cyclase in membrane preparation using over 40 serotonergic and non-serotonergic compounds demonstrated that the receptor mediating the response was distinct from previously described mammalian serotonin receptors. Agonist activity was only observed with tryptamine and ergoline derivatives. Potent antagonism was observed with several ergoline derivatives and with compounds such as mianserin and methiothepine. A comparison of the rank order of potency of a variety of compounds for the NCB.20 cell receptor with well characterized mammalian and non-mammalian serotonin receptors showed a pharmacological similarity, but not identity, with the mammalian 5-HT{sub 1C} receptor, which modulates phosphatidylinositol metabolism, and with serotonin receptors in the parasitic trematodes Fasciola hepatica and Schistosoma mansoni, which are coupled to adenylate cyclase. Equilibrium binding analysis utilizing ({sup 3}H)serotonin, ({sup 3}H)lysergic acid diethylamide or ({sup 3}H)dihydroergotamine demonstrated that there are no abundant high affinity serotonergic sites, which implies that the serotonin activation of adenylate cyclase is mediated by receptors present in low abundance. Incubation of intact NCB.20 cells with serotinin resulted in a time and concentration dependent desensitization of the serotonin receptor.

  7. Forskolin- and dihydroalprenolol (DHA) binding sites and adenylate cyclase activity in heart of rats fed diets containing different oils

    SciTech Connect

    Alam, S.Q.; Ren, Y.F.; Alam, B.S.

    1987-05-01

    The purpose of the present investigation was to determine if dietary lipids can induce changes in the adenylate cyclase system in rat heart. Three groups of male young Sprague-Dawley rats were fed for 6 weeks diets containing 10% corn oil (I), 8% coconut oil + 2% corn oil (II) or 10% menhaden oil (III). Adenylate cyclase activity (basal, fluoride-, isoproterenol-, and forskolin-stimulated) was higher in heart homogenates of rats in group III than in the other two groups. Concentration of the (/sup 3/H)-forskolin binding sites in the cardiac membranes were significantly higher in rats fed menhaden oil. The values (pmol/mg protein) were 4.8 +/- 0.2 (I), 4.5 +/- 0.7 (II) and 8.4 +/- 0.5 (III). There was no significant difference in the affinity of the forskolin binding sites among the 3 dietary groups. When measured at different concentrations of forskolin, the adenylate cyclase activity in cardiac membranes of rats fed menhaden oil was higher than in the other 2 groups. Concentrations of the (/sup 3/H)DHA binding sites were slightly higher but their affinity was lower in cardiac membranes of rats fed menhaden oil. The results suggest that diets containing fish oil increase the concentration of the forskolin binding sites and may also affect the characteristics of the ..beta..-adrenergic receptor in rat heart.

  8. High throughput synthetic lethality screen reveals a tumorigenic role of adenylate cyclase in fumarate hydratase-deficient cancer cells

    PubMed Central

    2014-01-01

    Background Synthetic lethality is an appealing technique for selectively targeting cancer cells which have acquired molecular changes that distinguish them from normal cells. High-throughput RNAi-based screens have been successfully used to identify synthetic lethal pathways with well-characterized tumor suppressors and oncogenes. The recent identification of metabolic tumor suppressors suggests that the concept of synthetic lethality can be applied to selectively target cancer metabolism as well. Results Here, we perform a high-throughput RNAi screen to identify synthetic lethal genes with fumarate hydratase (FH), a metabolic tumor suppressor whose loss-of-function has been associated with hereditary leiomyomatosis and renal cell carcinoma (HLRCC). Our unbiased screen identified synthetic lethality between FH and several genes in heme metabolism, in accordance with recent findings. Furthermore, we identified an enrichment of synthetic lethality with adenylate cyclases. The effects were validated in an embryonic kidney cell line (HEK293T) and in HLRCC-patient derived cells (UOK262) via both genetic and pharmacological inhibition. The reliance on adenylate cyclases in FH-deficient cells is consistent with increased cyclic-AMP levels, which may act to regulate cellular energy metabolism. Conclusions The identified synthetic lethality of FH with adenylate cyclases suggests a new potential target for treating HLRCC patients. PMID:24568598

  9. Bordetella adenylate cyclase toxin is a unique ligand of the integrin complement receptor 3

    PubMed Central

    Osicka, Radim; Osickova, Adriana; Hasan, Shakir; Bumba, Ladislav; Cerny, Jiri; Sebo, Peter

    2015-01-01

    Integrins are heterodimeric cell surface adhesion and signaling receptors that are essential for metazoan existence. Some integrins contain an I-domain that is a major ligand binding site. The ligands preferentially engage the active forms of the integrins and trigger signaling cascades that alter numerous cell functions. Here we found that the adenylate cyclase toxin (CyaA), a key virulence factor of the whooping cough agent Bordetella pertussis, preferentially binds an inactive form of the integrin complement receptor 3 (CR3), using a site outside of its I-domain. CyaA binding did not trigger downstream signaling of CR3 in human monocytes and CyaA-catalyzed elevation of cAMP effectively blocked CR3 signaling initiated by a natural ligand. This unprecedented type of integrin-ligand interaction distinguishes CyaA from all other known ligands of the I-domain-containing integrins and provides a mechanistic insight into the previously observed central role of CyaA in the pathogenesis of B. pertussis. DOI: http://dx.doi.org/10.7554/eLife.10766.001 PMID:26650353

  10. Characterization of the thermoregulatory response to pituitary adenylate cyclase-activating polypeptide in rodents.

    PubMed

    Banki, Eszter; Pakai, Eszter; Gaszner, Balazs; Zsiboras, Csaba; Czett, Andras; Bhuddi, Paras Rahul Parkash; Hashimoto, Hitoshi; Toth, Gabor; Tamas, Andrea; Reglodi, Dora; Garami, Andras

    2014-11-01

    Administration of the long form (38 amino acids) of pituitary adenylate cyclase-activating polypeptide (PACAP38) into the central nervous system causes hyperthermia, suggesting that PACAP38 plays a role in the regulation of deep body temperature (T b). In this study, we investigated the thermoregulatory role of PACAP38 in details. First, we infused PACAP38 intracerebroventricularly to rats and measured their T b and autonomic thermoeffector responses. We found that central PACAP38 infusion caused dose-dependent hyperthermia, which was brought about by increased thermogenesis and tail skin vasoconstriction. Compared to intracerebroventricular administration, systemic (intravenous) infusion of the same dose of PACAP38 caused significantly smaller hyperthermia, indicating a central site of action. We then investigated the thermoregulatory phenotype of mice lacking the Pacap gene (Pacap (-/-)). Freely moving Pacap (-/-) mice had higher locomotor activity throughout the day and elevated deep T b during the light phase. When the Pacap (-/-) mice were loosely restrained, their metabolic rate and T b were lower compared to their wild-type littermates. We conclude that PACAP38 causes hyperthermia via activation of the autonomic cold-defense thermoeffectors through central targets. Pacap (-/-) mice express hyperkinesis, which is presumably a compensatory mechanism, because under restrained conditions, these mice are hypometabolic and hypothermic compared to controls. PMID:24994541

  11. Effects of forskolin on cerebral blood flow: implications for a role of adenylate cyclase

    SciTech Connect

    Wysham, D.G.; Brotherton, A.F.; Heistad, D.D.

    1986-11-01

    We have studied cerebral vascular effects of forskolin, a drug which stimulates adenylate cyclase and potentiates dilator effects of adenosine in other vascular beds. Our goals were to determine whether forskolin is a cerebral vasodilator and whether it potentiates cerebral vasodilator responses to adenosine. We measured cerebral blood flow with microspheres in anesthetized rabbits. Forskolin (10 micrograms/kg per min) increased blood flow (ml/min per 100 gm) from 39 +/- 5 (mean +/- S.E.) to 56 +/- 9 (p less than 0.05) in cerebrum, and increased flow to myocardium and kidney despite a decrease in mean arterial pressure. Forskolin did not alter cerebral oxygen consumption, which indicates that the increase in cerebral blood flow is a direct vasodilator effect and is not secondary to increased metabolism. We also examined effects of forskolin on the response to infusion of adenosine. Cerebral blood flow was measured during infusion of 1-5 microM/min adenosine into one internal carotid artery, under control conditions and during infusion of forskolin at 3 micrograms/kg per min i.v. Adenosine alone increased ipsilateral cerebral blood flow from 32 +/- 3 to 45 +/- 5 (p less than 0.05). Responses to adenosine were not augmented during infusion of forskolin. We conclude that forskolin is a direct cerebral vasodilator and forskolin does not potentiate cerebral vasodilator responses to adenosine.

  12. Bordetella adenylate cyclase toxin is a unique ligand of the integrin complement receptor 3.

    PubMed

    Osicka, Radim; Osickova, Adriana; Hasan, Shakir; Bumba, Ladislav; Cerny, Jiri; Sebo, Peter

    2015-01-01

    Integrins are heterodimeric cell surface adhesion and signaling receptors that are essential for metazoan existence. Some integrins contain an I-domain that is a major ligand binding site. The ligands preferentially engage the active forms of the integrins and trigger signaling cascades that alter numerous cell functions. Here we found that the adenylate cyclase toxin (CyaA), a key virulence factor of the whooping cough agent Bordetella pertussis, preferentially binds an inactive form of the integrin complement receptor 3 (CR3), using a site outside of its I-domain. CyaA binding did not trigger downstream signaling of CR3 in human monocytes and CyaA-catalyzed elevation of cAMP effectively blocked CR3 signaling initiated by a natural ligand. This unprecedented type of integrin-ligand interaction distinguishes CyaA from all other known ligands of the I-domain-containing integrins and provides a mechanistic insight into the previously observed central role of CyaA in the pathogenesis of B. pertussis. PMID:26650353

  13. Pituitary Adenylate Cyclase-Activating Polypeptide Reverses Ammonium Metavanadate-Induced Airway Hyperresponsiveness in Rats

    PubMed Central

    Tlili, Mounira; Rouatbi, Sonia; Sriha, Badreddine; Ben Rhouma, Khémais; Sakly, Mohsen; Vaudry, David; Wurtz, Olivier; Tebourbi, Olfa

    2015-01-01

    The rate of atmospheric vanadium is constantly increasing due to fossil fuel combustion. This environmental pollution favours vanadium exposure in particular to its vanadate form, causing occupational bronchial asthma and bronchitis. Based on the well admitted bronchodilator properties of the pituitary adenylate cyclase-activating polypeptide (PACAP), we investigated the ability of this neuropeptide to reverse the vanadate-induced airway hyperresponsiveness in rats. Exposure to ammonium metavanadate aerosols (5 mg/m3/h) for 15 minutes induced 4 hours later an array of pathophysiological events, including increase of bronchial resistance and histological alterations, activation of proinflammatory alveolar macrophages, and increased oxidative stress status. Powerfully, PACAP inhalation (0.1 mM) for 10 minutes alleviated many of these deleterious effects as demonstrated by a decrease of bronchial resistance and histological restoration. PACAP reduced the level of expression of mRNA encoding inflammatory chemokines (MIP-1α, MIP-2, and KC) and cytokines (IL-1α and TNF-α) in alveolar macrophages and improved the antioxidant status. PACAP reverses the vanadate-induced airway hyperresponsiveness not only through its bronchodilator activity but also by counteracting the proinflammatory and prooxidative effects of the metal. Then, the development of stable analogs of PACAP could represent a promising therapeutic alternative for the treatment of inflammatory respiratory disorders. PMID:26199679

  14. Posttraumatic administration of pituitary adenylate cyclase activating polypeptide in central fluid percussion injury in rats.

    PubMed

    Kövesdi, Erzsébet; Tamás, Andrea; Reglodi, Dóra; Farkas, Orsolya; Pál, József; Tóth, Gábor; Bukovics, Péter; Dóczi, Tamás; Büki, András

    2008-04-01

    Several in vitro and in vivo experiments have demonstrated the neuroprotective effects of pituitary adenylate cyclase activating polypeptide (PACAP) in focal cerebral ischemia, Parkinson's disease and traumatic brain injury (TBI). The aim of the present study was to analyze the effect of PACAP administration on diffuse axonal injury (DAI), an important contributor to morbidity and mortality associated with TBI, in a central fluid percussion (CFP) model of TBI. Rats were subjected to moderate (2 Atm) CFP injury. Thirty min after injury, 100 microg PACAP was administered intracerebroventricularly. DAI was assessed by immunohistochemical detection of beta-amyloid precursor protein, indicating impaired axoplasmic transport, and RMO-14 antibody, representing foci of cytoskeletal alterations (neurofilament compaction), both considered classical markers of axonal damage. Analysis of damaged, immunoreactive axonal profiles revealed significant axonal protection in the PACAP-treated versus vehicle-treated animals in the corticospinal tract, as far as traumatically induced disturbance of axoplasmic transport and cytoskeletal alteration were considered. Similarly to our former observations in an impact acceleration model of diffuse TBI, the present study demonstrated that PACAP also inhibits DAI in the CFP injury model. The finding indicates that PACAP and derivates can be considered potential candidates for further experimental studies, or purportedly for clinical trials in the therapy of TBI. PMID:18515209

  15. Galanin stimulates cortisol secretion from human adrenocortical cells through the activation of galanin receptor subtype 1 coupled to the adenylate cyclase-dependent signaling cascade.

    PubMed

    Belloni, Anna S; Malendowicz, Ludwik K; Rucinski, Marcin; Guidolin, Diego; Nussdorfer, Gastone G

    2007-12-01

    Previous studies showed that galanin receptors are expressed in the rat adrenal, and galanin modulates glucocorticoid secretion in this species. Hence, we investigated the expression of the various galanin receptor subtypes (GAL-R1, GAL-R2 and GAL-R3) in the human adrenocortical cells, and the possible involvement of galanin in the control of cortisol secretion. Reverse transcription-polymerase chain reaction detected the expression of GAL-R1 (but not GAL-R2 and GAL-R3) in the inner zones of the human adrenal cortex. The galanin concentration dependently enhanced basal, but not ACTH-stimulated secretion of cortisol from dispersed inner adrenocortical cells (maximal effective concentration, 10(-8) M). The cortisol response to 10(-8) M galanin was abrogated by GAL-R1 immunoneutralization, and unaffected by GAL-R2 or GAL-R3 immunoneutralization. Galanin (10(-8) M) and ACTH (10(-9) M) enhanced cyclic-AMP production from dispersed cells, and the response was suppressed by the adenylate cyclase inhibitor SQ-22536 (10(-4) M). Galanin did not affect inositol triphosphate release, which, in contrast, was raised by angiotensin-II (10(-8) M). SQ-22536 and the protein kinase (PK)A inhibitor H-89 (10(-5) M) abolished the cortisol response to 10(-8) M galanin, while the phospholipase C inhibitor U-73122 and the PKC inhibitor calphostin-C were ineffective. Preincubation with pertussis toxin (Ptx) (0.5 microg/ml) partially inhibited the cortisol response to galanin. We conclude that galanin stimulates cortisol secretion from human inner adrenocortical cells, acting through GAL-R1 coupled to the adenylate cyclase/PKA-dependent signaling cascade via a Ptx-sensitive Galpha protein. PMID:17982695

  16. Pituitary Adenylate Cyclase-activating Polypeptide (PACAP)/PAC1HOP1 Receptor Activation Coordinates Multiple Neurotrophic Signaling Pathways

    PubMed Central

    May, Victor; Lutz, Eve; MacKenzie, Christopher; Schutz, Kristin C.; Dozark, Kate; Braas, Karen M.

    2010-01-01

    MAPK and Akt pathways are predominant mediators of trophic signaling for many neuronal systems. Among the vasoactive intestinal peptide/secretin/glucagon family of related peptides, pituitary adenylate cyclase-activating polypeptide (PACAP) binding to specific PAC1 receptor isoforms can engage multiple signaling pathways and promote neuroprotection through mechanisms that are not well understood. Using a primary sympathetic neuronal system, the current studies demonstrate that PACAP activation of PAC1HOP1 receptors engages both MAPK and Akt neurotrophic pathways in an integrated program to facilitate neuronal survival after growth factor withdrawal. PACAP not only stimulated prosurvival ERK1/2 and ERK5 activation but also abrogated SAPK/JNK and p38 MAPK signaling in parallel. In contrast to the potent and rapid effects of PACAP in ERK1/2 phosphorylation, PACAP stimulated Akt phosphorylation in a late phase of PAC1HOP1 receptor signaling. From inhibitor and immunoprecipitation analyses, the PACAP/PAC1HOP1 receptor-mediated Akt responses did not represent transactivation mechanisms but appeared to depend on Gαq/phosphatidylinositol 3-kinase γ activity and vesicular internalization pathways. Phosphatidylinositol 3-kinase γ-selective inhibitors blocked PACAP-stimulated Akt phosphorylation in primary neuronal cultures and in PAC1HOP1-overexpressing cell lines; RNA interference-mediated knockdown of the receptor effectors attenuated PACAP-mediated Akt activation. Similarly, perturbation of endocytic pathways also blocked Akt phosphorylation. Between ERK and Akt pathways, PACAP-stimulated Akt signaling was the primary cascade that attenuated cultured neuron apoptosis after growth factor withdrawal. The partitioning of PACAP-mediated Akt signaling in endosomes may be a key mechanism contributing to the high spatial and temporal specificity in signal transduction necessary for survival pathways. PMID:20093365

  17. Potentiation of P1075-induced K+ channel opening by stimulation of adenylate cyclase in rat isolated aorta.

    PubMed Central

    Linde, C.; Quast, U.

    1995-01-01

    1. The effects of analogues and stimulators of cyclic AMP on the 86Rb+ efflux-stimulating and binding properties of P1075, an opener of ATP-dependent potassium channels, were studied in rat aortic rings. The increase in 86Rb+ efflux stimulated by P1075 was taken as a qualitative measure of K+ channel opening. 2. Forskolin, a direct activator of adenylate cyclase, isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, and dibutyryl-cyclic AMP (db-cyclic AMP), a membrane permeant cyclic AMP-analogue, relaxed rat aortic rings contracted by noradrenaline with EC50 values of 0.06, 2 and 10 microM, respectively. 3. Forskolin, IBMX and db-cyclic AMP produced concentration-dependent increases of the 86Rb+ efflux induced by P1075 (50 nM) by up to twofold with EC50 values of about 0.1, 1.7 and 81 microM. At these concentrations the agents had little effect on the basal rate of 86Rb+ efflux. 4. The 86Rb+ efflux produced by P1075 in the presence of the cyclic AMP stimulators was inhibited by glibenclamide, a blocker of ATP-sensitive potassium channels. 5. IBMX (100 microM) induced a leftward shift of the concentration-86Rb+ efflux curve of P1075 without increasing the maximum. The enhancements of P1075-stimulated 86Rb+ efflux produced by combinations of forskolin and IBMX were either additive or less than additive. 6. The protein kinase A inhibitor, H-89, inhibited P1075-stimulated 86Rb+ efflux in the presence of IBMX significantly more than in the absence of IBMX, suggesting that the effect of increased cyclic AMP levels is mediated by protein kinase A.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7582466

  18. Heterologous desensitization of adenylate cyclase from pigeon erythrocytes under the action of the catalytic subunit of cAMP-dependent protein kinase

    SciTech Connect

    Popov, K.M.; Bulargina, T.V.; Severin, E.S.

    1985-09-20

    Preincubation of the plasma membranes from pigeon erythrocytes with the catalytic subunit of cAMP-dependent protein kinase leads to desensitization of adenylate cyclase of the erythrocytes. The adenylate cyclase activity, measured in the presence of 10 ..mu..M isoproterenol and 50 ..mu..M GTP-..gamma..-S, is decreased by 40% in 10 min of incubation, while the activity in the presence of 50 ..mu..M GTP-..gamma..-S is decreased by 35% in 20 min. The decrease in the adenylate cyclase activity is due to an increase in the lag phase of activation of the enzyme in the presence of a GTP analog stable to hydrolysis and a decrease in the activity in the steady-state phase of activation. Heterologous desensitization of adenylate cyclase under the action of cAMP-dependent protein kinase is coupled with a decrease in the number of ..beta..-adrenoreceptors capable of passing into a state of high affinity for antagonists in the absence of guanylic nucleotides. The influence of the catalytic subunit on adenylate cyclase entirely models the process of desensitization of the enzyme absorbed in the influence of isoproterenol or cAMP on erythrocytes.

  19. Dopamine inhibition of anterior pituitary adenylate cyclase is mediated through the high-affinity state of the D/sub 2/ receptor

    SciTech Connect

    Borgundvaag, B.; George, S.R.

    1985-07-29

    The diterpinoid forskolin stimulated adenylate cyclase activity (measured by conversion of (/sup 3/H)-ATP to (/sup 3/H)-cAMP) in anterior pituitary from male and female rats. Inhibition of stimulated adenylate cyclase activity by potent dopaminergic agonists was demonstrable only in female anterior pituitary. The inhibition of adenylate cyclase activity displayed a typically dopaminergic rank order of agonist potencies and could be completely reversed by a specific dopamine receptor antagonist. The IC/sub 50/ values of dopamine agonist inhibition of adenylate cyclase activity correlated with equal molarity with the dissociation constant of the high-affinity dopamine agonist-detected receptor binding site and with the IC/sub 50/ values for inhibition of prolactin secretion. These findings support the hypothesis that it is the high-affinity form of the D/sub 2/ dopamine receptor in anterior pituitary which is responsible for mediating the dopaminergic function of attenuating adenylate cyclase activity. 12 references, 4 figures, 1 table.

  20. Functional characterization of neural-restrictive silencer element in mouse pituitary adenylate cyclase-activating polypeptide (PACAP) gene expression.

    PubMed

    Sugawara, Hideki; Tominaga, Aiko; Inoue, Kazuhiko; Takeda, Yasuo; Yamada, Katsushi; Miyata, Atsuro

    2014-11-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is predominantly localized in the nervous system, but the underlying mechanism in its neuron-specific expression remains unclear. In addition to two neural-restrictive silencer-like element (NRSLE1 and 2), as reported previously, we have identified the third element in -1,601 to -1,581 bp from the translational initiation site of mouse PACAP gene and termed it as NRSLE3, of which, the sequence and location were highly conserved among mouse, rat, and human PACAP genes. In luciferase reporter assay, the deletion or site-directed mutagenesis of NRSLE3 in the reporter gene construct, driven by heterologous SV40 promoter, cancelled the repression of luciferase activity in non-neuronal Swiss-3T3 cells. Furthermore, its promoter activity was significantly repressed in Swiss-3T3 cells, but not in neuronal-differentiated PC12 cells. The electrophoretic mobility shift assay (EMSA) with nuclear extracts of Swiss-3T3 cells demonstrated a specific complex with NRSLE3 probe that exhibited the same migration with the neural-restrictive silencer element (NRSE) probe of rat type II sodium channel gene. During neuronal differentiation of PC12 cells, the increment of PACAP mRNA exhibited the correlation with that of REST4 mRNA, which is a neuron-specific variant form of neural-restrictive silencer factor (NRSF). In undifferentiated PC12 cells, trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, which indirectly inhibits NRSF-mediated gene silencing, increased PACAP mRNA level and attenuated the repression of promoter activity of 5' flanking region of mouse PACAP gene containing NRSLEs. These suggest that the NRSE-NRSF system implicates in the regulatory mechanism of neuron-specific expression of PACAP gene. PMID:24939248

  1. Pituitary Adenylate-Cyclase Activating Polypeptide Regulates Hunger- and Palatability-Induced Binge Eating

    PubMed Central

    Hurley, Matthew M.; Maunze, Brian; Block, Megan E.; Frenkel, Mogen M.; Reilly, Michael J.; Kim, Eugene; Chen, Yao; Li, Yan; Baker, David A.; Liu, Qing-Song; Choi, SuJean

    2016-01-01

    While pituitary adenylate cyclase activating polypeptide (PACAP) signaling in the hypothalamic ventromedial nuclei (VMN) has been shown to regulate feeding, a challenge in unmasking a role for this peptide in obesity is that excess feeding can involve numerous mechanisms including homeostatic (hunger) and hedonic-related (palatability) drives. In these studies, we first isolated distinct feeding drives by developing a novel model of binge behavior in which homeostatic-driven feeding was temporally separated from feeding driven by food palatability. We found that stimulation of the VMN, achieved by local microinjections of AMPA, decreased standard chow consumption in food-restricted rats (e.g., homeostatic feeding); surprisingly, this manipulation failed to alter palatable food consumption in satiated rats (e.g., hedonic feeding). In contrast, inhibition of the nucleus accumbens (NAc), through local microinjections of GABA receptor agonists baclofen and muscimol, decreased hedonic feeding without altering homeostatic feeding. PACAP microinjections produced the site-specific changes in synaptic transmission needed to decrease feeding via VMN or NAc circuitry. PACAP into the NAc mimicked the actions of GABA agonists by reducing hedonic feeding without altering homeostatic feeding. In contrast, PACAP into the VMN mimicked the actions of AMPA by decreasing homeostatic feeding without affecting hedonic feeding. Slice electrophysiology recordings verified PACAP excitation of VMN neurons and inhibition of NAc neurons. These data suggest that the VMN and NAc regulate distinct circuits giving rise to unique feeding drives, but that both can be regulated by the neuropeptide PACAP to potentially curb excessive eating stemming from either drive. PMID:27597817

  2. Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium

    PubMed Central

    Ohki, Mio; Sugiyama, Kanako; Kawai, Fumihiro; Tanaka, Hitomi; Nihei, Yuuki; Unzai, Satoru; Takebe, Masumi; Matsunaga, Shigeru; Adachi, Shin-ichi; Shibayama, Naoya; Zhou, Zhiwen; Koyama, Ryuta; Takahashi, Tetsuo; Tame, Jeremy R. H.; Iseki, Mineo; Park, Sam-Yong

    2016-01-01

    Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit. PMID:27247413

  3. Region-Specific Disruption of Adenylate Cyclase Type 1 Gene Differentially Affects Somatosensorimotor Behaviors in Mice

    PubMed Central

    Arakawa, Hiroyuki; Akkentli, Fatih; Erzurumlu, Reha S.

    2015-01-01

    Adenylate cyclase type I (AC1) is primarily, and, abundantly, expressed in the brain. Intracellular calcium/ calmodulin increases regulate AC1 in an activity-dependent manner. Upon stimulation, AC1 produces cAMP and it is involved in the patterning and the refinement of neural circuits. In mice, spontaneous mutations or targeted deletion of the Adcy1 gene, which encodes AC1, resulted in neuronal pattern formation defects. Neural modules in the primary somatosensory (SI) cortex, the barrels, which represent the topographic distribution of the whiskers on the snout, failed to form (Welker et al., 1996; Abdel-Majid et al., 1998). Cortex- or thalamus-specific Adcy1 deletions led to different cortical pattern phenotypes, with thalamus-specific disruption phenotype being more severe (Iwasato et al., 2008; Suzuki et al., 2013). Despite the absence of barrels in the “barrelless”/Adcy1 null mice, thalamocortical terminal bouton density and activation of cortical zones following whisker stimulation were roughly topographic (Abdel-Majid et al., 1998; Gheorghita et al., 2006). To what extent does patterning of the cortical somatosensory body map play a role in sensorimotor behaviors? In this study, we tested mice with global, cortical, or thalamic loss of AC1 function in a battery of sensorimotor and social behavior tests and compared them to mice with all of the whiskers clipped. Contrary to intuitive expectations that any region-specific or global disruption of the AC1 function would lead to similar behavioral phenotypes, we found significant differences in the degree of impairment between these strains. PMID:26023682

  4. Pituitary Adenylate-Cyclase Activating Polypeptide Regulates Hunger- and Palatability-Induced Binge Eating.

    PubMed

    Hurley, Matthew M; Maunze, Brian; Block, Megan E; Frenkel, Mogen M; Reilly, Michael J; Kim, Eugene; Chen, Yao; Li, Yan; Baker, David A; Liu, Qing-Song; Choi, SuJean

    2016-01-01

    While pituitary adenylate cyclase activating polypeptide (PACAP) signaling in the hypothalamic ventromedial nuclei (VMN) has been shown to regulate feeding, a challenge in unmasking a role for this peptide in obesity is that excess feeding can involve numerous mechanisms including homeostatic (hunger) and hedonic-related (palatability) drives. In these studies, we first isolated distinct feeding drives by developing a novel model of binge behavior in which homeostatic-driven feeding was temporally separated from feeding driven by food palatability. We found that stimulation of the VMN, achieved by local microinjections of AMPA, decreased standard chow consumption in food-restricted rats (e.g., homeostatic feeding); surprisingly, this manipulation failed to alter palatable food consumption in satiated rats (e.g., hedonic feeding). In contrast, inhibition of the nucleus accumbens (NAc), through local microinjections of GABA receptor agonists baclofen and muscimol, decreased hedonic feeding without altering homeostatic feeding. PACAP microinjections produced the site-specific changes in synaptic transmission needed to decrease feeding via VMN or NAc circuitry. PACAP into the NAc mimicked the actions of GABA agonists by reducing hedonic feeding without altering homeostatic feeding. In contrast, PACAP into the VMN mimicked the actions of AMPA by decreasing homeostatic feeding without affecting hedonic feeding. Slice electrophysiology recordings verified PACAP excitation of VMN neurons and inhibition of NAc neurons. These data suggest that the VMN and NAc regulate distinct circuits giving rise to unique feeding drives, but that both can be regulated by the neuropeptide PACAP to potentially curb excessive eating stemming from either drive. PMID:27597817

  5. Reconstitution of beta-adrenergic receptor with components of adenylate cyclase.

    PubMed Central

    Hekman, M; Feder, D; Keenan, A K; Gal, A; Klein, H W; Pfeuffer, T; Levitzki, A; Helmreich, E J

    1984-01-01

    Beta 1-Adrenergic receptor proteins were extracted from turkey erythrocyte membranes with lauroyl sucrose and digitonin and purified by affinity chromatography on a column of alprenolol agarose Affi-gel 10 or 15. The 5000-fold purified receptor is able to couple functionally with the stimulatory GTP-binding protein (GS) from either turkey or duck erythrocytes. Functional coupling was achieved by three different approaches. (i) Purified beta-receptor polypeptides were coupled in phospholipid (asolectin) vesicles with GS from a crude cholate or lauroyl sucrose extract of turkey erythrocyte membranes. The detergent was removed and vesicles were formed with SM-2 beads. (ii) Purified beta-receptor was reconstituted with pure, homogeneous GS in asolectin vesicles. (iii) Purified beta-receptors were either coupled in asolectin vesicles with a mixture of pure, homogeneous Gpp(NH)p-activated GS and a lauroyl sucrose extract of turkey erythrocyte membranes, or with pure, homogeneous Gpp(NH)p-activated GS alone. The decay of activity was measured on addition of GTP and hormone. In (ii) and (iii), the detergent was removed and vesicles were formed by gel filtration on Sephadex G-50 columns. In each of the three different experimental conditions, the beta-receptor was activated with l-isoproterenol and activation was blocked with d,l-propranolol. Activated GS were measured separately by means of their capacity to activate a crude Lubrol PX-solubilized adenylate cyclase preparation from rabbit myocardial membrane. The kinetics of GS activation by purified beta-receptors occupied by l-isoproterenol was first order and activation was linearly dependent on receptor concentration.(ABSTRACT TRUNCATED AT 250 WORDS) Images Fig. 1. PMID:6098472

  6. Quantification of the Adenylate Cyclase Toxin of Bordetella pertussis In Vitro and during Respiratory Infection

    PubMed Central

    Eby, Joshua C.; Gray, Mary C.; Warfel, Jason M.; Paddock, Christopher D.; Jones, Tara F.; Day, Shandra R.; Bowden, James; Poulter, Melinda D.; Donato, Gina M.; Merkel, Tod J.

    2013-01-01

    Whooping cough results from infection of the respiratory tract with Bordetella pertussis, and the secreted adenylate cyclase toxin (ACT) is essential for the bacterium to establish infection. Despite extensive study of the mechanism of ACT cytotoxicity and its effects over a range of concentrations in vitro, ACT has not been observed or quantified in vivo, and thus the concentration of ACT at the site of infection is unknown. The recently developed baboon model of infection mimics the prolonged cough and transmissibility of pertussis, and we hypothesized that measurement of ACT in nasopharyngeal washes (NPW) from baboons, combined with human and in vitro data, would provide an estimate of the ACT concentration in the airway during infection. NPW contained up to ∼108 CFU/ml B. pertussis and 1 to 5 ng/ml ACT at the peak of infection. Nasal aspirate specimens from two human infants with pertussis contained bacterial concentrations similar to those in the baboons, with 12 to 20 ng/ml ACT. When ∼108 CFU/ml of a laboratory strain of B. pertussis was cultured in vitro, ACT production was detected in 60 min and reached a plateau of ∼60 ng/ml in 6 h. Furthermore, when bacteria were brought into close proximity to target cells by centrifugation, intoxication was increased 4-fold. Collectively, these data suggest that at the bacterium-target cell interface during infection of the respiratory tract, the concentration of ACT can exceed 100 ng/ml, providing a reference point for future studies of ACT and pertussis pathogenesis. PMID:23429530

  7. Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium.

    PubMed

    Ohki, Mio; Sugiyama, Kanako; Kawai, Fumihiro; Tanaka, Hitomi; Nihei, Yuuki; Unzai, Satoru; Takebe, Masumi; Matsunaga, Shigeru; Adachi, Shin-Ichi; Shibayama, Naoya; Zhou, Zhiwen; Koyama, Ryuta; Ikegaya, Yuji; Takahashi, Tetsuo; Tame, Jeremy R H; Iseki, Mineo; Park, Sam-Yong

    2016-06-14

    Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit. PMID:27247413

  8. Activation of adenylate cyclase by dopamine, GTP, NaF and forskolin in striatal membranes of neonatal, adult and senescent rats.

    PubMed

    Nomura, Y; Makihata, J; Segawa, T

    1984-11-13

    Dopamine (DA) caused a significant activation of striatal adenylate cyclase in neonatal and adult but not in senescent rats. GTP activated cyclase at the adult stage but not at both neonatal and senescent stages. NaF and forskolin activated cyclase at every stage. The coupling mechanism between DA1 receptors and catalytic units of cyclase seems to become functional at the neonatal stage but GTP recognition and/or binding sites lack in stimulatory GTP binding protein in neonatal and senescent membranes. PMID:6543337

  9. Mutation in the β-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin

    SciTech Connect

    Springer, Tzvia I.; Goebel, Erich; Hariraju, Dinesh; Finley, Natosha L.

    2014-10-10

    Highlights: • Bordetella pertussis adenylate cyclase toxin modulates bi-lobal structure of CaM. • The structure and stability of the complex rely on intermolecular associations. • A novel mode of CaM-dependent activation of the adenylate cyclase toxin is proposed. - Abstract: Bordetella pertussis, causative agent of whooping cough, produces an adenylate cyclase toxin (CyaA) that is an important virulence factor. In the host cell, the adenylate cyclase domain of CyaA (CyaA-ACD) is activated upon association with calmodulin (CaM), an EF-hand protein comprised of N- and C-lobes (N-CaM and C-CaM, respectively) connected by a flexible tether. Maximal CyaA-ACD activation is achieved through its binding to both lobes of intact CaM, but the structural mechanisms remain unclear. No high-resolution structure of the intact CaM/CyaA-ACD complex is available, but crystal structures of isolated C-CaM bound to CyaA-ACD shed light on the molecular mechanism by which this lobe activates the toxin. Previous studies using molecular modeling, biochemical, and biophysical experiments demonstrate that CyaA-ACD’s β-hairpin participates in site-specific interactions with N-CaM. In this study, we utilize nuclear magnetic resonance (NMR) spectroscopy to probe the molecular association between intact CaM and CyaA-ACD. Our results indicate binding of CyaA-ACD to CaM induces large conformational perturbations mapping to C-CaM, while substantially smaller structural changes are localized primarily to helices I, II, and IV, and the metal-binding sites in N-CaM. Site-specific mutations in CyaA-ACD’s β-hairpin structurally modulate N-CaM, resulting in conformational perturbations in metal binding sites I and II, while no significant structural modifications are observed in C-CaM. Moreover, dynamic light scattering (DLS) analysis reveals that mutation of the β-hairpin results in a decreased hydrodynamic radius (R{sub h}) and reduced thermal stability in the mutant complex. Taken

  10. Kinetic Evidence for the Presence of Two Postaglandin Receptor Sites Regulating the Activity of Intestinal Adenylate Cyclase Sensitive to Escherichia coli Enterotoxin

    PubMed Central

    Kantor, Harvey S.; Tao, Pearl; Kiefer, Helen Chilton

    1974-01-01

    Kinetic behavior most consistent with the presence of two independent, but simultaneously acting, regulatory effector sites for prostaglandins has been presented for adenylate cyclase (EC 4.6.1.1) of rabbit intestinal epithelial cells. One site regulates activation of the catalytic site, while the other site regulates inhibition. A synthetic prostaglandin analogue, 7-oxa-13-prostynoic acid, is recognized at both sites in a concentration-dependent manner. At concentrations of 7-oxa-13-prostynoic acid less than 45 μg/ml, activation is seen, while at higher concentrations, inhibition is seen. Different naturally occurring prostaglandins appear to be site-specific. Prostaglandin E1 gives only activation of the cyclase, while prostaglandin A1 gives only inhibition of the activated cyclase. When saturating concentrations of prostaglandin E1 are used to activate adenylate cyclase, no further activation by 7-oxa-13-prostynoic acid can be elicited, indicating that both molecules activate at the same site. The similarity of inhibition constants for both 7-oxa-13-prostynoic acid and prostaglandin A1 suggests that the mode of binding is the same for both compounds and that they probably inhibit by acting at the same site. The inhibition by 7-oxa-13-prostynoic acid and by prostaglandin A1 overrides enzyme activation produced by either Escherichia coli enterotoxin, prostaglandin E1, or sodium fluoride, suggesting that in intestinal adenylate cyclase this site is the primary regulatory site (i.e., primary allosteric effector site) for enzyme activity. These data suggest that sites exist on adenylate cyclase which would allow prostaglandins to serve as the intracellular messengers by which the cell controls its adenylate-cyclase-mediated response to extracellular stimulation, as with hormones. PMID:4208548

  11. Investigation and characterization of receptors for pituitary adenylate cyclase-activating polypeptide in human brain by radioligand binding and chemical cross-linking

    SciTech Connect

    Suda, K.; Smith, D.M.; Ghatei, M.A.; Murphy, J.K.; Bloom, S.R. )

    1991-05-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel peptide of hypothalamic origin which increases adenylate cyclase activity in rat anterior pituitary cell cultures. The 38-amino acid peptide shows a close sequence homology to vasoactive intestinal peptide (VIP). Binding sites for PACAP in membranes from postmortem human brain tissue were studied using ({sup 125}I)PACAP27 as the radioligand. High specific binding sites (amount of specific binding measured at 0.25 nM ({sup 125}I)PACAP27 in femtomoles per mg protein +/- SEM; n = 4) were present in hypothalamus (344.5 +/- 13.0), brain stem (343.0 +/- 29.3), cerebellum (292.0 +/- 21.1), cortex (259.6 +/- 19.8), and basal ganglia (259.2 +/- 50.3). Specific binding sites in pituitary, although present, were less abundant (35.0 +/- 8.9). Binding of ({sup 125}I)PACAP27 was reversible and time, pH, and temperature dependent. Despite the homology with VIP, VIP was a poor inhibitor of ({sup 125}I)PACAP27 binding (IC50, greater than 1 microM) compared with PACAP27 (IC50, 0.5-1.3 nM) and PACAP38 (IC50, 0.2-1.3 nM). Scatchard plots of ({sup 125}I)PACAP27 binding showed the presence of both high and lower affinity sites. Chemical cross-linking of PACAP-binding sites revealed that ({sup 125}I)PACAP27 was bound to polypeptide chains of 67,000 and 48,000 mol wt. Thus, we have demonstrated the presence of PACAP-specific receptors in human brain which are not VIP receptors. This opens the possibility of PACAP functioning as a novel neurotransmitter/neuromodulator in human brain.

  12. Adrenalectomy mediated alterations in adrenergic activation of adenylate cyclase in rat liver

    SciTech Connect

    El-Refai, M.; Chan, T.

    1986-05-01

    Adrenalectomy caused a large increase in the number of ..beta..-adrenergic binding sites on liver plasma membranes as measured by /sup 125/I-iodocyanopindolol (22 and 102 fmol/mg protein for control and adrenalectomized (ADX) rats). Concomitantly an increase in the number of binding sites for /sup 3/H-yohimbine was also observed (104 and 175 fmol/mg protein for control and adx membranes). Epinephrine-stimulated increase in cyclic AMP accumulation in isolated hepatocytes were greater in cells from ADX rats. This increase in ..beta..-adrenergic mediated action was much less than what may be expected as a result of the increase in the ..beta..-adrenergic binding in ADX membranes. In addition phenoxybenzamine (10 ..mu..M) further augmented this action of epinephrine in both control and ADX cells. To test the hypothesis that the increase in the number of the inhibitory ..cap alpha../sub 2/-adrenergic receptors in adrenalectomy is responsible for the muted ..beta..-adrenergic response, the authors injected rats with pertussis toxin (PT). This treatment may cause the in vivo ribosylation of the inhibitory binding protein (Ni). Adenylate cyclase (AC) activity in liver plasma membranes prepared from treated and untreated animals was measured. In contrast with control rats, treatment of ADX rats with PT resulted in a significant increase in the basal activity of AC (5.5 and 7.7 pmol/mg protein/min for untreated and treated rats respectively). Isoproterenol (10 ..mu..M), caused AC activity to increase to 6.5 and 8.4 pmol/mg protein/min for membranes obtained from ADX untreated and ADX treated rats respectively. The ..cap alpha..-adrenergic antagonists had no significant effect on the ..beta..-adrenergic-mediated activation of AC in liver plasma membranes from PT treated control and ADX rats. The authors conclude that the ..beta..-adrenergic activation of AC is attenuated by Ni protein both directly and as a result of activation of ..cap alpha..-adrenergic receptors.

  13. Involvement of a Membrane-Bound Class III Adenylate Cyclase in Regulation of Anaerobic Respiration in Shewanella oneidensis MR-1

    SciTech Connect

    Charania, M.; Brockman, K.; Zhang, Yang; Banerjee, A.; Pinchuk, Grigoriy; Fredrickson, Jim K.; Beliaev, Alex S.; Saffarini, Daad

    2009-07-01

    Unlike other bacteria that use FNR to regulate anaerobic respiration, S. oneidensis MR-1 uses the cAMP receptor protein, CRP, for this purpose. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an E. coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, DMSO, or Fe(III), whereas the deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III), and to a lesser extent with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and the cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagella biosynthesis, and electron transport, were differentially expressed in the cyaC mutant, but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration, and may contribute to additional signaling pathways independent of CRP.

  14. Involvement of a Membrane-Bound Class III Adenylate Cyclase in Regulation of Anaerobic Respiration in Shewanella oneidensis MR-1

    SciTech Connect

    Charania, M.; Brockman, K. L.; Zhang, Y.; Banerjee, A.; Pinchuk, Grigoriy E.; Fredrickson, Jim K.; Beliaev, Alex S.; Saffarini, Daad

    2009-07-01

    Unlike other bacteria that use FNR to regulate anaerobic respiration, Shewanella oneidensis MR-1 uses the cyclic AMP receptor protein (CRP) for this purpose. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases, respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an Escherichia coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, dimethyl sulfoxide (DMSO), or Fe(III), whereas deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III) and, to a lesser extent, with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways, such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagellum biosynthesis, and electron transport were differentially expressed in the cyaC mutant but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration and may contribute to additional signaling pathways independent of CRP.

  15. Adenylate cyclase 1 promotes strengthening and experience-dependent plasticity of whisker relay synapses in the thalamus

    PubMed Central

    Wang, Hao; Liu, Hong; Storm, Daniel R; Zhang, Zhong-wei

    2011-01-01

    Abstract Synaptic refinement, a process that involves elimination and strengthening of immature synapses, is critical for the development of neural circuits and behaviour. The present study investigates the role of adenylate cyclase 1 (AC1) in developmental refinement of excitatory synapses in the thalamus at the single-cell level. In the mouse, thalamic relay synapses of the lemniscal pathway undergo extensive remodelling during the second week after birth, and AC1 is highly expressed in both pre- and postsynaptic neurons during this period. Synaptic connectivity was analysed by patch-clamp recording in acute slices obtained from mice carrying a targeted null mutation of the adenylate cyclase 1 gene (AC1-KO) and wild-type littermates. We found that deletion of AC1 had no effect on the number of relay inputs received by thalamic neurons during development. In contrast, there was a selective reduction of AMPA-receptor-mediated synaptic responses in mutant thalamic neurons, and the effect increased with age. Furthermore, experience-dependent plasticity was impaired in thalamic neurons of AC1-KO mice. Whisker deprivation during early life altered the number and properties of relay inputs received by thalamic neurons in wild-type mice, but had no effects in AC1-KO mice. Our findings underline a role for AC1 in experience-dependent plasticity of excitatory synapses. PMID:21930601

  16. Vasoactive intestinal polypeptide requires parallel changes in adenylate cyclase and phospholipase C to entrain circadian rhythms to a predictable phase

    PubMed Central

    An, Sungwon; Irwin, Robert P.; Allen, Charles N.; Tsai, Connie

    2011-01-01

    Circadian oscillations in the suprachiasmatic nucleus (SCN) depend on transcriptional repression by Period (PER)1 and PER2 proteins within single cells and on vasoactive intestinal polypeptide (VIP) signaling between cells. Because VIP is released by SCN neurons in a circadian pattern, and, after photic stimulation, it has been suggested to play a role in the synchronization to environmental light cycles. It is not known, however, if or how VIP entrains circadian gene expression or behavior. Here, we tested candidate signaling pathways required for VIP-mediated entrainment of SCN rhythms. We found that single applications of VIP reset PER2 rhythms in a time- and dose-dependent manner that differed from light. Unlike VIP-mediated signaling in other cell types, simultaneous antagonism of adenylate cyclase and phospholipase C activities was required to block the VIP-induced phase shifts of SCN rhythms. Consistent with this, VIP rapidly increased intracellular cAMP in most SCN neurons. Critically, daily VIP treatment entrained PER2 rhythms to a predicted phase angle within several days, depending on the concentration of VIP and the interval between VIP applications. We conclude that VIP entrains circadian timing among SCN neurons through rapid and parallel changes in adenylate cyclase and phospholipase C activities. PMID:21389307

  17. Calpain-Mediated Processing of Adenylate Cyclase Toxin Generates a Cytosolic Soluble Catalytically Active N-Terminal Domain

    PubMed Central

    Ostolaza, Helena

    2013-01-01

    Bordetella pertussis, the whooping cough pathogen, secretes several virulence factors among which adenylate cyclase toxin (ACT) is essential for establishment of the disease in the respiratory tract. ACT weakens host defenses by suppressing important bactericidal activities of the phagocytic cells. Up to now, it was believed that cell intoxication by ACT was a consequence of the accumulation of abnormally high levels of cAMP, generated exclusively beneath the host plasma membrane by the toxin N-terminal catalytic adenylate cyclase (AC) domain, upon its direct translocation across the lipid bilayer. Here we show that host calpain, a calcium-dependent Cys-protease, is activated into the phagocytes by a toxin-triggered calcium rise, resulting in the proteolytic cleavage of the toxin N-terminal domain that releases a catalytically active “soluble AC”. The calpain-mediated ACT processing allows trafficking of the “soluble AC” domain into subcellular organella. At least two strategic advantages arise from this singular toxin cleavage, enhancing the specificity of action, and simultaneously preventing an indiscriminate activation of cAMP effectors throughout the cell. The present study provides novel insights into the toxin mechanism of action, as the calpain-mediated toxin processing would confer ACT the capacity for a space- and time-coordinated production of different cAMP “pools”, which would play different roles in the cell pathophysiology. PMID:23840759

  18. Structure of the RNA 30-Phosphate Cyclase-Adenylate Intermediate Illuminates Nucleotide Specificity and Covalent Nucleotidyl Transfer

    SciTech Connect

    Tanaka, N.; Smith, P; Shuman, S

    2010-01-01

    RNA 3-phosphate cyclase (RtcA) synthesizes RNA 2,3 cyclic phosphate ends via three steps: reaction with ATP to form a covalent RtcA-AMP intermediate; transfer of adenylate to an RNA 3-phosphate to form RNA(3)pp(5)A; and attack of the vicinal O2 on the 3-phosphorus to form a 2,3 cyclic phosphate. Here we report the 1.7 {angstrom} crystal structure of the RtcA-AMP intermediate, which reveals the mechanism of nucleotidyl transfer. Adenylate is linked via a phosphoamide bond to the His309 N{var_epsilon} atom. A network of hydrogen bonds to the ribose O2 and O3 accounts for the stringent ribonucleotide preference. Adenine is sandwiched in a hydrophobic pocket between Tyr284 and Pro131 and the preference for adenine is enforced by Phe135, which packs against the purine C2 edge. Two sulfates bound near the adenylate plausibly mimic the 3-terminal and penultimate phosphates of RNA. The structure illuminates how the four {alpha}2/{beta}4 domains contribute to substrate binding and catalysis.

  19. Regulation of follitropin-sensitive adenylate cyclase by stimulatory and inhibitory forms of the guanine nucleotide regulatory protein in immature rat Sertoli cells

    SciTech Connect

    Johnson, G.P.

    1987-01-01

    Studies have been designed to examine the role of guanine nucleotides in mediating FSH-sensitive adenylate cyclase activity in Sertoli cell plasma membranes. Analysis of ({sup 3}H)GDP binding to plasma membranes suggested a single high affinity site with a K{sub d} = 0.24 uM. Competition studies indicated that GTP{sub {gamma}}S was 7-fold more potent than GDP{sub {beta}}S. Bound GDP could be released by FSH in the presence of GTP{sub {gamma}}S, but not by FSH alone. Adenylate cyclase activity was enhanced 5-fold by FSH in the presence of GTP. Addition of GDP{sub {beta}}S to the activated enzyme (FSH plus GTP) resulted in a time-dependent decay to basal activity within 20 sec. GDP{sub {beta}}S competitively inhibited GTP{sub {gamma}}S-stimulated adenylate cyclase activity with a K{sub i} = 0.18 uM. Adenylate cyclase activity was also demonstrated to be sensitive to the nucleotide bound state. In the presence of FSH, only the GTP{sub {gamma}}S-bound form persisted even if GDP{sub {beta}}S previously occupied all available binding sites. Two membrane proteins, M{sub r} = 43,000 and 48,000, were ADP{centered dot}ribosylated using cholera toxin and labeling was enhanced 2 to 4-fold by GTP{sub {gamma}}S but not by GDP{sub {beta}}S. The M{sub r} = 43,000 and 48,000 proteins represented variant forms of G{sub S}. A single protein of M{sub r} = 40,000 (G{sub i}) was ADP-ribosylated by pertussis toxin in vitro. GTP inhibited forskolin-stimulated adenylate cyclase activity with an IC{sub 50} = 0.1 uM. The adenosine analog, N{sup 6}{centered dot}phenylisopropyl adenosine enhanced GTP inhibition of forskolin-stimulated adenylate cyclase activity by an additional 15%. GTP-dependent inhibition of forskolin-sensitive adenylate cyclase activity was abolished in membranes prepared from Sertoli cells treated in culture with pertussis toxin.

  20. Pituitary adenylate cyclase-activating polypeptide-like compounds could modulate the activity of coelomocytes in the earthworm.

    PubMed

    Somogyi, Ildiko; Boros, Akos; Engelmann, Peter; Varhalmi, Eszter; Nemeth, Jozsef; Lubics, Andrea; Tamas, Andrea; Kiss, Peter; Reglodi, Dora; Pollak, Edit; Molnar, Laszlo

    2009-04-01

    By means of radioimmunoassay, we studied the concentration of pituitary adenylate cyclase-activating polypeptide (PACAP)-like proteins in intact and regenerating earthworms. Transection of animals increased the concentration of PACAP-like compounds in coelomocytes, and a decreasing rostrocaudal gradient was detected in the regenerating animals. Western blot analysis revealed a range of PAC1-receptor proteins with molecular weights from 40 to 80 kDa. Electron microscopic immunocytochemistry showed that PAC1 receptors were located on distinct sets of coelomocytes (mainly on amebocytes and on some granulocytes). Based on our results we hypothesize a link between PACAP and coelomocytes, suggesting that PACAP modulates the function of amebocytes and certain granulocytes that play a role in tissue remodeling of regenerating earthworms. PMID:19456404

  1. Pituitary adenylate cyclase activating polypeptide in stress-related disorders: data convergence from animal and human studies

    PubMed Central

    May, Victor

    2014-01-01

    The maladaptive expression and function of several stress-associated hormones have been implicated in pathological stress- and anxiety-related disorders. Among these, recent evidence has suggested that pituitary adenylate cyclase activating polypeptide (PACAP) has critical roles in central neurocircuits mediating stress-related emotional behaviors. We describe the PACAPergic systems, the data implicating PACAP in stress biology and how altered PACAP expression and signaling may result in psychopathologies. We include our work implicating PACAP signaling within the bed nucleus of the stria terminalis (BNST) in mediating the consequences of stressor exposure and relatedly, describe more recent studies suggesting that PACAP in the central nucleus of the amygdala (CeA) may impact the emotional aspects of chronic pain states. In aggregate, these results are consistent with data suggesting that PACAP dysregulation is associated with post-traumatic stress disorder (PTSD) in humans. PMID:25636177

  2. Insect Stage-Specific Receptor Adenylate Cyclases Are Localized to Distinct Subdomains of the Trypanosoma brucei Flagellar Membrane

    PubMed Central

    Saada, Edwin A.; Kabututu, Z. Pius; Lopez, Miguel; Shimogawa, Michelle M.; Langousis, Gerasimos; Oberholzer, Michael; Riestra, Angelica; Jonsson, Zophonias O.; Wohlschlegel, James A.

    2014-01-01

    Increasing evidence indicates that the Trypanosoma brucei flagellum (synonymous with cilium) plays important roles in host-parasite interactions. Several studies have identified virulence factors and signaling proteins in the flagellar membrane of bloodstream-stage T. brucei, but less is known about flagellar membrane proteins in procyclic, insect-stage parasites. Here we report on the identification of several receptor-type flagellar adenylate cyclases (ACs) that are specifically upregulated in procyclic T. brucei parasites. Identification of insect stage-specific ACs is novel, as previously studied ACs were constitutively expressed or confined to bloodstream-stage parasites. We show that procyclic stage-specific ACs are glycosylated, surface-exposed proteins that dimerize and possess catalytic activity. We used gene-specific tags to examine the distribution of individual AC isoforms. All ACs examined localized to the flagellum. Notably, however, while some ACs were distributed along the length of the flagellum, others specifically localized to the flagellum tip. These are the first transmembrane domain proteins to be localized specifically at the flagellum tip in T. brucei, emphasizing that the flagellum membrane is organized into specific subdomains. Deletion analysis reveals that C-terminal sequences are critical for targeting ACs to the flagellum, and sequence comparisons suggest that differential subflagellar localization might be specified by isoform-specific C termini. Our combined results suggest insect stage-specific roles for a subset of flagellar adenylate cyclases and support a microdomain model for flagellar cyclic AMP (cAMP) signaling in T. brucei. In this model, cAMP production is compartmentalized through differential localization of individual ACs, thereby allowing diverse cellular responses to be controlled by a common signaling molecule. PMID:24879126

  3. Subtyping of Salmonella enterica subspecies I using single nucleotide polymorphisms in adenylate cyclase (cyaA)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methods to rapidly identify serotypes of Salmonella enterica subspecies I are of vital importance for protecting the safety of food. To supplement the serotyping method dkgB-linked intergenic sequence ribotyping (ISR), single nucleotide polymorphisms (SNPs) were characterized within adenylate cyclas...

  4. Involvement of endogenous antioxidant systems in the protective activity of pituitary adenylate cyclase-activating polypeptide against hydrogen peroxide-induced oxidative damages in cultured rat astrocytes.

    PubMed

    Douiri, Salma; Bahdoudi, Seyma; Hamdi, Yosra; Cubì, Roger; Basille, Magali; Fournier, Alain; Vaudry, Hubert; Tonon, Marie-Christine; Amri, Mohamed; Vaudry, David; Masmoudi-Kouki, Olfa

    2016-06-01

    Astroglial cells possess an array of cellular defense mechanisms, including superoxide dismutase (SOD) and catalase antioxidant enzymes, to prevent damages caused by oxidative stress. Nevertheless, astroglial cell viability and functionality can be affected by significant oxidative stress. We have previously shown that pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent glioprotective agent that prevents hydrogen peroxide (H2 O2 )-induced apoptosis in cultured astrocytes. The purpose of this study was to investigate the potential protective effect of PACAP against oxidative-generated alteration of astrocytic antioxidant systems. Incubation of cells with subnanomolar concentrations of PACAP inhibited H2 O2 -evoked reactive oxygen species accumulation, mitochondrial respiratory burst, and caspase-3 mRNA level increase. PACAP also stimulated SOD and catalase activities in a concentration-dependent manner, and counteracted the inhibitory effect of H2 O2 on the activity of these two antioxidant enzymes. The protective action of PACAP against H2 O2 -evoked inhibition of antioxidant systems in astrocytes was protein kinase A, PKC, and MAP-kinase dependent. In the presence of H2 O2 , the SOD blocker NaCN and the catalase inhibitor 3-aminotriazole, both suppressed the protective effects of PACAP on SOD and catalase activities, mitochondrial function, and cell survival. Taken together, these results indicate that the anti-apoptotic effect of PACAP on astroglial cells can account for the activation of endogenous antioxidant enzymes and reduction in respiration rate, thus preserving mitochondrial integrity and preventing caspase-3 expression provoked by oxidative stress. Considering its powerful anti-apoptotic and anti-oxidative properties, the PACAPergic signaling system should thus be considered for the development of new therapeutical approaches to cure various pathologies involving oxidative neurodegeneration. We propose the following cascade for the

  5. Adenylate cyclase toxin is critical for colonization and pertussis toxin is critical for lethal infection by Bordetella pertussis in infant mice.

    PubMed Central

    Goodwin, M S; Weiss, A A

    1990-01-01

    Proliferation of Bordetella pertussis in the lungs of infant mice challenged by the intranasal route was examined. The bacteria rapidly proliferated in the lungs of mice challenged with a sublethal dose of a wild-type strain (BP338) or a filamentous hemagglutinin mutant (BPM409) from 500 at day 0 to 10(7) at day 15. The infection cleared in about 40 days. Pertussis toxin-deficient mutant BP357 gave a similar profile; however, the number of bacteria recovered was slightly reduced, suggesting that pertussis toxin is not essential for bacterial growth in the lungs. In contrast, adenylate cyclase toxin mutant BP348 was rapidly cleared from the lungs, with no viable bacteria remaining 10 days postchallenge, suggesting that the adenylate cyclase toxin is a colonization factor required for the bacteria to initiate infection. PMID:2401570

  6. (/sup 3/H)forskolin- and (/sup 3/H)dihydroalprenolol-binding sites and adenylate cyclase activity in heart of rats fed diets containing different oils

    SciTech Connect

    Alam, S.Q.; Ren, Y.F.; Alam, B.S.

    1988-03-01

    The characteristics of the cardiac adenylate cyclase system were studied in rats fed diets containing fish oil (menhaden oil) and other oils. Adenylate cyclase activity generally was higher in cardiac homogenates and membranes of rats fed diet containing 10% menhaden oil than in the other oils. The increase in enzyme activity, especially in forskolin-stimulated activity, was associated with an increase in the concentration of the (/sup 3/H) forskolin-binding sites in cardiac membranes of rats fed menhaden oil. The beta-adrenergic receptor concentration was not significantly altered although the affinity for (/sup 3/H)dihydroalprenolol-binding was lower in membranes of rats fed menhaden oil than those fed the other oils. omega-3 fatty acids from menhaden oil were incorporated into the cardiac membrane phospholipids. The results suggest that the observed increase in myocardial adenylate cyclase activity of rats fed menhaden oil may be due to an increase in the number of the catalytic subunits of the enzyme or due to a greater availability of the forskolin-binding sites.

  7. Corticotropin-releasing factor binding to peripheral tissue and activation of the adenylate cyclase-adenosine 3',5'-monophosphate system

    SciTech Connect

    Dave, J.R.; Eiden, L.E.; Eskay, R.L.

    1985-06-01

    Specific binding sites for rat corticotropin-releasing factor (rCRF) are present in rat adrenal medulla, ventral prostate, spleen, liver, kidney, and testis and bovine chromaffin cells in culture. Maximal binding of (/sup 125/I)rCRF occurred within 25 min at 4 C and was saturable. Scatchard analysis of rCRF binding to rat adrenal membranes and bovine chromaffin cells revealed the existence of two classes of binding sites. One class had a relatively higher apparent affinity and lower number of binding sites, whereas the other class had a relatively lower affinity and higher number of binding sites. CRF induced a dose-related increase in rat adrenal membrane adenylate cyclase activity and cAMP levels in bovine chromaffin cells. Nanomolar concentrations of rCRF maximally stimulated adenylate cyclase activity in rat adrenal membranes and maximally increased cAMP levels in bovine chromaffin cells to 86% and 130% above control values, respectively. The demonstration of specific CRF-binding sites in a variety of peripheral tissues and the finding that activation of specific CRF-binding sites in adrenal tissue stimulates the adenylate cyclase-cAMP system suggest that CRF may have an important regulatory role in various peripheral tissues.

  8. The turkey erythrocyte beta-adrenergic receptor couples to both adenylate cyclase and phospholipase C via distinct G-protein alpha subunits.

    PubMed Central

    James, S R; Vaziri, C; Walker, T R; Milligan, G; Downes, C P

    1994-01-01

    By contrast with mammalian beta-adrenergic receptors, the avian isoform elicits two distinct effector responses, activation of adenylate cyclase and polyphosphoinositide-specific phospholipase C (PLC) leading to the accumulation of both cyclic adenosine monophosphate (cyclic AMP) and inositol phosphates. We have investigated the mechanisms of beta-adrenergic receptor signalling in turkey erythrocytes. Stimulation of adenylate cyclase by the beta-adrenergic-receptor agonist isoprenaline exhibits a 30-fold lower EC50 than that for PLC activation, which may indicate a marked receptor reserve for the former effector. Similar Ki values were obtained for the inhibition of both responses by four beta-adrenergic antagonists, arguing that a single receptor population is responsible for both effects. Antibodies raised against G-protein peptide sequences were used to show that the identity of the G-protein mediating the PLC response was an avian homologue of G11, the level of expression of which was very similar to that of the stimulatory G-protein of adenylate cyclase, Gs. Thus a single population of beta-adrenergic receptors apparently interacts with distinct G-proteins to activate different effectors. The stoichiometries of the receptor-G-protein-effector interactions are therefore similar for both second-messenger responses and the data are discussed in terms of the different efficacies observed for each response. Images Figure 4 PMID:7998968

  9. Desensitization of adenylate cyclase in a human keratinocyte cell line by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

    SciTech Connect

    Choi, E.J.; Young, M.J.; Toscano, D.L.; Greenlee, W.F.; Toscano, W.A. Jr.

    1987-05-01

    Regulation of adenylate cyclase in human keratinocyte cell line SCC 12 is altered after TCDD exposure. TCDD-treated cells show a 50% decrease in isoproterenol - stimulated adenylate cyclase activity. The reduced responsiveness of these cells to isoproterenol was concentration dependent on TCDD. The inactive TCDD analog, 2,7-dibenzo-p-dioxin did not affect isoproterenol activation. Altered hormone stimulation of adenylate cyclase can result from decreased receptor number or affinity, a defect in coupling of receptors via G/sub s/, or modification of the catalytic subunit. To distinguish between these possibilities, enzyme activity was assayed in the presence of different site-specific activators of this enzyme system. Cells exposed to TCDD for 24 hr showed a reduced response to the GTP analog, Gpp(NH)p. Forskolin stimulation was not affected by TCDD treatment. (/sup 125/I)-iodocyanopindolol (ICP) binding to ..beta..-adrenergic receptors was examined after TCDD treatment. The equilibrium dissociation constant (K/sub d/) for ICP was unaffected by TCDD treatment, whereas, the total number of specific ICP-binding sites was reduced from 1080 in control cells to 780 sites per cell in TCDD (10 nM) exposed cells.

  10. Delta-opioid-receptor-mediated inhibition of adenylate cyclase is transduced specifically by the guanine-nucleotide-binding protein Gi2.

    PubMed Central

    McKenzie, F R; Milligan, G

    1990-01-01

    Mouse neuroblastoma x rat glioma hybrid cells (NG108-15) express an opioid receptor of the delta subclass which both stimulates high-affinity GTPase activity and inhibits adenylate cyclase by interacting with a pertussis-toxin-sensitive guanine-nucleotide-binding protein(s) (G-protein). Four such G-proteins have now been identified without photoreceptor-containing tissues. We have generated anti-peptide antisera against synthetic peptides which correspond to the C-terminal decapeptides of the alpha-subunit of each of these G-proteins and also to the stimulatory G-protein of the adenylate cyclase cascade (Gs). Using these antisera, we demonstrate the expression of three pertussis-toxin-sensitive G-proteins in these cells, which correspond to the products of the Gi2, Gi3 and Go genes, as well as Gs. Gi1, however, is not expressed in detectable amounts. IgG fractions from each of these antisera and from normal rabbit serum were used to attempt to interfere with the interaction of the opioid receptor with the G-protein system by assessing ligand stimulation of high-affinity GTPase activity, inhibition of adenylate cyclase activity and conversion of the receptor to a state which displays reduced affinity for agonists. The IgG fraction from the antiserum (AS7) which specifically identifies Gi2 in these cells attenuated the effects of the opioid receptor. This effect was complete and was not mimicked by any of the other antisera. We conclude that the delta-opioid receptor of these cells interacts directly and specifically with Gi2 to cause inhibition of adenylate cyclase, and that Gi2 represents the true Gi of the adenylate cyclase cascade. The ability to measure alterations in agonist affinity for receptors following the use of specific antisera against a range of G-proteins implies that such techniques should be applicable to investigations of the molecular identity of the G-protein(s) which interacts with any receptor. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID

  11. Beta-adrenergic receptor density and adenylate cyclase activity in lead-exposed rat brain after cessation of lead exposure.

    PubMed

    Chang, Huoy-Rou; Tsao, Der-An; Yu, Hsin-Su; Ho, Chi-Kung

    2005-01-01

    To understanding the reversible or irreversible harm to the beta-adrenergic system in the brain of lead-exposed rats, this study sets up an animal model to estimate the change in the sympathetic nervous system of brain after lead exposure was withdrawn. We address the following topics in this study: (a) the relationship between withdrawal time of lead exposure and brain beta-adrenergic receptor, blood lead level, and brain lead level in lead-exposed rats after lead exposure was stopped; and (b) the relationship between lead level and beta-adrenergic receptor and cyclic AMP (c-AMP) in brain. Wistar rats were chronically fed with 2% lead acetate and water for 2 months. Radioligand binding was assayed by a method that fulfilled strict criteria of beta-adrenergic receptor using the ligand [125I]iodocyanopindolol. The levels of lead were determined by electrothermal atomic absorption spectrometry. The c-AMP level was determined by radioimmunoassay. The results showed a close relationship between decreasing lead levels and increasing numbers of brain beta-adrenergic receptors and brain adenylate cyclase activity after lead exposure was withdrawn. The effect of lead exposure on the beta-adrenergic system of the brain is a partly reversible condition. PMID:15502967

  12. The Pseudomonas aeruginosa Chp Chemosensory System Regulates Intracellular cAMP Levels by Modulating Adenylate Cyclase Activity

    PubMed Central

    Fulcher, Nanette B.; Holliday, Phillip M.; Klem, Erich; Cann, Martin J.; Wolfgang, Matthew C.

    2010-01-01

    Summary Multiple virulence systems in the opportunistic pathogen Pseudomonas aeruginosa are regulated by the second messenger signaling molecule adenosine 3’, 5’-cyclic monophosphate (cAMP). Production of cAMP by the putative adenylate cyclase enzyme CyaB represents a critical control point for virulence gene regulation. To identify regulators of CyaB, we screened a transposon insertion library for mutants with reduced intracellular cAMP. The majority of insertions resulting in reduced cAMP mapped to the Chp gene cluster encoding a putative chemotaxis-like chemosensory system. Further genetic analysis of the Chp system revealed that it has both positive and negative effects on intracellular cAMP and that it regulates cAMP levels by modulating CyaB activity. The Chp system was previously implicated in the production and function of type IV pili (TFP). Given that cAMP and the cAMP-dependent transcriptional regulator Vfr control TFP biogenesis gene expression, we explored the relationship between cAMP, the Chp system and TFP regulation. We discovered that the Chp system controls TFP production through modulation of cAMP while control of TFP-dependent twitching motility is cAMP-independent. Overall, our data define a novel function for a chemotaxis-like system in controlling cAMP production and establish a regulatory link between the Chp system, TFP and other cAMP-dependent virulence systems. PMID:20345659

  13. Regulation by the quorum sensor from Vibrio indicates a receptor function for the membrane anchors of adenylate cyclases

    PubMed Central

    Beltz, Stephanie; Bassler, Jens; Schultz, Joachim E

    2016-01-01

    Adenylate cyclases convert intra- and extracellular stimuli into a second messenger cAMP signal. Many bacterial and most eukaryotic ACs possess membrane anchors with six transmembrane spans. We replaced the anchor of the AC Rv1625c by the quorum-sensing receptor from Vibrio harveyi which has an identical 6TM design and obtained an active, membrane-anchored AC. We show that a canonical class III AC is ligand-regulated in vitro and in vivo. At 10 µM, the cholera-autoinducer CAI-1 stimulates activity 4.8-fold. A sequence based clustering of membrane domains of class III ACs and quorum-sensing receptors established six groups of potential structural and functional similarities. The data support the notion that 6TM AC membrane domains may operate as receptors which directly regulate AC activity as opposed and in addition to the indirect regulation by GPCRs in eukaryotic congeners. This adds a completely novel dimension of potential AC regulation in bacteria and vertebrates. DOI: http://dx.doi.org/10.7554/eLife.13098.001 PMID:26920221

  14. Oxidative Stress Tolerance, Adenylate Cyclase, and Autophagy Are Key Players in the Chronological Life Span of Saccharomyces cerevisiae during Winemaking

    PubMed Central

    Orozco, Helena; Matallana, Emilia

    2012-01-01

    Most grape juice fermentation takes place when yeast cells are in a nondividing state called the stationary phase. Under such circumstances, we aimed to identify the genetic determinants controlling longevity, known as the chronological life span. We identified commercial strains with both short (EC1118) and long (CSM) life spans in laboratory growth medium and compared them under diverse conditions. Strain CSM shows better tolerance to stresses, including oxidative stress, in the stationary phase. This is reflected during winemaking, when this strain has an increased maximum life span. Compared to EC1118, CSM overexpresses a mitochondrial rhodanese gene-like gene, RDL2, whose deletion leads to increased reactive oxygen species production at the end of fermentation and a correlative loss of viability at this point. EC1118 shows faster growth and higher expression of glycolytic genes, and this is related to greater PKA activity due to the upregulation of the adenylate cyclase gene. This phenotype has been linked to the presence of a δ element in its promoter, whose removal increases the life span. Finally, EC1118 exhibits a higher level of protein degradation by autophagy, which might help achieve fast growth at the expense of cellular structures and may be relevant for long-term survival under winemaking conditions. PMID:22327582

  15. Defective dopamine-1 receptor adenylate cyclase coupling in the proximal convoluted tubule from the spontaneously hypertensive rat.

    PubMed Central

    Kinoshita, S; Sidhu, A; Felder, R A

    1989-01-01

    The natriuretic effect of DA-1 agonists is less in the spontaneously hypertensive rat (SHR) than its normotensive control, the Wistar-Kyoto rat (WKY). To determine a mechanism of the decreased effect of DA-1 agonists on sodium transport, DA-1 receptors in renal proximal convoluted tubule (PCT) were studied by radioligand binding and by adenylate cyclase (AC) determinations. Specific binding of 125I-SCH 23982 (defined by 10 microM SCH 23390, a DA-1 antagonist) was concentration dependent, saturable, and stereoselective. The dissociation constant, maximum receptor density, and DA-1 antagonist inhibition constant were similar in SHR and WKY. The apparent molecular weight of the DA-1 receptor determined by the photoaffinity D1 probe 125I-MAB was also similar in WKY and SHR. However, DA-1 agonists competed more effectively for specific 125I-SCH 23982 binding sites in WKY than in SHR. Basal as well as forskolin, parathyroid hormone, GTP and Gpp(NH)p-stimulated-AC activities were similar. In contrast DA-1 agonists (fenoldopam, SKF 38393, SND 911C12) stimulated AC activity to a lesser extent in SHR. GTP and Gpp(NH)p enhanced the ability of DA-1 agonists to stimulate AC activity in WKY but not in SHR. These data suggest a defect in the DA-1 receptor-second messenger coupling mechanism in the PCT of the SHR. Images PMID:2574187

  16. Pharmacological characterization of the dopamine-sensitive adenylate cyclase in cockroach brain: evidence for a distinct dopamine receptor

    SciTech Connect

    Orr, G.L.; Gole, J.W.D.; Notman, H.J.; Downer, R.G.H.

    1987-12-21

    Dopamine increases cyclic AMP production in crude membrane preparations of cockroach brain with plateaus in cyclic AMP production occurring between 1-10 ..mu..M and 10 mM. Maximal production of cyclic AMP is 2.25 fold greater than that of control values. Octopamine also increases cyclic AMP production with a Ka of 1.4 ..mu..M and maximal production 3.5 fold greater than that of control. 5-Hydroxytryptamine does not increase cyclic AMP production. The effects of octopamine and dopamine are fully additive. The vertebrate dopamine agonists ADTN and epinine stimulate the dopamine-sensitive adenylate cyclase (AC) with Ka values of 4.5 and 0.6 ..mu..M respectively and with maximal effectiveness 1.7 fold greater than that of control. The selective D/sub 2/-dopamine agonist LY-171555 stimulates cyclic AMP production to a similar extent with a Ka of 50 ..mu..M. Other dopamine agonists have no stimulatory effects. With the exception of mianserin, /sup 3/H-piflutixol is displaced from brain membranes by dopamine antagonists with an order of potency similar to that observed for the inhibition of dopamine-sensitive AC. The results indicate that the octopamine- and dopamine-sensitive AC in cockroach brain can be distinguished pharmacologically and the dopamine receptors coupled to AC have pharmacological characteristics distinct from vertebrate D/sup 1/- and D/sup 2/-dopamine receptors. 33 references, 3 figures, 2 tables.

  17. The biological role of pituitary adenylate cyclase-activating polypeptide (PACAP) in growth and feeding behavior in juvenile fish.

    PubMed

    Lugo, Juana Maria; Oliva, Aymé; Morales, Antonio; Reyes, Osvaldo; Garay, Hilda Elisa; Herrera, Fidel; Cabrales, Ania; Pérez, Ever; Estrada, Mario Pablo

    2010-11-01

    To date, many technologies have been developed to increase efficiency in aquaculture, but very few successful biotechnology molecules have arrived on the market. In this context, marine biotechnology has an opportunity to develop products to improve the output of fish in aquaculture. Published in vivo studies on the action of the pituitary adenylate cyclase-activating polypeptide (PACAP) in fish are scarce. Recently, our group, for the first time, demonstrated the biological role of this neuropeptide administrated by immersion baths in the growth and development of larval fish. In this work, we have evaluated the effects of recombinant Clarias gariepinus PACAP administration by intraperitoneal injection on growth performance and feeding behavior in juvenile fish. Our results showed the physiological role of this peptide for growth control in fish, including the juvenile stage, and confirm that its biological functions are well conserved in fish, since C. gariepinus PACAP stimulated growth in juvenile tilapia Oreochromis niloticus. In addition, we have observed that the growth-promoting effect of PACAP in juvenile tilapia was correlated with higher GH concentration in serum. With regard to the neuroendocrine regulation of growth control by PACAP, it was demonstrated that PACAP stimulates food intake in juvenile tilapia. In general, PACAP appears to act in the regulation of the growth control in juvenile fish. These findings propose that PACAP is a prominent target with the potential to stimulate fish growth in aquaculture. PMID:20853308

  18. A new small molecule inhibitor of soluble guanylate cyclase

    PubMed Central

    Mota, Filipa; Gane, Paul; Hampden-Smith, Kathryn; Allerston, Charles K.; Garthwaite, John; Selwood, David L.

    2015-01-01

    Soluble guanylate cyclase (sGC) is a haem containing enzyme that regulates cardiovascular homeostasis and multiple mechanisms in the central and peripheral nervous system. Commonly used inhibitors of sGC activity act through oxidation of the haem moiety, however they also bind haemoglobin and this limits their bioavailability for in vivo studies. We have discovered a new class of small molecule inhibitors of sGC and have characterised a compound designated D12 (compound 10) which binds to the catalytic domain of the enzyme with a KD of 11 μM in a SPR assay. PMID:26264842

  19. Region-Specific Disruption of Adenylate Cyclase Type 1 Gene Differentially Affects Somatosensorimotor Behaviors in Mice1,2,3

    PubMed Central

    Arakawa, Hiroyuki; Akkentli, Fatih

    2014-01-01

    Abstract Cover Figure Region-specific adenylyl cyclase 1 (AC1) loss of function differentially affects both patterning and sensorimotor behaviors in mice. AC1 is expressed at all levels of the somatosensory pathway and plays a major role in refinement and patterning of topographic sensory maps. Cortex-specific AC1 loss of function (CxAC1KO mice) does not affect barrel patterning and activation of specific barrels corresponding to stimulated whiskers and does not impair sensorimotor behaviors. While global (AC1KO) and thalamus-specific (ThAC1KO) AC1 loss of function leads to absence of barrel patterns, selective whisker stimulation activates topographically aligned cortical loci. Despite functional topography of the whisker-barrel cortex, sensorimotor and social behaviors are impaired, indicating the importance of patterning of topographical sensory maps in the neocortex. Adenylate cyclase type I (AC1) is primarily, and, abundantly, expressed in the brain. Intracellular calcium/calmodulin increases regulate AC1 in an activity-dependent manner. Upon stimulation, AC1 produces cAMP and it is involved in the patterning and the refinement of neural circuits. In mice, spontaneous mutations or targeted deletion of the Adcy1 gene, which encodes AC1, resulted in neuronal pattern formation defects. Neural modules in the primary somatosensory (SI) cortex, the barrels, which represent the topographic distribution of the whiskers on the snout, failed to form (Welker et al., 1996; Abdel-Majid et al., 1998). Cortex- or thalamus-specific Adcy1 deletions led to different cortical pattern phenotypes, with thalamus-specific disruption phenotype being more severe (Iwasato et al., 2008; Suzuki et al., 2013). Despite the absence of barrels in the “barrelless”/Adcy1 null mice, thalamocortical terminal bouton density and activation of cortical zones following whisker stimulation were roughly topographic (Abdel-Majid et al., 1998; Gheorghita et al., 2006). To what extent does patterning

  20. Region-Specific Disruption of Adenylate Cyclase Type 1 Gene Differentially Affects Somatosensorimotor Behaviors in Mice(1,2,3).

    PubMed

    Arakawa, Hiroyuki; Akkentli, Fatih; Erzurumlu, Reha S

    2014-01-01

    Cover FigureRegion-specific adenylyl cyclase 1 (AC1) loss of function differentially affects both patterning and sensorimotor behaviors in mice. AC1 is expressed at all levels of the somatosensory pathway and plays a major role in refinement and patterning of topographic sensory maps. Cortex-specific AC1 loss of function (CxAC1KO mice) does not affect barrel patterning and activation of specific barrels corresponding to stimulated whiskers and does not impair sensorimotor behaviors. While global (AC1KO) and thalamus-specific (ThAC1KO) AC1 loss of function leads to absence of barrel patterns, selective whisker stimulation activates topographically aligned cortical loci. Despite functional topography of the whisker-barrel cortex, sensorimotor and social behaviors are impaired, indicating the importance of patterning of topographical sensory maps in the neocortex. Adenylate cyclase type I (AC1) is primarily, and, abundantly, expressed in the brain. Intracellular calcium/calmodulin increases regulate AC1 in an activity-dependent manner. Upon stimulation, AC1 produces cAMP and it is involved in the patterning and the refinement of neural circuits. In mice, spontaneous mutations or targeted deletion of the Adcy1 gene, which encodes AC1, resulted in neuronal pattern formation defects. Neural modules in the primary somatosensory (SI) cortex, the barrels, which represent the topographic distribution of the whiskers on the snout, failed to form (Welker et al., 1996; Abdel-Majid et al., 1998). Cortex- or thalamus-specific Adcy1 deletions led to different cortical pattern phenotypes, with thalamus-specific disruption phenotype being more severe (Iwasato et al., 2008; Suzuki et al., 2013). Despite the absence of barrels in the "barrelless"/Adcy1 null mice, thalamocortical terminal bouton density and activation of cortical zones following whisker stimulation were roughly topographic (Abdel-Majid et al., 1998; Gheorghita et al., 2006). To what extent does patterning of the

  1. Characteristics of muscarinic receptors that selectively couple to inhibition of adenylate cyclase or stimulation of phospholipase C on NG108-15 and 1321N1 cells

    SciTech Connect

    Liang, M.

    1988-01-01

    The purpose of this dissertation was to establish whether different muscarinic receptor proteins selectively couple to different second messenger response system. Although both second messenger response systems are fully functional in both cell lines, activation of muscarinic cholinergic receptors only results in inhibition of adenylate cyclase in NG108-15 neuroblastoma {times} glioma cells and stimulation of phosphoinositide hydrolysis in 1321N1 human astrocytoma cells. Muscarinic receptors on both cell types were covalently labeled with ({sup 3}H)Propylbenzilylcholine mustard (({sup 3}H)PBCM) and the mobilities of the ({sup 3}H)PBCM-labelled species of both cells were compared by SDS-PAGE. 1321N1 and NG108-15 cells each primarily expressed a single ({sup 3}H)PBCM-labelled species with an apparent size of approximately 92,000 and 66,000 Da, respectively. ({sup 3}H)PBCM labelling was completely inhibited by 1 {mu}M atropine or by down-regulation of muscarinic receptors by an overnight incubation with carbachol. The apparent size of the ({sup 3}H)PBCM-labelled species of both cell lines was not altered by treatment with a series of protease inhibitors or by treatment with dithiothreitol and iodoacetamide. Another approach for determining differences in the muscarinic receptors of 2 cells lines was to study agonist-induced alteration of muscarinic receptor number. Exposure of both cell types to agonists resulted in rapid loss of muscarinic receptors from cell surface without change of total cellular muscarinic receptors followed by subsequently loss of receptors from cells. Muscarinic receptors on both cell lines were regulated by agonist with similar properties.

  2. Pituitary Adenylate Cyclase-Activating Peptide in the Central Amygdala Causes Anorexia and Body Weight Loss via the Melanocortin and the TrkB Systems.

    PubMed

    Iemolo, Attilio; Ferragud, Antonio; Cottone, Pietro; Sabino, Valentina

    2015-07-01

    Growing evidence suggests that the pituitary adenylate cyclase-activating polypeptide (PACAP)/PAC1 receptor system represents one of the main regulators of the behavioral, endocrine, and autonomic responses to stress. Although induction of anorexia is a well-documented effect of PACAP, the central sites underlying this phenomenon are poorly understood. The present studies addressed this question by examining the neuroanatomical, behavioral, and pharmacological mechanisms mediating the anorexia produced by PACAP in the central nucleus of the amygdala (CeA), a limbic structure implicated in the emotional components of ingestive behavior. Male rats were microinfused with PACAP (0-1 μg per rat) into the CeA and home-cage food intake, body weight change, microstructural analysis of food intake, and locomotor activity were assessed. Intra-CeA (but not intra-basolateral amygdala) PACAP dose-dependently induced anorexia and body weight loss without affecting locomotor activity. PACAP-treated rats ate smaller meals of normal duration, revealing that PACAP slowed feeding within meals by decreasing the regularity and maintenance of feeding from pellet-to-pellet; postprandial satiety was unaffected. Intra-CeA PACAP-induced anorexia was blocked by coinfusion of either the melanocortin receptor 3/4 antagonist SHU 9119 or the tyrosine kinase B (TrKB) inhibitor k-252a, but not the CRF receptor antagonist D-Phe-CRF(12-41). These results indicate that the CeA is one of the brain areas through which the PACAP system promotes anorexia and that PACAP preferentially lessens the maintenance of feeding in rats, effects opposite to those of palatable food. We also demonstrate that PACAP in the CeA exerts its anorectic effects via local melanocortin and the TrKB systems, and independently from CRF. PMID:25649277

  3. Convergent phosphomodulation of the major neuronal dendritic potassium channel Kv4.2 by pituitary adenylate cyclase-activating polypeptide.

    PubMed

    Gupte, Raeesa P; Kadunganattil, Suraj; Shepherd, Andrew J; Merrill, Ronald; Planer, William; Bruchas, Michael R; Strack, Stefan; Mohapatra, Durga P

    2016-02-01

    The endogenous neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is secreted by both neuronal and non-neuronal cells in the brain and spinal cord, in response to pathological conditions such as stroke, seizures, chronic inflammatory and neuropathic pain. PACAP has been shown to exert various neuromodulatory and neuroprotective effects. However, direct influence of PACAP on the function of intrinsically excitable ion channels that are critical to both hyperexcitation as well as cell death, remain largely unexplored. The major dendritic K(+) channel Kv4.2 is a critical regulator of neuronal excitability, back-propagating action potentials in the dendrites, and modulation of synaptic inputs. We identified, cloned and characterized the downstream signaling originating from the activation of three PACAP receptor (PAC1) isoforms that are expressed in rodent hippocampal neurons that also exhibit abundant expression of Kv4.2 protein. Activation of PAC1 by PACAP leads to phosphorylation of Kv4.2 and downregulation of channel currents, which can be attenuated by inhibition of either PKA or ERK1/2 activity. Mechanistically, this dynamic downregulation of Kv4.2 function is a consequence of reduction in the density of surface channels, without any influence on the voltage-dependence of channel activation. Interestingly, PKA-induced effects on Kv4.2 were mediated by ERK1/2 phosphorylation of the channel at two critical residues, but not by direct channel phosphorylation by PKA, suggesting a convergent phosphomodulatory signaling cascade. Altogether, our findings suggest a novel GPCR-channel signaling crosstalk between PACAP/PAC1 and Kv4.2 channel in a manner that could lead to neuronal hyperexcitability. PMID:26456351

  4. Pituitary Adenylate cyclase-activating polypeptide orchestrates neuronal regulation of the astrocytic glutamate-releasing mechanism system xc (.).

    PubMed

    Kong, Linghai; Albano, Rebecca; Madayag, Aric; Raddatz, Nicholas; Mantsch, John R; Choi, SuJean; Lobner, Doug; Baker, David A

    2016-05-01

    Glutamate signaling is achieved by an elaborate network involving neurons and astrocytes. Hence, it is critical to better understand how neurons and astrocytes interact to coordinate the cellular regulation of glutamate signaling. In these studies, we used rat cortical cell cultures to examine whether neurons or releasable neuronal factors were capable of regulating system xc (-) (Sxc), a glutamate-releasing mechanism that is expressed primarily by astrocytes and has been shown to regulate synaptic transmission. We found that astrocytes cultured with neurons or exposed to neuronal-conditioned media displayed significantly higher levels of Sxc activity. Next, we demonstrated that the pituitary adenylate cyclase-activating polypeptide (PACAP) may be a neuronal factor capable of regulating astrocytes. In support, we found that PACAP expression was restricted to neurons, and that PACAP receptors were expressed in astrocytes. Interestingly, blockade of PACAP receptors in cultures comprised of astrocytes and neurons significantly decreased Sxc activity to the level observed in purified astrocytes, whereas application of PACAP to purified astrocytes increased Sxc activity to the level observed in cultures comprised of neurons and astrocytes. Collectively, these data reveal that neurons coordinate the actions of glutamate-related mechanisms expressed by astrocytes, such as Sxc, a process that likely involves PACAP. A critical gap in modeling excitatory signaling is how distinct components of the glutamate system expressed by neurons and astrocytes are coordinated. In these studies, we found that system xc (-) (Sxc), a glutamate release mechanism expressed by astrocytes, is regulated by releasable neuronal factors including PACAP. This represents a novel form of neuron-astrocyte communication, and highlights the possibility that pathological changes involving astrocytic Sxc may stem from altered neuronal activity. PMID:26851652

  5. Hepatic adenylate cyclase 3 is upregulated by Liraglutide and subsequently plays a protective role in insulin resistance and obesity

    PubMed Central

    Liang, Y; Li, Z; Liang, S; Li, Y; Yang, L; Lu, M; Gu, H F; Xia, N

    2016-01-01

    Objective: Recent studies have demonstrated that adenylate cyclase 3 (AC3) has a protective role in obesity. This gene resides at the pathway with glucagon-like peptide (GLP)-1. Liraglutide is a GLP-1 analog and has independent glucose and body weight (BW)-reducing effects. In the present study, we aimed to examine whether hepatic AC3 activity was regulated by Liraglutide and to further understand the effect of AC3 in reduction of BW and insulin resistance. Subjects: The diabesity and obese mice were induced from db/db and C57BL/6 J mice, respectively, by high-fat diet. Liraglutide (0.1 mg kg−1 per 12 h) was given to the mice twice daily for 12 weeks. C57BL/6 J mice fed with chow diet and obese or diabesity mice treated with saline were used as the controls. Hepatic AC3 gene expression at mRNA and protein levels was analyzed with real-time reverse transcription-PCR and western blot. Fasting blood glucose and serum insulin levels were measured and followed insulin resistance index (HOMA-IR) was evaluated according to the homeostasis model assessment. Results: After administration of Liraglutide, BW and HOMA-IR in obese and diabesity mice were decreased, whereas hepatic AC3 mRNA and protein expression levels were upregulated. The AC3 gene expression was negatively correlated with BW, HOMA-IR and the area ratio of hepatic fat deposition in the liver. Conclusions: The present study thus provides the evidence that hepatic AC3 gene expression is upregulated by Liraglutide. The reduction of BW and improvement of insulin resistance with Liraglutide may be partially explained by AC3 activation. PMID:26807509

  6. Discovery of Pituitary Adenylate Cyclase-Activating Polypeptide-Regulated Genes through Microarray Analyses in Cell Culture and In Vivo

    PubMed Central

    Eiden, Lee E.; Samal, Babru; Gerdin, Matthew J.; Mustafa, Tomris; Vaudry, David; Stroth, Nikolas

    2010-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is an evolutionarily well conserved neuropeptide with multiple functions in the nervous, endocrine, and immune systems. PACAP provides neuroprotection from ischemia and toxin exposure, is anti-inflammatory in gastric inflammatory disease and sepsis, controls proliferative signaling pathways involved in neural cell transformation, and modulates glucohomeostasis. PACAP-based, disease-targeted therapeutics might thus be both effective and benign, enhancing homeostatic responses to behavioral, metabolic, oncogenic, and inflammatory stressors. PACAP signal transduction employs synergistic regulation of calcium and cyclic adenosine monophosphate (cAMP), and noncanonical activation of both calcium- and cAMP-dependent processes. Pharmacological activation of PACAP signaling should consequently have highly specific effects even in vivo. Here, a combined cellular biochemical, pharmacologic, transcriptomic, and bioinformatic approach to understanding PACAP signal transduction by identifying PACAP target genes with oligonucleotide- and cDNA-based microarray is described. Calcium- and cAMP-dependent PACAP signaling pathways for regulation of genes encoding proteins required for neuritogenesis, changes in cell morphology, and cell survival have been traced in PC12 cells. Pharmacological experiments have linked gene expression to cell physiological responses in this system, in which gene silencing can also be employed to confirm the functional significance of induction of specific transcripts. Differential transcriptional responses to metabolic, ischemic, and other stressors in wild type compared to PACAP-deficient mice establish in principle which PACAP-responsive transcripts in culture are PACAP-dependent in vivo. Bioinformatic approaches aid in creating a pipeline for identifying neuropeptide-regulated genes, validating their cellular functions, and defining their expression in the context of neuropeptide signaling

  7. Cloning, tissue distribution and effects of fasting on pituitary adenylate cyclase-activating polypeptide in largemouth bass

    NASA Astrophysics Data System (ADS)

    Li, Shengjie; Han, Linqiang; Bai, Junjie; Ma, Dongmei; Quan, Yingchun; Fan, Jiajia; Jiang, Peng; Yu, Lingyun

    2015-03-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) has a wide range of biological functions. We cloned the full-length cDNAs encoding PACAP and PACAP-related peptide (PRP) from the brain of largemouth bass ( Micropterus salmoides) and used real-time quantitative PCR to detect PRP-PACAP mRNA expression. The PRP-PACAP cDNA has two variants expressed via alternative splicing: a long form, which encodes both PRP and PACAP, and a short form, which encodes only PACAP. Sequence analysis results are consistent with a higher conservation of PACAP than PRP peptide sequences. The expression of PACAP-long and PACAP-short transcripts was highest in the forebrain, followed by the medulla, midbrain, pituitary, stomach, cerebellum, intestine, and kidney; however, these transcripts were either absent or were weakly expressed in the muscle, spleen, gill, heart, fatty tissue, and liver. The level of PACAP-short transcript expression was significantly higher than expression of the long transcript in the forebrain, cerebella, pituitary and intestine, but lower than that of the long transcript in the stomach. PACAP-long and PACAP-short transcripts were first detected at the blastula stage of embryogenesis, and the level of expression increased markedly between the muscular contraction stage and 3 d post hatch (dph). The expression of PACAP-long and PACAP-short transcripts decreased significantly in the brain following 4 d fasting compared with the control diet group. The down-regulation effect was enhanced as fasting continued. Conversely, expression levels increased significantly after 3 d of re-feeding. Our results suggest that PRP-PACAP acts as an important factor in appetite regulation in largemouth bass.

  8. Stimulation of the hypothalamic ventromedial nuclei by pituitary adenylate cyclase-activating polypeptide induces hypophagia and thermogenesis

    PubMed Central

    Resch, Jon M.; Boisvert, Joanne P.; Hourigan, Allison E.; Mueller, Christopher R.; Yi, Sun Shin

    2011-01-01

    Numerous studies have demonstrated that the hypothalamic ventromedial nuclei (VMN) regulate energy homeostasis by integrating and utilizing behavioral and metabolic mechanisms. The VMN heavily express pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptors (PAC1R). Despite the receptor distribution, most PACAP experiments investigating affects on feeding have focused on intracerebroventricular administration or global knockout mice. To identify the specific contribution of PACAP signaling in the VMN, we injected PACAP directly into the VMN and measured feeding behavior and indices of energy expenditure. Following an acute injection of PACAP, nocturnal food intake was significantly reduced for 6 h after injections without evidence of malaise. In addition, PACAP-induced suppression of feeding also occurred following an overnight fast and could be blocked by a specific PAC1R antagonist. Metabolically, VMN-specific injections of PACAP significantly increased both core body temperature and spontaneous locomotor activity with a concurrent increase in brown adipose uncoupling protein 1 mRNA expression. To determine which signaling pathways were responsive to PACAP administration into the VMN, we measured mRNA expression of well-characterized hypothalamic neuropeptide regulators of feeding. One hour after PACAP administration, expression of pro-opiomelanocortin mRNA was significantly increased in the arcuate nuclei (ARC), with no changes in neuropeptide Y and agouti-related polypeptide mRNA levels. This suggests that PAC1R expressing VMN neurons projecting to pro-opiomelanocortin neurons contribute to hypophagia by involving melanocortin signaling. While the VMN also abundantly express PACAP protein, the present study demonstrates that PACAP input to the VMN can influence the control of energy homeostasis. PMID:21957159

  9. Centrally acting hypotensive agents with affinity for 5-HT1A binding sites inhibit forskolin-stimulated adenylate cyclase activity in calf hippocampus.

    PubMed Central

    Schoeffter, P.; Hoyer, D.

    1988-01-01

    1. A number of centrally acting hypotensive agents and other ligands with high affinity for 5-hydroxytryptamine1A (5-HT1A) recognition sites have been tested on forskolin-stimulated adenylate cyclase activity in calf hippocampus, a functional model for 5-HT1A-receptors. 2. Concentration-dependent inhibition of forskolin-stimulated adenylate cyclase activity was elicited by the reference 5-HT1-receptor agonists (mean EC50 value, nM): 5-HT (22), 5-carboxamidotryptamine (5-CT, 3.2), 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT, 8.6), N,N-dipropyl-5-carboxamidotryptamine (DP-5-CT, 2.3), 1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)-piperazine (PAPP or LY 165163, 20), 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H indole (RU 24969, 20), buspirone (65) and ipsapirone (56). Emax amounted to 18-20% inhibition for all but the latter two agonists (14%). 3. The following hypotensive agents with high affinity for 5-HT1A sites were potent agonists in this system (mean EC50 value, nM): flesinoxan (24), indorenate (99), erythro-1-(1-[2-(1,4-benzodioxan-2-yl)-2-hydroxyethyl]-4-piperidyl )- 2-benzimidazolinone (R 28935, 2.5), urapidil (390) and 5-methyl-urapidil (3.5). The first two agents were full agonists, whereas the latter three acted as partial agonists with 60-80% efficacy. 4. Metergoline and methysergide behaved as full agonists and cyanopindolol as a partial agonist with low efficacy. Spiroxatrine and 2-(2,6-dimethoxyphenoxyethyl)aminomethyl- 1,4-benzodioxane (WB 4101) which bind to 5-HT1A sites with nanomolar affinity, were agonists and inhibited potently forskolin-stimulated adenylate cyclase in calf hippocampus, showing mean EC50 values of 23 and 15 nM, respectively. Spiroxatrine and WB 4101 yielded 90% and 50% efficacy, respectively. 5. Spiperone and methiothepin (each 1 microM) caused rightward shifts of the concentration-effect curve to 8-OH-DPAT, without loss of the maximal effect, as did the partial agonist cyanopindolol (0.1 microM) and the

  10. Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin

    SciTech Connect

    Murayama, T.; Ui, M.

    1985-06-25

    Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased /sup 45/Ca/sup 2 +/ uptake into the cell monolayer, and (f) increased /sup 86/Rb/sup +/ uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separate effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca/sup 2 +/ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca/sup 2 +/-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca/sup 2 +/ gating.

  11. The Adenylate Cyclase Toxins of Bacillus anthracis and Bordetella pertussis Promote Th2 Cell Development by Shaping T Cell Antigen Receptor Signaling

    PubMed Central

    Rossi Paccani, Silvia; Benagiano, Marisa; Capitani, Nagaja; Zornetta, Irene; Ladant, Daniel; Montecucco, Cesare; D'Elios, Mario M.; Baldari, Cosima T.

    2009-01-01

    The adjuvanticity of bacterial adenylate cyclase toxins has been ascribed to their capacity, largely mediated by cAMP, to modulate APC activation, resulting in the expression of Th2–driving cytokines. On the other hand, cAMP has been demonstrated to induce a Th2 bias when present during T cell priming, suggesting that bacterial cAMP elevating toxins may directly affect the Th1/Th2 balance. Here we have investigated the effects on human CD4+ T cell differentiation of two adenylate cyclase toxins, Bacillus anthracis edema toxin (ET) and Bordetella pertussis CyaA, which differ in structure, mode of cell entry, and subcellular localization. We show that low concentrations of ET and CyaA, but not of their genetically detoxified adenylate cyclase defective counterparts, potently promote Th2 cell differentiation by inducing expression of the master Th2 transcription factors, c-maf and GATA-3. We also present evidence that the Th2–polarizing concentrations of ET and CyaA selectively inhibit TCR–dependent activation of Akt1, which is required for Th1 cell differentiation, while enhancing the activation of two TCR–signaling mediators, Vav1 and p38, implicated in Th2 cell differentiation. This is at variance from the immunosuppressive toxin concentrations, which interfere with the earliest step in TCR signaling, activation of the tyrosine kinase Lck, resulting in impaired CD3ζ phosphorylation and inhibition of TCR coupling to ZAP-70 and Erk activation. These results demonstrate that, notwithstanding their differences in their intracellular localization, which result in focalized cAMP production, both toxins directly affect the Th1/Th2 balance by interfering with the same steps in TCR signaling, and suggest that their adjuvanticity is likely to result from their combined effects on APC and CD4+ T cells. Furthermore, our results strongly support the key role of cAMP in the adjuvanticity of these toxins. PMID:19266022

  12. Photo-dynamics of the lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain

    NASA Astrophysics Data System (ADS)

    Penzkofer, A.; Tanwar, M.; Veetil, S. K.; Kateriya, S.; Stierl, M.; Hegemann, P.

    2013-09-01

    The absorption and emission spectroscopic behavior of lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain consisting of a BLUF domain (BLUF = Blue Light sensor Using Flavin) and a cyclase homology domain was studied in the dark, during blue-light exposure and after blue-light exposure at a temperature of 4 °C. The BLUF domain photo-cycle dynamics observed for snap-frozen NgPAC2 was lost by lyophilization (no signaling state formation with flavin absorption red-shift). Instead, blue-light photo-excitation of lyophilized NgPAC2 caused sterically restricted Tyr-Tyr cross-linking (o,o‧-ditysosine formation) and partial flavin cofactor reduction.

  13. Alpha 2-adrenergic receptor stimulation of phospholipase A2 and of adenylate cyclase in transfected Chinese hamster ovary cells is mediated by different mechanisms

    SciTech Connect

    Jones, S.B.; Halenda, S.P.; Bylund, D.B. )

    1991-02-01

    The effect of alpha 2-adrenergic receptor activation on adenylate cyclase activity in Chinese hamster ovary cells stably transfected with the alpha 2A-adrenergic receptor gene is biphasic. At lower concentrations of epinephrine forskolin-stimulated cyclic AMP production is inhibited, but at higher concentrations the inhibition is reversed. Both of these effects are blocked by the alpha 2 antagonist yohimbine but not by the alpha 1 antagonist prazosin. Pretreatment with pertussis toxin attenuates inhibition at lower concentrations of epinephrine and greatly potentiates forskolin-stimulated cyclic AMP production at higher concentrations of epinephrine. alpha 2-Adrenergic receptor stimulation also causes arachidonic acid mobilization, presumably via phospholipase A2. This effect is blocked by yohimbine, quinacrine, removal of extracellular Ca2+, and pretreatment with pertussis toxin. Quinacrine and removal of extracellular Ca2+, in contrast, have no effect on the enhanced forskolin-stimulated cyclic AMP production. Thus, it appears that the alpha 2-adrenergic receptor in these cells can simultaneously activate distinct signal transduction systems; inhibition of adenylate cyclase and stimulation of phospholipase A2, both via G1, and potentiation of cyclic AMP production by a different (pertussis toxin-insensitive) mechanism.

  14. Negatively charged residues of the segment linking the enzyme and cytolysin moieties restrict the membrane-permeabilizing capacity of adenylate cyclase toxin.

    PubMed

    Masin, Jiri; Osickova, Adriana; Sukova, Anna; Fiser, Radovan; Halada, Petr; Bumba, Ladislav; Linhartova, Irena; Osicka, Radim; Sebo, Peter

    2016-01-01

    The whooping cough agent, Bordetella pertussis, secretes an adenylate cyclase toxin-hemolysin (CyaA) that plays a crucial role in host respiratory tract colonization. CyaA targets CR3-expressing cells and disrupts their bactericidal functions by delivering into their cytosol an adenylate cyclase enzyme that converts intracellular ATP to cAMP. In parallel, the hydrophobic domain of CyaA forms cation-selective pores that permeabilize cell membrane. The invasive AC and pore-forming domains of CyaA are linked by a segment that is unique in the RTX cytolysin family. We used mass spectrometry and circular dichroism to show that the linker segment forms α-helical structures that penetrate into lipid bilayer. Replacement of the positively charged arginine residues, proposed to be involved in target membrane destabilization by the linker segment, reduced the capacity of the toxin to translocate the AC domain across cell membrane. Substitutions of negatively charged residues then revealed that two clusters of negative charges within the linker segment control the size and the propensity of CyaA pore formation, thereby restricting the cell-permeabilizing capacity of CyaA. The 'AC to Hly-linking segment' thus appears to account for the smaller size and modest cell-permeabilizing capacity of CyaA pores, as compared to typical RTX hemolysins. PMID:27581058

  15. Negatively charged residues of the segment linking the enzyme and cytolysin moieties restrict the membrane-permeabilizing capacity of adenylate cyclase toxin

    PubMed Central

    Masin, Jiri; Osickova, Adriana; Sukova, Anna; Fiser, Radovan; Halada, Petr; Bumba, Ladislav; Linhartova, Irena; Osicka, Radim; Sebo, Peter

    2016-01-01

    The whooping cough agent, Bordetella pertussis, secretes an adenylate cyclase toxin-hemolysin (CyaA) that plays a crucial role in host respiratory tract colonization. CyaA targets CR3-expressing cells and disrupts their bactericidal functions by delivering into their cytosol an adenylate cyclase enzyme that converts intracellular ATP to cAMP. In parallel, the hydrophobic domain of CyaA forms cation-selective pores that permeabilize cell membrane. The invasive AC and pore-forming domains of CyaA are linked by a segment that is unique in the RTX cytolysin family. We used mass spectrometry and circular dichroism to show that the linker segment forms α-helical structures that penetrate into lipid bilayer. Replacement of the positively charged arginine residues, proposed to be involved in target membrane destabilization by the linker segment, reduced the capacity of the toxin to translocate the AC domain across cell membrane. Substitutions of negatively charged residues then revealed that two clusters of negative charges within the linker segment control the size and the propensity of CyaA pore formation, thereby restricting the cell-permeabilizing capacity of CyaA. The ‘AC to Hly-linking segment’ thus appears to account for the smaller size and modest cell-permeabilizing capacity of CyaA pores, as compared to typical RTX hemolysins. PMID:27581058

  16. Transmembrane segments of complement receptor 3 do not participate in cytotoxic activities but determine receptor structure required for action of Bordetella adenylate cyclase toxin.

    PubMed

    Wald, Tomas; Osickova, Adriana; Masin, Jiri; Liskova, Petra M; Petry-Podgorska, Inga; Matousek, Tomas; Sebo, Peter; Osicka, Radim

    2016-04-01

    Adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) of the whooping cough agent Bordetella pertussis penetrates phagocytes expressing the integrin complement receptor 3 (CR3, CD11b/CD18, α(M)β(2) or Mac-1). CyaA translocates its adenylate cyclase (AC) enzyme domain into cell cytosol and catalyzes unregulated conversion of ATP to cAMP, thereby subverting cellular signaling. In parallel, CyaA forms small cation-selective membrane pores that permeabilize cells for potassium efflux, contributing to cytotoxicity of CyaA and eventually provoking colloid-osmotic cell lysis. To investigate whether the single-pass α-helical transmembrane segments of CR3 subunits CD11b and CD18 do directly participate in AC domain translocation and/or pore formation by the toxin, we expressed in CHO cells variants of CR3 that contained artificial transmembrane segments, or lacked the transmembrane segment(s) at all. The results demonstrate that the transmembrane segments of CR3 are not directly involved in the cytotoxic activities of CyaA but serve for maintaining CR3 in a conformation that is required for efficient toxin binding and action. PMID:26802078

  17. Thyroid-stimulating immunoglobulins in Hashimoto's thyroiditis measured by radioreceptor assay and adenylate cyclase stimulation and their relationship to HLA-D alleles

    SciTech Connect

    Bliddal, H.; Bech, K.; Feldt-Rasmussen, U.; Thomsen, M.; Ryder, L.P.; Hansen, J.M.; Siersbaek-Nielsen, K.; Friis, T.

    1982-11-01

    The relationship between thyroid-stimulating immunoglobulins, measured by both radioreceptor assay and adenylate cyclase stimulation, and the HLA alleles was studied in 41 patients with Hashimoto's thyroiditis. TSH binding-inhibiting immunoglobulins (TBII) were detected in 9 (22%) patients, and human thyroid adenylate cyclase-stimulating immunoglobulins (HTACS) were found in 21 (51%) patients. Only 2 patients were positive in both assays, and an inverse relationship was observed between TBII and HTACS. In the 21 HTACS-positive patients, HLA-Dw5 was found in 1 subject, compared to 8 of the 20 HTACS-negative patients (P < 0.01), while 4 of the 9 TBII-positive patients had HLA-Dw5 compared to 5 of the 32 TBII-negative subjects (P = 0.09).No significant relations were observed between the presence of HTACS or TBII and HLA-Dw3 or HLA-B8. It is concluded that TBII and HTACS are produced independently in Hashimoto's thyroiditis, and that the production of these autoantibodies seems to be related to the HLA-D region in this disease.

  18. Guanine nucleotide binding regulatory proteins and adenylate cyclase in livers of streptozotocin- and BB/Wor-diabetic rats. Immunodetection of Gs and Gi with antisera prepared against synthetic peptides.

    PubMed Central

    Lynch, C J; Blackmore, P F; Johnson, E H; Wange, R L; Krone, P K; Exton, J H

    1989-01-01

    Adenylate cyclase in liver plasma membranes from streptozotocin-diabetic (STZ) or BB/Wor spontaneously diabetic rats showed increased responsiveness to GTP, glucagon, fluoroaluminate, and cholera toxin. Basal or forskolin-stimulated activity was unchanged in STZ rats, but increased in BB/Wor rats. No change in the alpha-subunit of Gi (alpha i) was observed in STZ or BB/Wor rats using pertussis toxin-stimulated [32P]ADP-ribosylation. Immunodetection using antibodies against the COOH-terminal decapeptides of alpha T and alpha i-3 showed no change in alpha i in STZ rats and a slight decrease in BB/Wor rats. Angiotensin II inhibition of hepatic adenylate cyclase was not altered in either diabetic rat. In both models of diabetes, Gs alpha-subunits were increased as measured by cholera toxin-stimulated [32P]-ADP-ribosylation of 43-47.5-kD peptides, reconstitution with membranes from S49 cyc- cells or immunoreactivity using antibodies against the COOH-terminal decapeptide of alpha s. These data indicate that STZ-diabetes increases hepatic Gs but does not change Gi or adenylate cyclase catalytic activity. In contrast, BB/Wor rats show increased hepatic Gs and adenylate cyclase. These changes could explain the increase in hepatic cAMP and related dysfunctions observed in diabetes. Images PMID:2498395

  19. Characterization of the adenosine receptor in cultured embryonic chick atrial myocytes: Coupling to modulation of contractility and adenylate cyclase activity and identification by direct radioligand binding

    SciTech Connect

    Liang, B.T.

    1989-06-01

    Adenosine receptors in a spontaneously contracting atrial myocyte culture from 14-day chick embryos were characterized by radioligand binding studies and by examining the involvement of G-protein in coupling these receptors to a high-affinity state and to the adenylate cyclase and the myocyte contractility. Binding of the antagonist radioligand (3H)-8-cyclopentyl-1,3-diproylxanthine ((3H)CPX) was rapid, reversible and saturable and was to a homogeneous population of sites with a Kd value of 2.1 +/- 0.2 nM and an apparent maximum binding of 26.2 +/- 3 fmol/mg of protein (n = 10, +/- S.E.). Guanyl-5-yl-(beta, gamma-imido)diphosphate had no effect on either the Kd or the maximum binding and CPX reversed the N6-R-phenyl-2-propyladenosine-induced inhibition of adenylate cyclase activity and contractility, indicating that (3H) CPX is an antagonist radioligand. Competition curves for (3H) CPX binding by a series of reference adenosine agonists were consistent with labeling of an A1 adenosine receptor and were better fit by a two-site model than by a one-site model. ADP-ribosylation of the G-protein by the endogenous NAD+ in the presence of pertussis toxin shifted the competition curves from bi to monophasic with Ki values similar to those of the KL observed in the absence of prior pertussis intoxication. The adenosine agonists were capable of inhibiting both the adenylate cyclase activity and myocyte contractility in either the absence or the presence of isoproterenol. The A1 adenosine receptor-selective antagonist CPX reversed these agonist effects. The order of ability of the reference adenosine receptor agonists in causing these inhibitory effects was similar to the order of potency of the same agonists in inhibiting the specific (3H)CPX binding (N6-R-phenyl-2-propyladenosine greater than N6-S-phenyl-2-propyladenosine or N-ethyladenosine-5'-uronic acid).

  20. Long-term morphine treatment enhances proteasome-dependent degradation of G beta in human neuroblastoma SH-SY5Y cells: correlation with onset of adenylate cyclase sensitization.

    PubMed

    Moulédous, Lionel; Neasta, Jérémie; Uttenweiler-Joseph, Sandrine; Stella, Alexandre; Matondo, Mariette; Corbani, Maïthé; Monsarrat, Bernard; Meunier, Jean-Claude

    2005-08-01

    The initial aim of this study was to identify protein changes associated with long-term morphine treatment in a recombinant human neuroblastoma SH-SY5Y clone (sc2) stably overexpressing the human mu-opioid (MOP) receptor. In MOP receptor-overexpressing sc2 cells, short-term morphine exposure was found to be much more potent and efficacious in inhibiting forskolin-elicited production of cAMP, and long-term morphine exposure was shown to induce a substantially higher degree of opiate dependence, as reflected by adenylate cyclase sensitization, than it did in wild-type neuroblastoma cells. Differential proteomic analysis of detergent-resistant membrane rafts isolated from untreated and chronically morphine-treated sc2 cells revealed long-term morphine exposure to have reliably induced a 30 to 40% decrease in the abundance of five proteins, subsequently identified by mass spectrometry as G protein subunits alphai(2), alphai(3), beta(1), and beta(2), and prohibitin. Quantitative Western blot analyses of whole-cell extracts showed that long-term morphine treatment-induced down-regulation of Gbeta but not of the other proteins is highly correlated (r(2) = 0.96) with sensitization of adenylate cyclase. Down-regulation of Gbeta and adenylate cyclase sensitization elicited by long-term morphine treatment were suppressed in the presence of carbobenzoxy-l-leucyl-l-leucyl-l-norvalinal (MG-115) or lactacystin. Thus, sustained activation of the MOP receptor by morphine in sc2 cells seems to promote proteasomal degradation of Gbeta to sensitize adenylate cyclase. Together, our data suggest that the long-term administration of opiates may elicit dependence by altering the neuronal balance of heterotrimeric G proteins and adenylate cyclases, with the ubiquitin-proteasome pathway playing a pivotal role. PMID:15901846

  1. Pituitary Adenylate Cyclase-activating Polypeptide (PACAP) Targets Down Syndrome Candidate Region 1 (DSCR1/RCAN1) to control Neuronal Differentiation*

    PubMed Central

    Lee, Eun Hye; Kim, Seon Sook; Lee, Seul; Baek, Kwan-Hyuck; Seo, Su Ryeon

    2015-01-01

    Pituitary adenylate cyclase-activating peptide (PACAP) is a neurotrophic peptide involved in a wide range of nervous functions, including development, differentiation, and survival, and various aspects of learning and memory. Here we report that PACAP induces the expression of regulator of calcineurin 1 (RCAN1, also known as DSCR1), which is abnormally expressed in the brains of Down syndrome patients. Increased RCAN1 expression is accompanied by activation of the PKA-cAMP response element-binding protein pathways. EMSA and ChIP analyses demonstrate the presence of a functional cAMP response element in the RCAN1 promoter. Moreover, we show that PACAP-dependent neuronal differentiation is significantly disturbed by improper RCAN1 expression. Our data provide the first evidence of RCAN1, a Down syndrome-related gene, as a novel target for control of the neurotrophic function of PACAP. PMID:26157140

  2. Neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) slows down Alzheimer's disease-like pathology in amyloid precursor protein-transgenic mice

    PubMed Central

    Rat, Dorothea; Schmitt, Ulrich; Tippmann, Frank; Dewachter, Ilse; Theunis, Clara; Wieczerzak, Ewa; Postina, Rolf; van Leuven, Fred; Fahrenholz, Falk; Kojro, Elzbieta

    2011-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) has neuroprotective and neurotrophic properties and is a potent α-secretase activator. As PACAP peptides and their specific receptor PAC1 are localized in central nervous system areas affected by Alzheimer's disease (AD), this study aims to examine the role of the natural peptide PACAP as a valuable approach in AD therapy. We investigated the effect of PACAP in the brain of an AD transgenic mouse model. The long-term intranasal daily PACAP application stimulated the nonamyloidogenic processing of amyloid precursor protein (APP) and increased expression of the brain-derived neurotrophic factor and of the antiapoptotic Bcl-2 protein. In addition, it caused a strong reduction of the amyloid β-peptide (Aβ) transporter receptor for advanced glycation end products (RAGE) mRNA level. PACAP, by activation of the somatostatin-neprilysin cascade, also enhanced expression of the Aβ-degrading enzyme neprilysin in the mouse brain. Furthermore, daily PAC1-receptor activation via PACAP resulted in an increased mRNA level of both the PAC1 receptor and its ligand PACAP. Our behavioral studies showed that long-term PACAP treatment of APP[V717I]-transgenic mice improved cognitive function in animals. Thus, nasal application of PACAP was effective, and our results indicate that PACAP could be of therapeutic value in treating AD.—Rat, D., Schmitt, U., Tippmann, F., Dewachter, I., Theunis, C., Wieczerzak, E, Postina, R., van Leuven, F., Fahrenholz, F., Kojro, E. Neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) slows down Alzheimer's disease-like pathology in amyloid precursor protein-transgenic mice. PMID:21593432

  3. Adenylate cyclase 1 (ADCY1) mutations cause recessive hearing impairment in humans and defects in hair cell function and hearing in zebrafish

    PubMed Central

    Santos-Cortez, Regie Lyn P.; Lee, Kwanghyuk; Giese, Arnaud P.; Ansar, Muhammad; Amin-Ud-Din, Muhammad; Rehn, Kira; Wang, Xin; Aziz, Abdul; Chiu, Ilene; Hussain Ali, Raja; Smith, Joshua D.; Shendure, Jay; Bamshad, Michael; Nickerson, Deborah A.; Ahmed, Zubair M.; Ahmad, Wasim; Riazuddin, Saima; Leal, Suzanne M.

    2014-01-01

    Cyclic AMP (cAMP) production, which is important for mechanotransduction within the inner ear, is catalyzed by adenylate cyclases (AC). However, knowledge of the role of ACs in hearing is limited. Previously, a novel autosomal recessive non-syndromic hearing impairment locus DFNB44 was mapped to chromosome 7p14.1-q11.22 in a consanguineous family from Pakistan. Through whole-exome sequencing of DNA samples from hearing-impaired family members, a nonsense mutation c.3112C>T (p.Arg1038*) within adenylate cyclase 1 (ADCY1) was identified. This stop-gained mutation segregated with hearing impairment within the family and was not identified in ethnically matched controls or within variant databases. This mutation is predicted to cause the loss of 82 amino acids from the carboxyl tail, including highly conserved residues within the catalytic domain, plus a calmodulin-stimulation defect, both of which are expected to decrease enzymatic efficiency. Individuals who are homozygous for this mutation had symmetric, mild-to-moderate mixed hearing impairment. Zebrafish adcy1b morphants had no FM1-43 dye uptake and lacked startle response, indicating hair cell dysfunction and gross hearing impairment. In the mouse, Adcy1 expression was observed throughout inner ear development and maturation. ADCY1 was localized to the cytoplasm of supporting cells and hair cells of the cochlea and vestibule and also to cochlear hair cell nuclei and stereocilia. Ex vivo studies in COS-7 cells suggest that the carboxyl tail of ADCY1 is essential for localization to actin-based microvilli. These results demonstrate that ADCY1 has an evolutionarily conserved role in hearing and that cAMP signaling is important to hair cell function within the inner ear. PMID:24482543

  4. Effect of peptides corresponding to extracellular domains of serotonin 1B/1D receptors and melanocortin 3 and 4 receptors on hormonal regulation of adenylate cyclase in rat brain.

    PubMed

    Shpakova, E A; Derkach, K V; Shpakov, A O

    2014-03-01

    The ligand-recognizing part of G protein-coupled receptors consists of their extracellular loops and N-terminal domain. Identification of these sites is essential for receptor mapping and for the development and testing of new hormone system regulators. The peptides corresponding by their structure to extracellular loop 2 of serotonin 1B/1D receptor (peptide 1), extracellular loop 3 of melanocortin 3 receptor (peptide 2), and N-terminal domain of melanocortin 4 (peptide 3) were synthesized by the solid-phase method. In synaptosomal membranes isolated from rat brain, peptide 1 (10(-5)-10(-4) M) attenuated the effects of 5-nonyloxytryptamine (selective agonist of serotonin 1B/1D receptor) and to a lesser extent serotonin and 5-methoxy-N,N-dimethyltryptamine acting on all the subtypes of serotonin receptor 1. Peptide 2 (10(-5)-10(-4) M) significantly reduced the adenylate cyclase-stimulating effect of γ-melanocyte-stimulating hormone (agonist of melanocortin receptor 3), but had no effect on the adenylate cyclase effect of THIQ (agonist melanocortin receptor 4). Peptide 3 reduced the adenylate cyclase-stimulating effects of THIQ and α-melanocyte-stimulating hormone (non-selective agonist of melanocortin receptors 3 and 4), but did not modulate the effect of γ-melanocyte-stimulating hormone. The effect of peptide 3 was weaker: it was observed at peptide 3 concentration of 10(-4) M. Peptides 1-3 did no change the adenylate cyclase-modulating effects of hormones acting through non-homologous receptors. Thus, the synthesized peptides specifically inhibited the regulatory effects of hormones acting through homologous receptors. This suggests that the corresponding extracellular domains are involved in ligand recognition and binding and determine functional activity of the receptor. PMID:24770752

  5. Structure-function studies of the adenylate cyclase toxin of Bordetella pertussis and the leukotoxin of Pasteurella haemolytica by heterologous C protein activation and construction of hybrid proteins.

    PubMed Central

    Westrop, G; Hormozi, K; da Costa, N; Parton, R; Coote, J

    1997-01-01

    The adenylate cyclase toxin (CyaA) from Bordetella pertussis and the leukotoxin (LktA) from Pasteurella haemolytica are members of the RTX (stands for repeats in toxin) family of cytolytic toxins. They have pore-forming activity and share significant amino acid homology but show marked differences in biological activity. CyaA is an invasive adenylate cyclase and a weak hemolysin which is active on a wide range of mammalian cells. LktA is a cytolytic protein with a high target cell specificity and is able to lyse only leukocytes and platelets from ruminants. Each toxin is synthesized as an inactive protoxin encoded by the A gene, and the product of the accessory C gene is required for posttranslational activation. Heterologous activation of LktA by CyaC did not result in a change in its specificity for nucleated cells, although the toxin showed a greater hemolytic-to-cytotoxic ratio. LktC was unable to activate CyaA. A hybrid toxin (Hyb1), which contained the N-terminal enzymic domain and the pore-forming domain from CyaA (amino acids [aa] 1 to 687), with the remainder of the protein derived from the C-terminal end of LktA (aa 379 to 953), showed no toxic activity. Replacement of part of the LktA C-terminal domain of Hyb1 by the CyaA C-terminal domain (aa 919 to 1706) to create hybrid toxin 2 (Hyb2) partially restored toxic activity. In contrast to CyaA, Hyb2 was activated more efficiently by LktC than by CyaC, showing the importance of the region between aa 379 and 616 of LktA for activation by LktC. LktC-activated Hyb2 was more active against ruminant than murine nucleated cells, whereas CyaC-activated Hyb2 displayed a similar, but lower, activity against both cell types. These data indicate that LktC and the region with which it interacts have an influence on the target cell specificity of the mature toxin. PMID:9006045

  6. Bordetella pertussis Commits Human Dendritic Cells to Promote a Th1/Th17 Response through the Activity of Adenylate Cyclase Toxin and MAPK-Pathways

    PubMed Central

    Palazzo, Raffaella; Nasso, Maria; Cheung, Gordon Yiu Chong; Coote, John Graham; Ausiello, Clara Maria

    2010-01-01

    The complex pathology of B. pertussis infection is due to multiple virulence factors having disparate effects on different cell types. We focused our investigation on the ability of B. pertussis to modulate host immunity, in particular on the role played by adenylate cyclase toxin (CyaA), an important virulence factor of B. pertussis. As a tool, we used human monocyte derived dendritic cells (MDDC), an ex vivo model useful for the evaluation of the regulatory potential of DC on T cell immune responses. The work compared MDDC functions after encounter with wild-type B. pertussis (BpWT) or a mutant lacking CyaA (BpCyaA−), or the BpCyaA− strain supplemented with either the fully functional CyaA or a derivative, CyaA*, lacking adenylate cyclase activity. As a first step, MDDC maturation, cytokine production, and modulation of T helper cell polarization were evaluated. As a second step, engagement of Toll-like receptors (TLR) 2 and TLR4 by B. pertussis and the signaling events connected to this were analyzed. These approaches allowed us to demonstrate that CyaA expressed by B. pertussis strongly interferes with DC functions, by reducing the expression of phenotypic markers and immunomodulatory cytokines, and blocking IL-12p70 production. B. pertussis-treated MDDC promoted a mixed Th1/Th17 polarization, and the activity of CyaA altered the Th1/Th17 balance, enhancing Th17 and limiting Th1 expansion. We also demonstrated that Th1 effectors are induced by B. pertussis-MDDC in the absence of IL-12p70 through an ERK1/2 dependent mechanism, and that p38 MAPK is essential for MDDC-driven Th17 expansion. The data suggest that CyaA mediates an escape strategy for the bacterium, since it reduces Th1 immunity and increases Th17 responses thought to be responsible, when the response is exacerbated, for enhanced lung inflammation and injury. PMID:20090944

  7. Multiple splice variants of the pituitary adenylate cyclase-activating polypeptide type 1 receptor detected by RT-PCR in single rat pituitary cells.

    PubMed

    Bresson-Bépoldin, L; Jacquot, M C; Schlegel, W; Rawlings, S R

    1998-10-01

    Alternative splicing of the rat type 1 pituitary adenylate cyclase-activating polypeptide (PACAP) receptor (PVR1) produces variants that couple either to both adenylyl cyclase (AC) and phospholipase C (PLC) (PVR1 short, PVR1 hop, PVR1 hiphop), or to AC alone (PVR1 hip). We have previously shown that populations of clonal alphaT3-1 gonadotrophs express PVR1 hop and PVR1 short mRNAs, whereas clonal GH4C1 somatotrophs do not. Here we have used the single cell RT-PCR technique to investigate whether normal rat gonadotrophs and somatotrophs express PVR1 mRNA, whether a single cell co-expresses multiple splice variant forms, and whether differential PVR1 mRNA expression correlates with differences in PACAP-stimulated Ca2+ signalling. We found that individual rat gonadotrophs expressed mRNA either for PVR1 hop, for PVR1 short, or co-expressed the two forms. Although we found no differences between the splice variant(s) expressed and the characteristics of PACAP-stimulated Ca2+ responses, the expression of PVR1 mRNA is consistent with the known PACAP stimulation of the PLC system in gonadotrophs. Individual rat somatotrophs also expressed PVR1 hop or PVR1 short (but not PVR1 hip) mRNAs although these forms were never co-expressed. The expression of PVR1 mRNA in somatotrophs can explain in part the activation by PACAP of the AC system in such cells. In conclusion, the single cell RT-PCR technique was used to demonstrate expression of multiple PVR1 splice variants in single identified pituitary cells. These findings open up important questions on the role of alternative splicing in cell biology. PMID:9801454

  8. Study of follitropin receptors in testis using a homologous system. Binding of porcine follitropin to plasma membranes from immature porcine testis and correlation with adenylate cyclase stimulation.

    PubMed

    Maghuin-Rogister, G; Closset, J; Combarnous, Y; Hennen, G; Dechenne, C; Ketelslegers, J M

    1978-05-01

    The properties of follitropin receptors in immature porcine testis were determined using highly purified porcine follitropin. 1. The characteristics of follitropin binding to a subcellular fraction rich in plasma membranes were studied using a 125I-labelled follitropin with high specific activity (75-100 Ci/g) and high binding activity. The binding is dependent on time, temperature and pH. It is specific to follitropin as demonstrated by the very low binding activity of the follitropin alpha and beta subunits and of the other glycoprotein hormones. Scatchard analysis of binding data indicated an equilibrium association constant of 2 x 10(10) M-1 and a concentration of high affinity binding sites of 500 fmol/mg membrane proteins. 2. A sensitive radio-ligand receptor assay was developed. Fifty percent inhibition of binding was obtained with as little as 2 ng of porcine follitropin. Ovine and bovine follitropins and pregnant mare serum gonadotropin gave binding inhibition curves parallel to that given by porcine follitropin. With equine and human follitropin, significantly different slopes were recorded. 3. Kinetics of dissociation of labelled follitropin from its testis receptors showed the presence of at least two compartments with fast and slow dissociation rate constants. The ratio between the sizes of the slow and fast compartments appeared dependent upon preincubation time. 4. A temporal correlation was observed between binding of follitropin to testis receptors and activation of membrane bound adenylate cyclase. PMID:207514

  9. AKAP79, PKC, PKA and PDE4 participate in a Gq-linked muscarinic receptor and adenylate cyclase 2 cAMP signalling complex

    PubMed Central

    Shen, Jia X.; Cooper, Dermot M. F.

    2014-01-01

    AC2 (adenylate cyclase 2) is stimulated by activation of Gq-coupled muscarinic receptors through PKC (protein kinase C) to generate localized cAMP in HEK (human embryonic kidney)-293 cells. In the present study, we utilized a sensitive live-cell imaging technique to unravel the proteins that play essential roles in a Gq-coupled muscarinic receptor-mediated cAMP signalling complex. We reveal that, upon agonist binding to the Gq-coupled muscarinic receptor, AKAP79 (A-kinase-anchoring protein 79) recruits PKC to activate AC2 to produce cAMP. The cAMP formed is degraded by PDE4 (phosphodiesterase 4) activated by an AKAP-anchored PKA (protein kinase A). Calcineurin, a phosphatase bound to AKAP79, is not involved in this regulation. Overall, a transient cAMP increase is generated from AC2 by Gq-coupled muscarinic receptor activation, subject to sophisticated regulation through AKAP79, PKC, PDE4 and PKA, which significantly enhances acetylcholine-mediated signalling. PMID:23889134

  10. Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Pathway Is Induced by Mechanical Load and Reduces the Activity of Hedgehog Signaling in Chondrogenic Micromass Cell Cultures

    PubMed Central

    Juhász, Tamás; Szentléleky, Eszter; Szűcs Somogyi, Csilla; Takács, Roland; Dobrosi, Nóra; Engler, Máté; Tamás, Andrea; Reglődi, Dóra; Zákány, Róza

    2015-01-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurohormone exerting protective function during various stress conditions either in mature or developing tissues. Previously we proved the presence of PACAP signaling elements in chicken limb bud-derived chondrogenic cells in micromass cell cultures. Since no data can be found if PACAP signaling is playing any role during mechanical stress in any tissues, we aimed to investigate its contribution in mechanotransduction during chondrogenesis. Expressions of the mRNAs of PACAP and its major receptor, PAC1 increased, while that of other receptors, VPAC1, VPAC2 decreased upon mechanical stimulus. Mechanical load enhanced the expression of collagen type X, a marker of hypertrophic differentiation of chondrocytes and PACAP addition attenuated this elevation. Moreover, exogenous PACAP also prevented the mechanical load evoked activation of hedgehog signaling: protein levels of Sonic and Indian Hedgehogs and Gli1 transcription factor were lowered while expressions of Gli2 and Gli3 were elevated by PACAP application during mechanical load. Our results suggest that mechanical load activates PACAP signaling and exogenous PACAP acts against the hypertrophy inducing effect of mechanical load. PMID:26230691

  11. Use of a genetically defined double mutant strain of Bordetella bronchiseptica lacking adenylate cyclase and type III secretion as a live vaccine.

    PubMed

    Mann, Paul; Goebel, Elizabeth; Barbarich, James; Pilione, Mylisa; Kennett, Mary; Harvill, Eric

    2007-07-01

    While most vaccines consisting of killed bacteria induce high serum antibody titers, they do not always confer protection as effective as that induced by infection, particularly against mucosal pathogens. Bordetella bronchiseptica is a gram-negative respiratory pathogen that is endemic in many nonhuman mammalian populations and causes substantial disease in a variety of animals. At least 14 different live attenuated vaccines against this pathogen are available for use in a variety of livestock and companion animals. However, there are few published data on the makeup or efficacy of these vaccines. Here we report the use of a genetically engineered double mutant of B. bronchiseptica, which lacks adenylate cyclase and type III secretion, as a vaccine candidate. This strain is safe at high doses, even for highly immunocompromised animals, and induces immune responses that are protective against highly divergent B. bronchiseptica strains, preventing colonization in the lower respiratory tract and decreasing the bacterial burden in the upper respiratory tract. This novel B. bronchiseptica vaccine candidate induces strong local immunity while eliminating damage caused by the two predominant cytotoxic mechanisms. PMID:17452472

  12. A direct pyrophosphatase-coupled assay provides new insights into the activation of the secreted adenylate cyclase from Bordetella pertussis by calmodulin.

    PubMed

    Lawrence, Anthony J; Coote, John G; Kazi, Yasmin F; Lawrence, Paul D; MacDonald-Fyall, Julia; Orr, Barbara M; Parton, Roger; Riehle, Mathis; Sinclair, James; Young, John; Price, Nicholas C

    2002-06-21

    Continuous recording of the activity of recombinant adenylate cyclase (CyaA) of Bordetella pertussis (EC ) by conductimetric determination of enzyme-coupled pyrophosphate cleavage has enabled us to define a number of novel features of the activation of this enzyme by calmodulin and establish conditions under which valid activation data can be obtained. Activation either in the presence or absence of calcium is characterized by a concentration-dependent lag phase. The rate of formation and breakdown of the activated complex can be determined from an analysis of the lag phase kinetics and is in good agreement with thermodynamic data obtained by measuring the dependence of activation on calmodulin concentration, which show that calcium increases k(on) by about 30-fold. The rate of breakdown of the activated complex, formed either in the presence or absence of calcium, has been determined by dilution experiments and has been shown to be independent of the presence of calcium. The coupled assay is established as a rapid, convenient and safe method which should be readily applicable to the continuous assays of most other enzymes that catalyze reactions in which inorganic pyrophosphate is liberated. PMID:11934879

  13. Recombinant novel pituitary adenylate cyclase-activating polypeptide from African catfish (Clarias gariepinus) authenticates its biological function as a growth-promoting factor in low vertebrates.

    PubMed

    Lugo, Juana Maria; Rodriguez, Alina; Helguera, Yusmila; Morales, Reynold; Gonzalez, Osmany; Acosta, Jannel; Besada, Vladimir; Sanchez, Aniel; Estrada, Mario Pablo

    2008-06-01

    Nowadays, the studies of pituitary adenylate cyclase-activating polypeptide (PACAP)-related peptide (PRP) and PACAP in non-mammalian vertebrates, especially in fish, have paid attention mainly to the localization, cloning, and structural evolution of the peptides, but very little is known about its biological functions as growth-promoting factors in low vertebrates. In this work, we have cloned and characterized the PRP/PACAP cDNA from the commercially important North African catfish Clarias gariepinus. The sequence obtained agrees with the higher conservation of PACAP than of PRP peptide sequences. We have reported for the first time the recombinant expression of fish PRP and PACAP in mammalian cells and bacteria and also demonstrated that the growth rate of fish is enhanced by both PRP and PACAP recombinant peptides. The results obtained in vivo in three different fish species, catfish (C. gariepinus), tilapia (Oreochromis niloticus), and carp (Cyprinus carpio) support the finding that PACAP rather than PRP plays a primordial role in growth control in teleost fish. This finding could help to elucidate the neuroendocrine axis proposed to explain the hypothalamic regulation of growth in non-mammalian vertebrates. PMID:18492822

  14. Buprenorphine-elicited alteration of adenylate cyclase activity in human embryonic kidney 293 cells coexpressing κ-, μ-opioid and nociceptin receptors

    PubMed Central

    Wang, Pei-Chen; Ho, Ing-Kang; Lee, Cynthia Wei-Sheng

    2015-01-01

    Buprenorphine, a maintenance drug for heroin addicts, exerts its pharmacological function via κ- (KOP), μ-opioid (MOP) and nociceptin/opioid receptor-like 1 (NOP) receptors. Previously, we investigated its effects in an in vitro model expressing human MOP and NOP receptors individually or simultaneously (MOP, NOP, and MOP+NOP) in human embryonic kidney 293 cells. Here, we expanded this cell model by expressing human KOP, MOP and NOP receptors individually or simultaneously (KOP, KOP+MOP, KOP+NOP and KOP+MOP+NOP). Radioligand binding with tritium-labelled diprenorphine confirmed the expression of KOP receptors. Immunoblotting and immunocytochemistry indicated that the expressed KOP, MOP and NOP receptors are N-linked glycoproteins and colocalized in cytoplasmic compartments. Acute application of the opioid receptor agonists— U-69593, DAMGO and nociceptin— inhibited adenylate cyclase (AC) activity in cells expressing KOP, MOP and NOP receptors respectively. Buprenorphine, when applied acutely, inhibited AC activity to ~90% in cells expressing KOP+MOP+NOP receptors. Chronic exposure to buprenorphine induced concentration-dependent AC superactivation in cells expressing KOP+NOP receptors, and the level of this superactivation was even higher in KOP+MOP+NOP-expressing cells. Our study demonstrated that MOP receptor could enhance AC regulation in the presence of coexpressed KOP and NOP receptors, and NOP receptor is essential for concentration-dependent AC superactivation elicited by chronic buprenorphine exposure. PMID:26153065

  15. Pituitary adenylate cyclase-activating polypeptide 6-38 blocks cocaine- and amphetamine-regulated transcript Peptide-induced hypophagia in rats.

    PubMed

    Burgos, Jonathan R; Iresjö, Britt-Marie; Smedh, Ulrika

    2013-01-01

    Cocaine- and amphetamine-regulated transcript peptides (CARTp) suppress nutritional intake after administration into the fourth intracerebral ventricle. Recent in vitro studies have shown that PACAP 6-38, a pituitary adenylate cyclase-activating polypeptide (PACAP) fragment, could act as a competitive antagonist against CARTp 55-102 on a common CARTp-sensitive receptor structure. Here, we show for the first time in vivo that the reduction in solid food intake induced by exogenous CARTp 55-102 (0.3 nmol: 1.5 µg) administered fourth i.c.v. is blocked by pretreatment with PACAP 6-38 (3 nmol). The PACAP 6-38 fragment had no effect by itself either when given into the fourth ventricle or subcutaneously. Although effective to block the CARTp-effect on feeding and short-term body weight, PACAP 6-38 failed to attenuate CARTp-associated gross motor behavioral changes suggesting at least two CARTp-sensitive receptor subtypes. In conclusion, PACAP 6-38 acts as a functional CARTp antagonist in vivo and blocks its effects on feeding and short term weight gain. PMID:23967296

  16. Effect of the pituitary adenylate cyclase-activating polypeptide on the autophagic activation observed in in vitro and in vivo models of Parkinson's disease.

    PubMed

    Lamine-Ajili, Asma; Fahmy, Ahmed M; Létourneau, Myriam; Chatenet, David; Labonté, Patrick; Vaudry, David; Fournier, Alain

    2016-04-01

    Parkinson's disease (PD) is a neurodegenerative disorder that leads to destruction of the midbrain dopaminergic (DA) neurons. This phenomenon is related to apoptosis and its activation can be blocked by the pituitary adenylate cyclase-activating polypeptide (PACAP). Growing evidence indicates that autophagy, a self-degradation activity that cleans up the cell, is induced during the course of neurodegenerative diseases. However, the role of autophagy in the pathogenesis of neuronal disorders is yet poorly understood and the potential ability of PACAP to modulate the related autophagic activation has never been significantly investigated. Hence, we explored the putative autophagy-modulating properties of PACAP in in vitro and in vivo models of PD, using the neurotoxic agents 1-methyl-4-phenylpyridinium (MPP(+)) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), respectively, to trigger alterations of DA neurons. In both models, following the toxin exposure, PACAP reduced the autophagic activity as evaluated by the production of LC3 II, the modulation of the p62 protein levels, and the formation of autophagic vacuoles. The ability of PACAP to inhibit autophagy was also observed in an in vitro cell assay by the blocking of the p62-sequestration activity produced with the autophagy inducer rapamycin. Thus, the results demonstrated that autophagy is induced in PD experimental models and that PACAP exhibits not only anti-apoptotic but also anti-autophagic properties. PMID:26769362

  17. Buprenorphine-elicited alteration of adenylate cyclase activity in human embryonic kidney 293 cells coexpressing κ-, μ-opioid and nociceptin receptors.

    PubMed

    Wang, Pei-Chen; Ho, Ing-Kang; Lee, Cynthia Wei-Sheng

    2015-11-01

    Buprenorphine, a maintenance drug for heroin addicts, exerts its pharmacological function via κ- (KOP), μ-opioid (MOP) and nociceptin/opioid receptor-like 1 (NOP) receptors. Previously, we investigated its effects in an in vitro model expressing human MOP and NOP receptors individually or simultaneously (MOP, NOP, and MOP+NOP) in human embryonic kidney 293 cells. Here, we expanded this cell model by expressing human KOP, MOP and NOP receptors individually or simultaneously (KOP, KOP+MOP, KOP+NOP and KOP+MOP+NOP). Radioligand binding with tritium-labelled diprenorphine confirmed the expression of KOP receptors. Immunoblotting and immunocytochemistry indicated that the expressed KOP, MOP and NOP receptors are N-linked glycoproteins and colocalized in cytoplasmic compartments. Acute application of the opioid receptor agonists- U-69593, DAMGO and nociceptin- inhibited adenylate cyclase (AC) activity in cells expressing KOP, MOP and NOP receptors respectively. Buprenorphine, when applied acutely, inhibited AC activity to ~90% in cells expressing KOP+MOP+NOP receptors. Chronic exposure to buprenorphine induced concentration-dependent AC superactivation in cells expressing KOP+NOP receptors, and the level of this superactivation was even higher in KOP+MOP+NOP-expressing cells. Our study demonstrated that MOP receptor could enhance AC regulation in the presence of coexpressed KOP and NOP receptors, and NOP receptor is essential for concentration-dependent AC superactivation elicited by chronic buprenorphine exposure. PMID:26153065

  18. Pituitary adenylate cyclase-activating polypeptide (PACAP) contributes to the proliferation of hematopoietic progenitor cells in murine bone marrow via PACAP-specific receptor

    PubMed Central

    Xu, Zhifang; Ohtaki, Hirokazu; Watanabe, Jun; Miyamoto, Kazuyuki; Murai, Norimitsu; Sasaki, Shun; Matsumoto, Minako; Hashimoto, Hitoshi; Hiraizumi, Yutaka; Numazawa, Satoshi; Shioda, Seiji

    2016-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP, encoded by adcyap1) plays an important role in ectodermal development. However, the involvement of PACAP in the development of other germ layers is still unclear. This study assessed the expression of a PACAP-specific receptor (PAC1) gene and protein in mouse bone marrow (BM). Cells strongly expressing PAC1+ were large in size, had oval nuclei, and merged with CD34+ cells, suggesting that the former were hematopoietic progenitor cells (HPCs). Compared with wild-type mice, adcyap1−/− mice exhibited lower multiple potential progenitor cell populations and cell frequency in the S-phase of the cell cycle. Exogenous PACAP38 significantly increased the numbers of colony forming unit-granulocyte/macrophage progenitor cells (CFU-GM) with two peaks in semi-solid culture. PACAP also increased the expression of cyclinD1 and Ki67 mRNAs. These increases were completely and partially inhibited by the PACAP receptor antagonists, PACAP6-38 and VIP6-28, respectively. Little or no adcyap1 was expressed in BM and the number of CFU-GM colonies was similar in adcyap1−/− and wild-type mice. However, PACAP mRNA and protein were expressed in paravertebral sympathetic ganglia, which innervate tibial BM, and in the sympathetic fibers of BM cavity. These results suggested that sympathetic nerve innervation may be responsible for PACAP-regulated hematopoiesis in BM, mainly via PAC1. PMID:26925806

  19. Molecular cloning and mRNA distribution of pituitary adenylate cyclase-activating polypeptide (PACAP)/PACAP-related peptide in the lungfish.

    PubMed

    Lee, L T O; Tam, J K V; Chan, D W; Chow, B K C

    2009-04-01

    In this article, we report the isolation of a full-length cDNA clone encoding pituitary adenylate cyclase-activating polypeptide (PACAP)/PACAP-related peptide (PRP) from lungfish Protopterus dolloi. When comparing the deduced amino acid sequences, the lungfish PACAP was found to be highly conserved with other vertebrates; however, the PRP shares only lower levels of sequence identity with known PRP sequences. Consistently in phylogenetic analysis, the lungfish PRP, similar to sturgeon PRP, fails to cluster with other PRPs. In addition to the full-length clone, another cDNA encoding a short precursor that lacks the first 32 amino acids of the PRP was also isolated. Interestingly, similar isoforms were also identified in several nonmammalian vertebrates, and it was suggested that exon skipping of PRP/PACAP transcripts was a mechanism that regulated the expression ratio of PACAP to PRP in nonmammalian vertebrates. By real-time PCR, both long and short PRP/PACAP transcripts were found almost exclusively in the brain, and the short isoform is the more abundant transcript (3.7 times more), indicating that PACAP is the major product produced in lungfish brain. The expression patterns of lungfish and previously studied frog PRP/PACAP suggest that the PRP/PACAP gene in the tetrapod lineage may first express in the central nervous system; in the process of evolution, the functions of these peptides diversified and were later found in other tissues. PMID:19456341

  20. Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Pathway Is Induced by Mechanical Load and Reduces the Activity of Hedgehog Signaling in Chondrogenic Micromass Cell Cultures.

    PubMed

    Juhász, Tamás; Szentléleky, Eszter; Somogyi, Csilla Szűcs; Takács, Roland; Dobrosi, Nóra; Engler, Máté; Tamás, Andrea; Reglődi, Dóra; Zákány, Róza

    2015-01-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurohormone exerting protective function during various stress conditions either in mature or developing tissues. Previously we proved the presence of PACAP signaling elements in chicken limb bud-derived chondrogenic cells in micromass cell cultures. Since no data can be found if PACAP signaling is playing any role during mechanical stress in any tissues, we aimed to investigate its contribution in mechanotransduction during chondrogenesis. Expressions of the mRNAs of PACAP and its major receptor, PAC1 increased, while that of other receptors, VPAC1, VPAC2 decreased upon mechanical stimulus. Mechanical load enhanced the expression of collagen type X, a marker of hypertrophic differentiation of chondrocytes and PACAP addition attenuated this elevation. Moreover, exogenous PACAP also prevented the mechanical load evoked activation of hedgehog signaling: protein levels of Sonic and Indian Hedgehogs and Gli1 transcription factor were lowered while expressions of Gli2 and Gli3 were elevated by PACAP application during mechanical load. Our results suggest that mechanical load activates PACAP signaling and exogenous PACAP acts against the hypertrophy inducing effect of mechanical load. PMID:26230691

  1. Regulation of the beta-adrenergic receptor-adenylate cyclase complex of 3T3-L1 fibroblasts by sodium butyrate

    SciTech Connect

    Stadel, J.M.; Poksay, K.S.; Nakada, M.T.; Crooke, S.T.

    1986-05-01

    Mouse 3T3-L1 fibroblasts contain beta-adrenergic receptors (BAR), predominantly of the B/sub 1/ subtype. Incubation of these cells with 2-10 mM sodium butyrate (SB) for 24-48 hr results in a switch in the BAR subtype from B/sub 1/ to B/sub 2/ and promotes a 1.5 to 2.5 fold increase in total BAR number. Other short chain acids were not as effective as SB in promoting changes in BAR. BAR were assayed in membranes prepared from the 3T3-L1 cells using the radiolabeled antagonist (/sup 125/I)-cyanopindolol and the B/sub 2/ selective antagonist ICI 118.551. BAR subtype switch was confirmed functionally by measuring cellular cAMP accumulation in response to agonists. The structure and amount of the alpha subunits of the guanine nucleotide regulatory proteins N/sub s/ and N/sub i/ were determined by ADP-ribosylation using /sup 32/P-NAD and either cholera toxin or pertussis toxin for labeling of the respective subunits. Preincubation of cells with 5 mM SB for 48 hr resulted in a 2-3 fold increase in the labeling of the alpha subunits of both N/sub s/ and N/sub i/. A protein of M/sub r/ = 44,000 showed enhanced labeling by cholera toxin following SB treatment of the cells. These data indicate SB concomitantly regulates expression of BAR subtype and components of the adenylate cyclase in 3T3-L1 cells.

  2. Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Impairs the Regulation of Apoptosis in Megakaryocytes by Activating NF-κB: a Proteomic Study*

    PubMed Central

    Di Michele, Michela; Peeters, Karen; Loyen, Serena; Thys, Chantel; Waelkens, Etienne; Overbergh, Lutgart; Hoylaerts, Marc; Van Geet, Christel; Freson, Kathleen

    2012-01-01

    We previously showed that the Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and its receptor VPAC1 are negative regulators of megakaryopoiesis and platelet function, but their downstream signaling pathway that inhibits this process still remained unknown. A combined proteomic, transcriptomic, and bioinformatic approach was here used to elucidate the molecular mechanisms underlying PACAP signaling via VPAC1 in megakaryocytes. Two-dimensional difference gel electrophoresis and tandem MS were applied to detect differentially expressed proteins in megakaryocytic CHRF cells stimulated with PACAP. The majority of the 120 proteins modulated by PACAP belong to the class of “cell cycle and apoptosis” proteins. The up- or down-regulated expression of some proteins was confirmed by immunoblot and immunohistochemical analysis. A meta-analysis of our data and 12 other published studies was performed to evaluate signaling pathways involved in different cellular models of PACAP response. From 2384 differentially expressed genes/proteins, 83 were modulated by PACAP in at least three independent studies and Ingenuity Pathway Analysis further identified apoptosis as the highest scored network with NF-κB as a key-player. PACAP inhibited serum depletion-induced apoptosis of CHRF cells via VPAC1 stimulation. In addition, PACAP switched on NF-κB dependent gene expression since higher nuclear levels of the active NF-κB p50/p65 heterodimer were found in CHRF cells treated with PACAP. Finally, a quantitative real time PCR apoptosis array was used to study RNA from in vitro differentiated megakaryocytes from a PACAP overexpressing patient, leading to the identification of 15 apoptotic genes with a 4-fold change in expression and Ingenuity Pathway Analysis again revealed NF-κB as the central player. In conclusion, our findings suggest that PACAP interferes with the regulation of apoptosis in megakaryocytes, probably via stimulation of the NF-κB pathway. PMID:21972247

  3. Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Regulates the Hypothalamo-Pituitary-Thyroid (HPT) Axis via Type 2 Deiodinase in Male Mice.

    PubMed

    Egri, P; Fekete, C; Dénes, Á; Reglődi, D; Hashimoto, H; Fülöp, B D; Gereben, Balázs

    2016-06-01

    The hypothalamic activation of thyroid hormones by type 2 deiodinase (D2), catalyzing the conversion of thyroxine to T3, is critical for the proper function of the hypothalamo-pituitary-thyroid (HPT) axis. Regulation of D2 expression in tanycytes alters the activity of the HPT axis. However, signals that regulate D2 expression in tanycytes are poorly understood. The pituitary adenylate cyclase-activating polypeptide (PACAP) increases intracellular cAMP level, a second messenger known to stimulate the DIO2 gene; however, its importance in tanycytes is not completely characterized. Therefore, we tested whether this ubiquitously expressed neuropeptide regulates the HPT axis through stimulation of D2 in tanycytes. PACAP increased the activity of human DIO2 promoter in luciferase reporter assay that was abolished by mutation of cAMP-response element. Furthermore, PAC1R receptor immunoreactivity was identified in hypothalamic tanycytes, suggesting that these D2-expressing cells could be regulated by PACAP. Intracerebroventricular PACAP administration resulted in increased D2 activity in the mediobasal hypothalamus, suppressed Trh expression in the hypothalamic paraventricular nucleus, and decreased Tshb expression in the pituitary demonstrating that PACAP affects the D2-mediated control of the HPT axis. To understand the role of endogenous PACAP in the regulation of HPT axis, the effect of decreased PACAP expression was studied in heterozygous Adcyap1 (PACAP) knockout mice. These animals were hypothyroid that may be the consequence of altered hypothalamic T3 degradation during set-point formation of the HPT axis. In conclusion, PACAP is an endogenous regulator of the HPT axis by affecting T3-mediated negative feedback via cAMP-induced D2 expression of tanycytes. PMID:27046436

  4. Structural and functional identification of the pituitary adenylate cyclase-activating polypeptide receptor VPAC2 from the frog Rana tigrina rugulosa.

    PubMed

    Hoo, R L; Alexandre, D; Chan, S M; Anouar, Y; Pang, R T; Vaudry, H; Chow, B K

    2001-10-01

    Recently, a frog pituitary adenylate cyclase-activating polypeptide (PACAP)/vasoactive intestinal peptide (VIP) receptor (fPVR) has been characterized, and interestingly, this receptor exhibits characteristics of both mammalian PACAP type II receptors VPAC(1)R and VPAC(2)R. In order to investigate the receptors responsible for mediating the actions of VIP and PACAP in amphibians, in this report, a frog VPAC(2) receptor (fVPAC(2)R) cDNA was isolated. fVPAC(2)R shares 47.7, 46.9 and 62.5% amino acid sequence identity with fPVR, human VPAC(1)R and human VPAC(2)R respectively. Functionally, fVPAC(2)R, when expressed in CHO cells, was responsive to both frog peptides including VIP, PACAP38 and PACAP27 where the EC(50) values of these peptides in intracellular cAMP production were 0.15, 0.18 and 0.16 microM respectively. The pharmacological profiles of human peptides (VIP, PACAP38 and peptide histidine methionine) to stimulate frog and human VPAC(2)Rs were compared, and it was found that these peptides could only activate the frog receptor at micromolar concentrations. fVPAC(2)R was found to be widely distributed in various peripheral tissues as well as several regions of the brain. The presence of the receptor transcripts suggests the functional roles of the receptor in mediating the actions of PACAP and/or VIP in these tissues. As VIP and particularly PACAP27 are highly conserved peptides in vertebrate evolution, comparative studies of these peptides and their receptors in non-mammalian vertebrates should provide clues to better understand the physiology of these important peptides in human and other vertebrates. PMID:11564605

  5. Pituitary adenylate cyclase-activating polypeptide enhances saliva secretion via direct binding to PACAP receptors of major salivary glands in mice.

    PubMed

    Matoba, Yuko; Nonaka, Naoko; Takagi, Yoshitoki; Imamura, Eisaku; Narukawa, Masayuki; Nakamachi, Tomoya; Shioda, Seiji; Banks, William A; Nakamura, Masanori

    2016-09-01

    Xerostomia, or dry mouth, is a common syndrome that is generally treated with artificial saliva; however, no other effective methods have yet been established. Saliva secretion is mainly under the control of the autonomic nervous system. Pituitary adenylate cyclase-activating polypeptide (PACAP) is recognized as a multifunctional neuropeptide in various organs. In this study, we examined the effect of PACAP on saliva secretion, and detected the distribution of the PACAP type 1 receptor (PAC1R) in major salivary glands, including the parotid, submandibular, and sublingual glands, in 9-week-old male C57BL/6 mice. Intranasal administration of PACAP 38 increased the amount of saliva secreted, which was not inhibited by atropine pretreatment. Immunohistochemical analysis showed that PAC1R was distributed in the three major salivary glands. In the parotid and sublingual glands, PAC1R was detected in striated duct cells, whereas in the submandibular gland, a strong PAC1R immunoreaction was detected in tall columnar epithelial cells in the granular ducts (i.e., pillar cells), as well as in some striated duct cells. PACAP significantly increased the concentration of epidermal growth factor in saliva. These results suggest that PACAP directly regulates saliva secretion by controlling the absorption activity in the ducts, and that pillar cells regulate the function of granular epithelial cells in the granular duct, such as the secretion of growth factors into the saliva. Collectively, these results suggest the possibility of PACAP as a new effective treatment of xerostomia. Anat Rec, 299:1293-1299, 2016. © 2016 Wiley Periodicals, Inc. PMID:27339371

  6. Epidermal growth factor (EGF) withdrawal masks gene expression differences in the study of pituitary adenylate cyclase-activating polypeptide (PACAP) activation of primary neural stem cell proliferation

    PubMed Central

    Sievertzon, Maria; Wirta, Valtteri; Mercer, Alex; Frisén, Jonas; Lundeberg, Joakim

    2005-01-01

    Background The recently discovered adult neural stem cells, which maintain continuous generation of new neuronal and glial cells throughout adulthood, are a promising and expandable source of cells for use in cell replacement therapies within the central nervous system. These cells could either be induced to proliferate and differentiate endogenously, or expanded and differentiated in culture before being transplanted into the damaged site of the brain. In order to achieve these goals effective strategies to isolate, expand and differentiate neural stem cells into the desired specific phenotypes must be developed. However, little is known as yet about the factors and mechanisms influencing these processes. It has recently been reported that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neural stem cell proliferation both in vivo and in vitro. Results We used cDNA microarrays with the aim of analysing the transcriptional changes underlying PACAP induced proliferation of neural stem cells. The primary neural stem/progenitor cells used were neurospheres, generated from the lateral ventricle wall of the adult mouse brain. The results were compared to both differentiation and proliferation controls, which revealed an unexpected and significant differential expression relating to withdrawal of epidermal growth factor (EGF) from the neurosphere growth medium. The effect of EGF removal was so pronounced that it masked the changes in gene expression patterns produced by the addition of PACAP. Conclusion Experimental models aiming at transcriptional analysis of induced proliferation in primary neural stem cells need to take into consideration the significant effect on transcription caused by removal of EGF. Alternatively, EGF-free culture conditions need to be developed. PMID:16124881

  7. [Differentially expressed genes identified in the main olfactory epithelium of mice with deficiency of adenylate cyclase 3 by using suppression subtractive hybridization approach].

    PubMed

    Zhenlong, Cao; Jiangye, Hao; Yanfen, Zhou; Zhe, Zhang; Zhihua, Ni; Yuanxiang, Hu; Weili, Liu; Yongchao, Li; Daniel, R Storm; Runlin, Z Ma; Zhenshan, Wang

    2014-06-01

    Adenylate cyclase 3 (AC3) is one of the major players in the olfactory signaling within the main olfactory epithelium (MOE) of mice. However, we are not ascertained whether deficiency of AC3 will lead to the differential expression of related genes in the MOE. Forward and reverse subtractive libraries were constructed by suppression subtractive hybridization (SSH) approach, with MOEs from AC3(-/-) and AC3(+/+) mice. These two libraries were primarily screened by Dot blot, differential expressed clones were sequenced and analyzed by bioinformatics, and differential expressed genes were verified by qRT-PCR. A total of 386 differentially expressed clones were picked out after Dot blot. The DNA sequences of 80 clones randomly selected were determined, and 62 clones were identified by blasting in GenBank. We found that 24 up-regulated clones were corresponded to genes of kcnk3, mapk7, megf11, and 38 down-regulated clones were corresponded to tmem88b, c-mip, skp1a, mlycd, etc. Their functions were annotated with Gene Ontology (GO) and found to be mainly focused on molecular binding, cell cycle, processes of biology and cells. Five genes (kcnk3, c-mip, mlycd, tmem88b and trappc5) were verified by qRT-PCR with individuals of AC3(+/+) and AC3(-/-) mice. The data indicate that kcnk3 gene is up-regulated significantly, increasing 1.27 folds compared to control mice, whereas c-mip, mlycd, tmem88b and trappc5 are down-regulated significantly, decreasing 20%, 7%, 32% and 29% compared to the AC3(+/+)mice. The functions of these genes are closely related with K(+) channels, cell differentiation, metabolism of fats, membrane transportation, and so on. It is tempting to speculate that these genes might work together with AC3 to orchestrate the olfactory transduction signaling in the MOE. PMID:24929516

  8. Stimulatory effect of pituitary adenylate cyclase-activating polypeptide 6-38, M65 and vasoactive intestinal polypeptide 6-28 on trigeminal sensory neurons.

    PubMed

    Sághy, É; Payrits, M; Helyes, Zs; Reglődi, D; Bánki, E; Tóth, G; Couvineau, A; Szőke, É

    2015-11-12

    Pituitary adenylate cyclase-activating polypeptide (PACAP) acts on G protein-coupled receptors: the specific PAC1 and VPAC1/VPAC2 receptors. PACAP6-38 was described as a potent PAC1/VPAC2 antagonist in several models, but recent studies reported its agonistic behaviors proposing novel receptorial mechanisms. Since PACAP in migraine is an important research tool, we investigated the effect of PACAP and its peptide fragments on trigeminal primary sensory neurons. Effect of the peptides was studied with ratiometric Ca-imaging technique using the fluorescent indicator fura-2 AM on primary cultures of rat and mouse trigeminal ganglia (TRGs) neurons. Specificity testing was performed on PAC1, VPAC1 and VPAC2 receptor-expressing cell lines with both fluorescent and radioactive Ca-uptake methods. Slowly increasing intracellular free calcium concentration [Ca(2+)]i was detected after PACAP1-38, PACAP1-27, vasoactive intestinal polypeptide (VIP) and the selective PAC1 receptor agonist maxadilan administration on TRG neurons, but interestingly, PACAP6-38, VIP6-28 and the PAC1 receptor antagonist M65 also caused similar activation. The VPAC2 receptor agonist BAY 55-9837 induced similar activation, while the VPAC1 receptor agonist Ala(11,22,28)VIP had no significant effect on [Ca(2+)]i. It was proven that the Ca(2+)-influx originated from intracellular stores using radioactive calcium-45 uptake experiment and Ca-free solution. On the specific receptor-expressing cell lines the antagonists inhibited the stimulating actions of the respective agonists, but had no effects by themselves. PACAP6-38, M65 and VIP6-28, which were described as antagonists in numerous studies in several model systems, act as agonists on TRG primary sensory neurons. Currently unknown receptors or splice variants linked to distinct signal transduction pathways might explain these differences. PMID:26321242

  9. Parabrachial nucleus (PBn) pituitary adenylate cyclase activating polypeptide (PACAP) signaling in the amygdala: implication for the sensory and behavioral effects of pain

    PubMed Central

    Missig, Galen A.; Roman, Carolyn W.; Vizzard, Margaret A.; Braas, Karen M.; May, Victor

    2015-01-01

    The intricate relationships that associate pain, stress responses and emotional behavior have been well established. Acute stressful situations can decrease nociceptive sensations and conversely, chronic pain can enhance other pain experiences and heighten the emotional and behavioral consequences of stress. Accordingly, chronic pain is comorbid with a number of behavioral disorders including depression, anxiety abnormalities and associated stress-related disorders including post traumatic stress disorder (PTSD). The central nucleus of the amygdala (CeA) represents a convergence of pathways for pain, stress and emotion, and we have identified pituitary adenylate cyclase activating polypeptide (PACAP) immunoreactivity in fiber elements in the lateral capsular division of the CeA (CeLC). The PACAP staining patterns colocalized in part with those for calcitonin gene related peptide (CGRP); anterograde fiber tracing and excitotoxic lesion studies demonstrated that the CeLC PACAP/CGRP immunoreactivities represented sensory fiber projections from the lateral parabrachial nucleus (LPBn) along the spino-parabrachioamygdaloid tract. The same PBn PACAP/CGRP fiber system also projected to the BNST. As in the BNST, CeA PACAP signaling increased anxiety-like behaviors accompanied by weight loss and decreased feeding. But in addition to heightened anxiety-like responses, CeA PACAP signaling also altered nociception as reflected by decreased latency and threshold responses in thermal and mechanical sensitivity tests, respectively. From PACAP expression in major pain pathways, the current observations are novel and suggest that CeA PACAP nociceptive signaling and resulting neuroplasticity via the spino-parabrachioamygdaloid tract may represent mechanisms that associate chronic pain with sensory hypersensitivity, fear memory consolidation and severe behavioral disorders. PMID:24998751

  10. Spinal astrocytic activation contributes to both induction and maintenance of pituitary adenylate cyclase-activating polypeptide type 1 receptor-induced long-lasting mechanical allodynia in mice

    PubMed Central

    Yokai, Masafumi; Miyata, Atsuro

    2016-01-01

    Background Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors are present in the spinal dorsal horn and dorsal root ganglia, suggesting an important role of PACAP–PACAP receptors signaling system in the modulation of spinal nociceptive transmission. We have previously reported that a single intrathecal injection of PACAP or a PACAP specific (PAC1) receptor selective agonist, maxadilan, in mice induced dose-dependent aversive behaviors, which lasted more than 30 min, and suggested that the maintenance of the nociceptive behaviors was associated with the spinal astrocytic activation. Results We found that a single intrathecal administration of PACAP or maxadilan also produced long-lasting hind paw mechanical allodynia, which persisted at least 84 days without affecting thermal nociceptive threshold. In contrast, intrathecal application of vasoactive intestinal polypeptide did not change mechanical threshold, and substance P, calcitonin gene-related peptide, or N-methyl-D-aspartate induced only transient mechanical allodynia, which disappeared within 21 days. Western blot and immunohistochemical analyses with an astrocytic marker, glial fibrillary acidic protein, revealed that the spinal PAC1 receptor stimulation caused sustained astrocytic activation, which also lasted more than 84 days. Intrathecal co-administration of L-α-aminoadipate, an astroglial toxin, with PACAP or maxadilan almost completely prevented the induction of the mechanical allodynia. Furthermore, intrathecal treatment of L-α-aminoadipate at 84 days after the PAC1 stimulation transiently reversed the mechanical allodynia accompanied by the reduction of glial fibrillary acidic protein expression level. Conclusion Our data suggest that spinal astrocytic activation triggered by the PAC1 receptor stimulation contributes to both induction and maintenance of the long-term mechanical allodynia. PMID:27175011

  11. Role for the beta-adrenoceptor-coupled adenylate cyclase in the ontogenetic subsensitivity to isoproterenol in the embryonic chick ventricle

    SciTech Connect

    Smith, C.J.

    1985-01-01

    Isoproterenol (ISO) increases contractility and cyclic AMP content in ventricles of embryonic and hatched chicks. A transient decrease in beta-agonist sensitivity for both effects is seen in 18 day embryos (10E). Beta-adrenoceptor-coupled adenylate cylase (AC) and receptor binding were characterized in 14,000xg particulates and purified membranes from the ventricles of 10-11E, 17-19E and week-old chicks (5-6H). In crude particulates, the K/sub act/ for ISO (+100 ..mu..M Gpp(NH)p)-stimulated AC is greatest in the 17-19E. Maximal (ISO + Gpp(NH)p)-AC of the 11E is two-fold greater and NaF-AC is 30% greater than those of the 17-19E and 5-6 H. All age groups have comparable catalytic AC. All age groups have comparable K/sub d/'s for /sup 3/H-dihydroalprenolol (5-11 nM), while the 18E has 40% fewer receptors than the 11E and 5-6H. In particulates or membranes, K/sub act/ values for Gpp(NH)p, NaF, MnCl> and forskolin are unchanged with age. In membranes, K/sub act/ values for ISO plus guanine nucleotide (G) and maximal (ISO + G)-AC are similar in all ages. The net effect of ISO ((ISO + G) minus G) is least while that of G (G minus basal) is greatest in the 18E. Whereas /sup 32/P-labeling of a 42 kd protein by cholera toxin is lowest (25% decrease) in particulates of the 18E, labeling of a 39-41 kd doublet by pertussis toxin decreases continuously (by 50%) with age. All age groups have comparable K/sub d/'s (10-13 pM) for (/sup 125/I)-cyanopindolol (CYP). These data indicate that a transient decrease in receptor number and receptor-N/sub s/ (guanine nucleotide-sensitive) coupling in the 18E contribute to the subsensitivity to beta-agonist.

  12. Direct interaction between the catalytic subunit of the calmodulin-sensitive adenylate cyclase from bovine brain with /sup 125/I-labeled wheat germ agglutinin and /sup 125/I-labeled calmodulin

    SciTech Connect

    Minocherhomjee, A.M.; Selfe, S.; Flowers, N.J.; Storm, D.R.

    1987-07-14

    A calmodulin-sensitive adenylate cyclase has been purified to apparent homogeneity from bovine cerebral cortex using calmodulin-Sepharose followed by forskolin-Sepharose and wheat germ agglutinin-Sepharose. The final product appeared as one major polypeptide of approximately 135,000 daltons on sodium dodecyl sulfate-polyacrylamide gels. This polypeptide was a major component of the protein purified through calmodulin-Sepharose. The catalytic subunit was stimulated 3-4-fold by calmodulin (CaM) with a turnover number greater than 1000 min/sup -1/ and was directly inhibited by adenosine. The catalytic subunit of the enzyme interacted directly with /sup 125/I-CaM on a sodium dodecyl sulfate-polyacrylamide gel overlay system, and this interaction was Ca/sup 2 +/ concentration dependent. In addition, the catalytic subunit was shown to directly bind /sup 125/I-labeled wheat germ agglutinin using a sodium dodecyl sulfate-polyacrylamide gel overlay technique, and N-acetylglucosamine inhibited binding of the lectin to the catalytic subunit. Calmodulin did not inhibit binding of wheat germ agglutinin to the catalytic subunit, and the binding of calmodulin was unaffected by wheat germ agglutinin. These data illustrate that the catalytic subunit of the calmodulin-sensitive adenylate cyclase is a glycoprotein which interacts directly with calmodulin and that adenosine can inhibit the enzyme without intervening receptors or G coupling proteins. It is concluded that the catalytic subunit of adenylate cyclase is a transmembrane protein with a domain accessible from the outer surface of the cell.

  13. Pituitary adenylate cyclase-activating polypeptide (PACAP) is an islet substance serving as an intra-islet amplifier of glucose-induced insulin secretion in rats.

    PubMed Central

    Yada, T; Sakurada, M; Ishihara, H; Nakata, M; Shioda, S; Yaekura, K; Hamakawa, N; Yanagida, K; Kikuchi, M; Oka, Y

    1997-01-01

    1. We examined whether pituitary adenylate cyclase-activating polypeptide with 38 or 27 residues (PACAP-38 or PACAP-27) serves as an intra-islet regulator of glucose-induced insulin secretion in rats. PACAP antiserum specific for PACAP-38 and PACAP-27 was used to neutralize the effect of endogenous PACAP in islets. PACAP release from islets was bioassayed using the response of cytosolic Ca2+ concentration ([Ca2+]i) in single beta-cells, monitored by dual-wavelength fura-2 microfluorometry. Expression of PACAP mRNA was studied by reverse transcription-polymerase chain reaction (RT-PCR), while expression of PACAP was studied by metabolic labelling and immunoblotting. Localization of PACAP receptors was studied immunohistochemically. 2. High glucose-stimulated insulin release from isolated islets was attenuated by PACAP antiserum but not by non-immune sera. 3. The islet incubation medium with high glucose (Med) possessed a capacity, which was neutralized by PACAP antiserum, to increase [Ca2+]i in beta-cells. PACAP antiserum also neutralized the [Ca2+]i-increasing action of synthetic PACAP-38 and PACAP-27, but not that of vasoactive intestinal polypeptide (VIP) and glucagon. 4. Both Med and synthetic PACAP increased [Ca2+]i in beta-cells only in the presence of stimulatory, but not basal, glucose concentrations. In contrast, ATP, a substance that is known to be released from beta-cells, increased [Ca2+]i in beta-cells at both and stimulatory glucose concentrations. 5. Expression of PACAP mRNA and biosynthesis of PACAP-38 were detected in islets and a beta-cell line, MIN6. 6. Immunoreactivity for PACAP-selective type-I receptor was observed in islets. 7. [Ca2+]i measurements combined with immunocytochemistry with insulin antiserum revealed a substantial population of glucose-unresponsive beta-cells, many of which were recruited by PACAP-38 into [Ca2+]i responses. 8. These results indicate that PACAP-38 is a novel islet substance that is synthesized and released by islet

  14. Distribution, characterization, and growth hormone-releasing activity of pituitary adenylate cyclase-activating polypeptide in the European eel, Anguilla anguilla.

    PubMed

    Montero, M; Yon, L; Rousseau, K; Arimura, A; Fournier, A; Dufour, S; Vaudry, H

    1998-10-01

    The complementary DNA encoding pituitary adenylate cyclase-activating polypeptide (PACAP) has been cloned from two species of teleost fishes, the Sockeye salmon and the Thai catfish, and the amino acid sequence of PACAP has been determined in another teleost, the stargazer. However, to date, the detailed distribution of PACAP immunoreactivity has never been investigated in the fish brain. In the present study, we have determined the localization of PACAP-immunoreactive neurons in the central nervous system of a primitive teleost fish, the European eel Anguilla anguilla, using an antiserum raised against PACAP27. PACAP-positive perikarya were exclusively observed in the diencephalon, i.e. in the preoptic nucleus of the hypothalamus and in the dorsal and ventral nuclei of the thalamus. PACAP-immunoreactive fibers were detected in various areas of the brain, notably in the ventral telencephalon, the diencephalon, the mesencephalon, the cerebellar valvula, and the medulla oblongata. In addition, a dense accumulation of PACAP-containing nerve terminals was found in the pars distalis of the pituitary. The PACAP-like immunoreactivity contained in the eel brain was characterized by HPLC analysis combined with RIA quantification. The major form of PACAP-immunoreactive material coeluted with mammalian PACAP38. Molecular cloning of the PACAP precursor has previously shown that in fish, PACAP and GH-releasing hormone (GHRH) originate from the same precursor. We have thus investigated the effects of PACAP and GHRH on GH secretion from eel pituitary cells in primary culture. Dose-response experiments revealed that PACAP27 and PACAP38 possessed the same efficacy, but PACAP38 was 12 times more potent than PACAP27 in stimulating GH release (ED50 = 4.3 x 10(-10) and 3.5 x 10(-9) M, respectively). In contrast, GHRH, even at a high concentration (10(-6) M), had no effect on GH release. Taken together, these data indicate that in the eel, PACAP may play a significant role in the

  15. Vasoactive intestinal polypeptide- and pituitary adenylate cyclase activating polypeptide-mediated control of catecholamine release from chromaffin tissue in the rainbow trout, Oncorhynchus mykiss.

    PubMed

    Montpetit, C J; Perry, S F

    2000-09-01

    The aim of the present investigation was to assess the relative contributions of cholinergic (acetylcholine) and non-cholinergic vasoactive intestinal polypeptide (VIP), and pituitary adenylate cyclase activating polypeptide (PACAP) neurotransmitters in the neuronal control of catecholamine secretion from the chromaffin tissue lining the posterior cardinal vein of the rainbow trout (Oncorhynchus mykiss). Using an in situ saline-perfused posterior cardinal vein preparation, it was demonstrated that exogenous administration of chicken VIP or human PACAP-27 caused a dose-dependent increase in adrenaline secretion; noradrenaline secretion was unaffected. Analysis of dose-response curves indicated that VIP and PACAP stimulated the secretion of adrenaline with a similar degree of potency (ED(50) for VIP=1.90x10(-11) mol/kg; ED(50) for PACAP=1.03x10(-11) mol/kg). The VIP/PACAP-elicited secretion was diminished in the presence of the VIP receptor antagonist, VIP 6-28, but was unaffected by the PACAP receptor antagonist, PACAP 6-27, or the cholinergic antagonists, hexamethonium and atropine. Thus, this is the first study to demonstrate a direct stimulatory role for VIP or PACAP in catecholamine secretion from piscine chromaffin cells. The relative contribution of cholinergic and non-cholinergic neurotransmitters in the neuronal control of catecholamine secretion from the chromaffin tissue was evaluated using an in situ nerve-stimulating technique previously validated by us in the rainbow trout. This was accomplished by comparing catecholamine secretion in the presence or absence of cholinergic and the VIP and PACAP receptor antagonists during different levels of electrical stimulation. The results demonstrated that cholinergic stimulation predominated during high frequency of electrical stimulation (20 Hz) while the non-cholinergic component prevailed at low frequency (1 Hz). Overall, the results of the present investigation demonstrate that VIP and/or PACAP may directly

  16. Pituitary adenylate cyclase-activating polypeptide (PACAP) inhibits the slow afterhyperpolarizing current sIAHP in CA1 pyramidal neurons by activating multiple signaling pathways

    PubMed Central

    Taylor, Ruth DT; Madsen, Marita Grønning; Krause, Michael; Sampedro-Castañeda, Marisol; Stocker, Martin; Pedarzani, Paola

    2014-01-01

    The slow afterhyperpolarizing current (sIAHP) is a calcium-dependent potassium current that underlies the late phase of spike frequency adaptation in hippocampal and neocortical neurons. sIAHP is a well-known target of modulation by several neurotransmitters acting via the cyclic AMP (cAMP) and protein kinase A (PKA)-dependent pathway. The neuropeptide pituitary adenylate cyclase activating peptide (PACAP) and its receptors are present in the hippocampal formation. In this study we have investigated the effect of PACAP on the sIAHP and the signal transduction pathway used to modulate intrinsic excitability of hippocampal pyramidal neurons. We show that PACAP inhibits the sIAHP, resulting in a decrease of spike frequency adaptation, in rat CA1 pyramidal cells. The suppression of sIAHP by PACAP is mediated by PAC1 and VPAC1 receptors. Inhibition of PKA reduced the effect of PACAP on sIAHP, suggesting that PACAP exerts part of its inhibitory effect on sIAHP by increasing cAMP and activating PKA. The suppression of sIAHP by PACAP was also strongly hindered by the inhibition of p38 MAP kinase (p38 MAPK). Concomitant inhibition of PKA and p38 MAPK indicates that these two kinases act in a sequential manner in the same pathway leading to the suppression of sIAHP. Conversely, protein kinase C is not part of the signal transduction pathway used by PACAP to inhibit sIAHP in CA1 neurons. Our results show that PACAP enhances the excitability of CA1 pyramidal neurons by inhibiting the sIAHP through the activation of multiple signaling pathways, most prominently cAMP/PKA and p38 MAPK. Our findings disclose a novel modulatory action of p38 MAPK on intrinsic excitability and the sIAHP, underscoring the role of this current as a neuromodulatory hub regulated by multiple protein kinases in cortical neurons. © 2013 The Authors. Hippocampus Published by Wiley Periodicals, Inc. PMID:23996525

  17. Saturated high-fat diet-induced obesity increases adenylate cyclase of myocardial β-adrenergic system and does not compromise cardiac function.

    PubMed

    Vileigas, Danielle F; de Deus, Adriana F; da Silva, Danielle C T; de Tomasi, Loreta C; de Campos, Dijon H S; Adorni, Caroline S; de Oliveira, Scarlet M; Sant'Ana, Paula G; Okoshi, Katashi; Padovani, Carlos R; Cicogna, Antonio C

    2016-09-01

    Obesity is a worldwide pandemic associated with high incidence of cardiovascular disease. The mechanisms by which the obesity leads cardiac dysfunction are not fully elucidated and few studies have evaluated the relationship between obesity and proteins involved in myocardial β-adrenergic (βA) system. The purpose of this study was to evaluate the cardiac function and βA pathway components in myocardium of obese rats. Male Wistar rats were distributed into two groups: control (n = 17; standard diet) and obese (n = 17; saturated high-fat diet) fed for 33 weeks. Nutritional profile and comorbidities were assessed. Cardiac structure and function was evaluated by macroscopic postmortem, echocardiographic and isolated papillary muscle analyzes. Myocardial protein expression of β1- and β2-adrenergic receptors, Gαs protein, adenylate cyclase (AC) and protein kinase A (PKA) was performed by Western blot. Cardiac cyclic adenosine monophosphate (cAMP) levels and PKA activity were assessed by ELISA Obese rats showed increased adiposity index (P < 0.001) and several comorbidities as hypertension, glucose intolerance, insulin resistance, and dyslipidemia compared with control rats. Echocardiographic assessment revealed increased left atrium diameter (C: 4.98 ± 0.38 vs. Ob: 5.47 ± 0.53, P = 0.024) and posterior wall shortening velocity (C: 37.1 ± 3.6 vs. Ob: 41.8 ± 3.8, P = 0.007) in obese group. Papillary muscle evaluation indicated that baseline data and myocardial responsiveness to isoproterenol stimulation were similar between the groups. Protein expression of myocardial AC was higher in obese group than in the control (C: 1.00 ± 0.21 vs. Ob: 1.25 ± 0.10, P = 0.025), whereas the other components were unchanged. These results suggest that saturated high-fat diet-induced obesity was not effective in triggering cardiac dysfunction and impair the beta-adrenergic signaling. PMID:27582064

  18. A new recombinant pituitary adenylate cyclase-activating peptide-derived peptide efficiently promotes glucose uptake and glucose-dependent insulin secretion.

    PubMed

    Ma, Yi; Luo, Tianjie; Xu, Wenna; Ye, Zulu; Hong, An

    2012-11-01

    The recombinant peptide, DBAYL, a promising therapeutic peptide for type 2 diabetes, is a new, potent, and highly selective agonist for VPAC2 generated through site-directed mutagenesis based on sequence alignments of pituitary adenylate cyclase-activating peptide (PACAP), vasoactive intestinal peptide (VIP), and related analogs. The recombinant DBAYL was used to evaluate its effect and mechanism in blood glucose metabolism and utilization. As much as 28.9 mg recombinant DBAYL peptide with purity over 98% can be obtained from 1 l of Luria-Bertani medium culture by the method established in this study and the prepared DBAYL with four mutations (N10Q, V18L, N29Q, and M added to the N-terminal) were much more stable than BAY55-9837. The half-life of recombinant DBAYL was about 25 folds compared with that of BAY55-9837 in vitro. The bioactivity assay of DBAYL showed that it displaced [(125)I]PACAP38 and [(125)I]VIP from VPAC2 with a half-maximal inhibitory concentration of 48.4 ± 6.9 and 47.1 ± 4.9 nM, respectively, which were significantly lower than that of BAY55-9837, one established VPAC2 agonists. DBAYL enhances the cAMP accumulation in CHO cells expressing human VPAC2 with a half-maximal stimulatory concentration (EC(50)) of 0.68 nM, whereas the receptor potency of DBAYL at human VPAC1 (EC(50) of 737 nM) was only 1/1083 of that at human VPAC2, and DBAYL had no activity toward human PAC1 receptor. Western blot analysis of the key proteins of insulin receptor signaling pathway: insulin receptor substrate 1 (IRS-1) and glucose transporter 4 (GLUT4) indicated that the DBAYL could significantly induce the insulin-stimulated IRS-1 and GLUT4 expression more efficiently than BAY55-9837 and VIP in adipocytes. Compared with BAY55-9837 and PACAP38, the recombinant peptide DBAYL can more efficiently promote insulin release and decrease plasma glucose level in Institute of Cancer Research (ICR) mice. These results suggested that DBAYL could efficiently improve glucose

  19. Pituitary adenylate cyclase-activating peptide induces long-lasting neuroprotection through the induction of activity-dependent signaling via the cyclic AMP response element-binding protein-regulated transcription co-activator 1

    PubMed Central

    Baxter, Paul S; Martel, Marc-Andre; McMahon, Aoife; Kind, Peter C; Hardingham, Giles E

    2011-01-01

    Pituitary adenylate cyclase-activating peptide (PACAP) is a neuroprotective peptide which exerts its effects mainly through the cAMP-protein kinase A (PKA) pathway. Here, we show that in cortical neurons, PACAP-induced PKA signaling exerts a major part of its neuroprotective effects indirectly, by triggering action potential (AP) firing. Treatment of cortical neurons with PACAP induces a rapid and sustained PKA-dependent increase in AP firing and associated intracellular Ca2+ transients, which are essential for the anti-apoptotic actions of PACAP. Transient exposure to PACAP induces long-lasting neuroprotection in the face of apoptotic insults which is reliant on AP firing and the activation of cAMP response element (CRE) binding protein (CREB)-mediated gene expression. Although direct, activity-independent PKA signaling is sufficient to trigger phosphorylation on CREB’s activating serine-133 site, this is insufficient for activation of CREB-mediated gene expression. Full activation is dependent on CREB-regulated transcription co-activator 1 (CRTC1), whose PACAP-induced nuclear import is dependent on firing activity-dependent calcineurin signaling. Over-expression of CRTC1 is sufficient to rescue PACAP-induced CRE-mediated gene expression in the face of activity-blockade, while dominant negative CRTC1 interferes with PACAP-induced, CREB-mediated neuroprotection. Thus, the enhancement of AP firing may play a significant role in the neuroprotective actions of PACAP and other adenylate cyclase-coupled ligands. PMID:21623792

  20. A mutation in CFTR modifies the effects of the adenylate kinase inhibitor Ap5A on channel gating.

    PubMed

    Dong, Qian; Randak, Christoph O; Welsh, Michael J

    2008-12-01

    Mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis. The CFTR anion channel is controlled by ATP binding and enzymatic activity at the two nucleotide-binding domains. CFTR exhibits two types of enzymatic activity: 1), ATPase activity in the presence of ATP and 2), adenylate kinase activity in the presence of ATP plus physiologic concentrations of AMP or ADP. Previous work showed that P(1),P(5)-di(adenosine-5')pentaphosphate (Ap(5)A), a specific adenylate kinases inhibitor, inhibited wild-type CFTR. In this study, we report that Ap(5)A increased activity of CFTR with an L1254A mutation. This mutation increased the EC50 for ATP by >10-fold and reduced channel activity by prolonging the closed state. Ap(5)A did not elicit current on its own nor did it alter ATP EC50 or maximal current. However, it changed the relationship between ATP concentration and current. At submaximal ATP concentrations, Ap(5)A stimulated current by stabilizing the channel open state. Whereas previous work indicated that adenylate kinase activity regulated channel opening, our data suggest that Ap(5)A binding may also influence channel closing. These results also suggest that a better understanding of the adenylate kinase activity of CFTR may be of value in developing new therapeutic strategies for cystic fibrosis. PMID:18805924

  1. A Mutation in CFTR Modifies the Effects of the Adenylate Kinase Inhibitor Ap5A on Channel Gating

    PubMed Central

    Dong, Qian; Randak, Christoph O.; Welsh, Michael J.

    2008-01-01

    Mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis. The CFTR anion channel is controlled by ATP binding and enzymatic activity at the two nucleotide-binding domains. CFTR exhibits two types of enzymatic activity: 1), ATPase activity in the presence of ATP and 2), adenylate kinase activity in the presence of ATP plus physiologic concentrations of AMP or ADP. Previous work showed that P1,P5-di(adenosine-5′)pentaphosphate (Ap5A), a specific adenylate kinases inhibitor, inhibited wild-type CFTR. In this study, we report that Ap5A increased activity of CFTR with an L1254A mutation. This mutation increased the EC50 for ATP by >10-fold and reduced channel activity by prolonging the closed state. Ap5A did not elicit current on its own nor did it alter ATP EC50 or maximal current. However, it changed the relationship between ATP concentration and current. At submaximal ATP concentrations, Ap5A stimulated current by stabilizing the channel open state. Whereas previous work indicated that adenylate kinase activity regulated channel opening, our data suggest that Ap5A binding may also influence channel closing. These results also suggest that a better understanding of the adenylate kinase activity of CFTR may be of value in developing new therapeutic strategies for cystic fibrosis. PMID:18805924

  2. Development of a High-Throughput Screening Paradigm for the Discovery of Small-Molecule Modulators of Adenylyl Cyclase: Identification of an Adenylyl Cyclase 2 Inhibitor

    PubMed Central

    Conley, Jason M.; Brand, Cameron S.; Bogard, Amy S.; Pratt, Evan P. S.; Xu, Ruqiang; Hockerman, Gregory H.; Ostrom, Rennolds S.; Dessauer, Carmen W.

    2013-01-01

    Adenylyl cyclase (AC) isoforms are implicated in several physiologic processes and disease states, but advancements in the therapeutic targeting of AC isoforms have been limited by the lack of potent and isoform-selective small-molecule modulators. The discovery of AC isoform-selective small molecules is expected to facilitate the validation of AC isoforms as therapeutic targets and augment the study of AC isoform function in vivo. Identification of chemical probes for AC2 is particularly important because there are no published genetic deletion studies and few small-molecule modulators. The present report describes the development and implementation of an intact-cell, small-molecule screening approach and subsequent validation paradigm for the discovery of AC2 inhibitors. The NIH clinical collections I and II were screened for inhibitors of AC2 activity using PMA-stimulated cAMP accumulation as a functional readout. Active compounds were subsequently confirmed and validated as direct AC2 inhibitors using orthogonal and counterscreening assays. The screening effort identified SKF-83566 [8-bromo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hydrobromide] as a selective AC2 inhibitor with superior pharmacological properties for selective modulation of AC2 compared with currently available AC inhibitors. The utility of SKF-83566 as a small-molecule probe to study the function of endogenous ACs was demonstrated in C2C12 mouse skeletal muscle cells and human bronchial smooth muscle cells. PMID:24008337

  3. Changes in brain mRNA levels of gonadotropin-releasing hormone, pituitary adenylate cyclase activating polypeptide, and somatostatin during ovulatory luteinizing hormone and growth hormone surges in goldfish.

    PubMed

    Canosa, Luis Fabián; Stacey, Norm; Peter, Richard Ector

    2008-12-01

    In goldfish, circulating LH and growth hormone (GH) levels surge at the time of ovulation. In the present study, changes in gene expression of salmon gonadotropin-releasing hormone (sGnRH), chicken GnRH-II (cGnRH-II), somatostatin (SS) and pituitary adenylate cyclase activating polypeptide (PACAP) were analyzed during temperature- and spawning substrate-induced ovulation in goldfish. The results demonstrated that increases in PACAP gene expression during ovulation are best correlated with the GH secretion profile. These results suggest that PACAP, instead of GnRH, is involved in the control of GH secretion during ovulation. Increases of two of the SS transcripts during ovulation are interpreted as the activation of a negative feedback mechanism triggered by high GH levels. The results showed a differential regulation of sGnRH and cGnRH-II gene expression during ovulation, suggesting that sGnRH controls LH secretion, whereas cGnRH-II correlates best with spawning behavior. This conclusion is further supported by the finding that nonovulated fish induced to perform spawning behavior by prostaglandin F2alpha treatment increased cGnRH-II expression in both forebrain and midbrain, but decreased sGnRH expression in the forebrain. PMID:18815210

  4. Microinfusion of pituitary adenylate cyclase-activating polypeptide into the central nucleus of amygdala of the rat produces a shift from an active to passive mode of coping in the shock-probe fear/defensive burying test.

    PubMed

    Legradi, Gabor; Das, Mahasweta; Giunta, Brian; Hirani, Khemraj; Mitchell, E Alice; Diamond, David M

    2007-01-01

    High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) nerve fibers are present in the central nucleus of amygdala (CeA), a brain region implicated in the control of fear-related behavior. This study evaluated PACAPergic modulation of fear responses at the CeA in male Sprague-Dawley rats. PACAP (50-100 pmol) microinfusion via intra-CeA cannulae produced increases in immobility and time the rats spent withdrawn into a corner opposite to the electrified probe compared to controls in the shock-probe fear/defensive burying test. Shock-probe burying and exploration, numbers of shocks received, locomotion distance, and velocity were all reduced by intra-CeA PACAP injection. Further, intra-CeA PACAP effects were manifested only when the animals were challenged by shock, as intra-CeA PACAP injections did not cause significant changes in the behaviors of unshocked rats. Thus, intra-CeA administration of PACAP produces a distinct reorganization of stress-coping behaviors from active (burying) to passive modes, such as withdrawal and immobility. These findings are potentially significant toward enhancing our understanding of the involvement of PACAP and the CeA in the neural basis of fear and anxiety. PMID:17641738

  5. Ca2+ Influx and Tyrosine Kinases Trigger Bordetella Adenylate Cyclase Toxin (ACT) Endocytosis. Cell Physiology and Expression of the CD11b/CD18 Integrin Major Determinants of the Entry Route

    PubMed Central

    Etxebarria, Aitor; González-Bullón, David; Gómez-Bilbao, Geraxane; Ostolaza, Helena

    2013-01-01

    Humans infected with Bordetella pertussis, the whooping cough bacterium, show evidences of impaired host defenses. This pathogenic bacterium produces a unique adenylate cyclase toxin (ACT) which enters human phagocytes and catalyzes the unregulated formation of cAMP, hampering important bactericidal functions of these immune cells that eventually cause cell death by apoptosis and/or necrosis. Additionally, ACT permeabilizes cells through pore formation in the target cell membrane. Recently, we demonstrated that ACT is internalised into macrophages together with other membrane components, such as the integrin CD11b/CD18 (CR3), its receptor in these immune cells, and GM1. The goal of this study was to determine whether ACT uptake is restricted to receptor-bearing macrophages or on the contrary may also take place into cells devoid of receptor and gain more insights on the signalling involved. Here, we show that ACT is rapidly eliminated from the cell membrane of either CR3-positive as negative cells, though through different entry routes, which depends in part, on the target cell physiology and characteristics. ACT-induced Ca2+ influx and activation of non-receptor Tyr kinases into the target cell appear to be common master denominators in the different endocytic strategies activated by this toxin. Very importantly, we show that, upon incubation with ACT, target cells are capable of repairing the cell membrane, which suggests the mounting of an anti-toxin cell repair-response, very likely involving the toxin elimination from the cell surface. PMID:24058533

  6. In vitro susceptibility of fungi to acyclic inhibitors of 2,3-oxidosqualene cyclases.

    PubMed

    Airaudi, D; Ceruti, M; Bianco, C; Filipello Marchisio, V

    1996-01-01

    In the present study we determine the antifungal properties of two acyclic inhibitors of 2,3-oxidosqualene cyclases: 22,23-epoxy-2-aza-2,3-dihydrosqualene (EAS) and azasqualene alcohol (ASA). Fungistatic and fungicidal activity towards dermatophytes and other fungi involved in cutaneous and systemic infections was tested (48 isolates from 10 species). The tests were carried out by inoculating 10 microliters of mycelial homogenate in 1 ml of Sabouraud glucose liquid medium containing serial dilutions of 100 to 0.25 micrograms ml-1 of the substance. For each isolate, the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) of both compounds were determined. EAS was more active (MIC range 1.5-25 micrograms ml-1) than ASA (MIC range 3-50 micrograms ml-1). At the highest concentration tested, EAS also showed fungicidal action towards some isolates of Trichophyton mentagrophytes, T. terrestre, Epidermophyton floccosum, Microsporum canis and Scopulariopsis brumptii. The most sensitive species was T. mentagrophytes, the most resistant T. rubrum. PMID:8786759

  7. In silico prediction of tyrosinase and adenylyl cyclase inhibitors from natural compounds.

    PubMed

    Fong, Pedro; Tong, Henry H Y; Chao, Chi M

    2014-02-01

    Although many herbal medicines are effective in the treatment of hyperpigmentation, the potency of different constituents remains unknown. In this work, more than 20,000 herbal ingredients from 453 herbs were docked into the crystal structures of adenylyl cyclase and a human homology tyrosinase model using Surflex-Dock. These two enzymes are responsible for melanin production and inhibition of them may attain a skin-whitening effect superior to currently available agents. The essential drug properties for topical formulation of the herbal ingredients, including skin permeability, sensitization, irritation, corrosive and carcinogenic properties were predicted by Dermwin, Skin Sensitization Alerts (SSA), Skin Irritation Corrosion Rules Estimation Tool (SICRET) and Benigni/Bossa rulebase module of Toxtree. Moreover, similarity ensemble and pharmacophore mapping approaches were used to forecast other potential targets for these herbal compounds by the software, SEArch and PharmMapper. Overall, this study predicted seven compounds to have advanced drug-like properties over the well-known effective tyrosinase inhibitors, arbutin and kojic acid. These seven compounds have the highest potential for further in vitro and in vivo investigation with the aim of developing safe and high-efficacy skin-whitening agents. PMID:24689287

  8. Analgesic effects of adenylyl cyclase inhibitor NB001 on bone cancer pain in a mouse model

    PubMed Central

    Kang, Wen-bo; Yang, Qi; Guo, Yan-yan; Wang, Lu; Wang, Dong-sheng; Cheng, Qiang; Li, Xiao-ming; Tang, Jun; Zhao, Jian-ning; Liu, Gang; Zhuo, Min

    2016-01-01

    Background Cancer pain, especially the one caused by metastasis in bones, is a severe type of pain. Pain becomes chronic unless its causes and consequences are resolved. With improvements in cancer detection and survival among patients, pain has been considered as a great challenge because traditional therapies are partially effective in terms of providing relief. Cancer pain mechanisms are more poorly understood than neuropathic and inflammatory pain states. Chronic inflammatory pain and neuropathic pain are influenced by NB001, an adenylyl cyclase 1 (AC1)-specific inhibitor with analgesic effects. In this study, the analgesic effects of NB001 on cancer pain were evaluated. Results Pain was induced by injecting osteolytic murine sarcoma cell NCTC 2472 into the intramedullary cavity of the femur of mice. The mice injected with sarcoma cells for four weeks exhibited significant spontaneous pain behavior and mechanical allodynia. The continuous systemic application of NB001 (30 mg/kg, intraperitoneally, twice daily for three days) markedly decreased the number of spontaneous lifting but increased the mechanical paw withdrawal threshold. NB001 decreased the concentrations of cAMP and the levels of GluN2A, GluN2B, p-GluA1 (831), and p-GluA1 (845) in the anterior cingulate cortex, and inhibited the frequency of presynaptic neurotransmitter release in the anterior cingulate cortex of the mouse models. Conclusions NB001 may serve as a novel analgesic to treat bone cancer pain. Its analgesic effect is at least partially due to the inhibition of AC1 in anterior cingulate cortex. PMID:27612915

  9. Characterizations of a synthetic pituitary adenylate cyclase-activating polypeptide analog displaying potent neuroprotective activity and reduced in vivo cardiovascular side effects in a Parkinson's disease model.

    PubMed

    Lamine, Asma; Létourneau, Myriam; Doan, Ngoc Duc; Maucotel, Julie; Couvineau, Alain; Vaudry, Hubert; Chatenet, David; Vaudry, David; Fournier, Alain

    2016-09-01

    Parkinson's disease (PD) is characterized by a steady loss of dopamine neurons through apoptotic, inflammatory and oxidative stress processes. In that line of view, the pituitary adenylate cyclase-activating polypeptide (PACAP), with its ability to cross the blood-brain barrier and its anti-apoptotic, anti-inflammatory and anti-oxidative properties, has proven to offer potent neuroprotection in various PD models. Nonetheless, its peripheral actions, paired with low metabolic stability, hampered its clinical use. We have developed Ac-[Phe(pI)(6), Nle(17)]PACAP(1-27) as an improved PACAP-derived neuroprotective compound. In vitro, this analog stimulated cAMP production, maintained mitochondrial potential and protected SH-SY5Y neuroblastoma cells from 1-methyl-4-phenylpyridinium (MPP(+)) toxicity, as potently as PACAP. Furthermore, contrasting with PACAP, it is stable in human plasma and against dipeptidyl peptidase IV activity. When injected intravenously to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, PACAP and Ac-[Phe(pI)(6), Nle(17)]PACAP(1-27) restored tyrosine hydoxylase expression into the substantia nigra and modulated the inflammatory response. Albeit falls of mean arterial pressure (MAP) were observed with both PACAP- and Ac-[Phe(pI)(6), Nle(17)]PACAP(1-27)-treated mice, the intensity of the decrease as well as its duration were significantly less marked after iv injections of the analog than after those of the native polypeptide. Moreover, no significant changes in heart rate were measured with the animals for both compounds. Thus, Ac-[Phe(pI)(6), Nle(17)]PACAP(1-27) appears as a promising lead molecule for the development of PACAP-derived drugs potentially useful for the treatment of PD or other neurodegenerative diseases. PMID:26006268

  10. Follicle-stimulating hormone receptor-mediated uptake of sup 45 Ca sup 2+ by cultured rat Sertoli cells does not require activation of cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding proteins or adenylate cyclase

    SciTech Connect

    Grasso, P.; Reichert, L.E. Jr. )

    1990-08-01

    We have previously reported that FSH stimulates flux of 45Ca2+ into cultured Sertoli cells from immature rats via voltage-sensitive and voltage-independent calcium channels. In the present study, we show that this effect of FSH does not require cholera toxin (CT)- or pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein or activation of adenylate cyclase (AC). Significant stimulation of 45Ca2+ influx was observed within 1 min, and maximal response (3.2-fold over basal levels) was achieved within 2 min after exposure to FSH. FSH-stimulated elevations in cellular cAMP paralleled increases in 45Ca2+ uptake, suggesting a possible coupling of AC activation to 45Ca2+ influx. (Bu)2cAMP, however, was not able to enhance 45Ca2+ uptake over basal levels at a final concentration of 1000 microM, although a concentration-related increase in androstenedione conversion to estradiol was evident. Exposure of Sertoli cells to CT (10 ng/ml) consistently stimulated basal levels of androstenedione conversion to estradiol but had no effect on basal levels of 45Ca2+ uptake. Similarly, CT had no effect on FSH-induced 45Ca2+ uptake, but potentiated FSH-stimulated estradiol synthesis. PT (10 ng/ml) augmented basal and FSH-stimulated estradiol secretion without affecting 45Ca2+ influx. The adenosine analog N6-phenylisopropyladenosine, which binds to Gi-coupled adenosine receptors on Sertoli cells, inhibited FSH-stimulated androgen conversion to estradiol in a dose-related (1-1000 nM) manner, but FSH-stimulated 45Ca2+ influx remained unchanged. Our results show that in contrast to FSH-stimulated estradiol synthesis, the flux of 45Ca2+ into Sertoli cells in response to FSH is not mediated either directly or indirectly by CT- or PT-sensitive G protein, nor does it require activation of AC. Our data further suggest that the FSH receptor itself may function as a calcium channel.

  11. Alterations in the expression of G-proteins and regulation of adenylate cyclase in human neuroblastoma SH-SY5Y cells chronically exposed to low-efficacy mu-opioids.

    PubMed

    Ammer, H; Schulz, R

    1993-10-01

    Western-blot analysis of human neuroblastoma SH-SY5Y cells (mu- and delta-receptors) revealed the presence of the following G-protein subunits: Gi alpha 1, Gi alpha 2, Gs alpha, G(o) alpha, Gz alpha, and G beta, a pattern resembling that observed in central nervous tissue. Chronic treatment of differentiated [all-trans-retinoic acid (10 microM; 6 days)] SH-SY5Y cells with D(-)-morphine (10 microM; 3 days) significantly increased the abundance of all G-protein subunits identified. Co-incubation of morphine-exposed cells together with naloxone (10 microM; 3 days) or the mu-selective opioid antagonist CTOP (10 microM; 3 days), but not with the delta-selective antagonist ICI-174,864 (10 microM; 3 days), completely abolished this effect, suggesting that the increase in G-protein abundance is specifically mediated by mu-receptors. Moreover, the biologically inactive enantiomer L(+)-morphine (10 microM; 3 days) failed to produce a similar effect. G-protein up-regulation developed in a time- and dose-dependent manner and is most likely due to enhanced protein synthesis de novo, since concomitant treatment of the cells with cycloheximide (100 micrograms/ml; 3 days) prevented this effect. Chronic treatment with the low-efficacy mu-selective opioid peptide morphiceptin (10 microM; 3 days), but not with the highly potent mu-agonist DAGO (0.1 microM; 3 days) produced a comparable increase in G-protein abundance. Coincident with quantitative effects on G-protein levels in morphine-tolerant/dependent SH-SY5Y cells, we found elevated levels of basal, forskolin (1 microM)- and prostaglandin-E1 (1 microM)-stimulated adenylate cyclase activities. Reconstitution experiments using S49 cyc- lymphoma-cell membranes suggest that this increase is most likely due to elevated levels of functionally intact Gs. Chronic treatment with both morphine and DAGO induces high degrees of tolerance in this cell line. However, the intrinsic activity of G1 was unchanged, as assessed in functional studies

  12. Regulation of the cytosolic adenylate ratio as determined by rapid fractionation of mesophyll protoplasts of oat : Effect of electron transfer inhibitors and uncouplers.

    PubMed

    Goller, M; Hampp, R; Ziegler, H

    1982-12-01

    Adenylate levels in chloroplasts, mitochondria and the cytosol of oat mesophyll protoplasts were determined under light and dark conditions, in the absence and presence of plasmalemma-permeable inhibitors of electron transfer and uncouplers of phosphorylation. This was achieved using a microgradient technique which allowed an integrated homogenization and fractionation of protoplasts within 60 s (Hampp et al. 1982, Plant Physiol. 69, 448-455), under conditions which quench bulk activities of metabolic interconversion in less than 2 s. In illuminated controls, ATP/ADP ratios were found to be 2.1 in chloroplasts, about unity in mitochondria, and 11 in the cytosol; whereas, in the dark, this ratio only showed a large drop in chloroplasts (0.4). None of the compounds used [carbonylcyanide m-chlorophenylhydrazone (CCCP), carbonylcyanide p-trifluoromethoxy-phenylhydrazone (FCCP), antimycin A, dibromothymoquinone (DBMIB), dichlorophenyldi-methylurea (DCMU), or salicylhydroxamic acid (SHAM)] affected the stroma adenylate ratio in the dark. Under illumination, however, the ATP/ADP ratios were partly reduced in the presence of antimycin (inhibitor of cyclic photophosphorylation) and of DCMU (inhibitor of linear electron flow), while in the presence of DBMIB, DCMU+ antimycin (inhibition of both cyclic and linear electron flow), and CCCP (uncoupling) the ratio obtained was the same as that occurring in the dark. In contrast, mitochondrial adenylate levels did not exhibit large variations under the various treatments. The cytosolic ATP/ADP ratio, however, showed dramatic changes: in darkened protoplasts, cytosolic values dropped to 0.2 and 0.1 in the presence of uncouplers and antimycin, respectively, while SHAM did not induce any significant alteration. In the light, a similar pronounced decrease in ATP levels was observed only after the application of uncouplers or inhibitors of both mitochondrial and photosynthetic electron transport, whereas selective inhibition of the

  13. REGULATION OF POSTNATAL B-ADRENERGIC RECEPTOR/ADENYLATE CYCLASE DEVELOPMENT BY PRENATAL AGONIST STIMULATION AND STEROIDS: ALTERATIONS IN RAT KIDNEY AND LUNG AFTER EXPOSURE TO TERBUTALINE OR DEXAMETHASONE

    EPA Science Inventory

    Glucocorticoids and adrenergic stimulation are both thought to control the development of adrenergic receptors/responses. n the current study, rats were exposed to dexamethasone or terbutaline during late gestation and the development of B-binding capabilities and adenylate cycla...

  14. Comparative theoretical study of the binding of luciferyl-adenylate and dehydroluciferyl-adenylate to firefly luciferase

    NASA Astrophysics Data System (ADS)

    Pinto da Silva, Luís; Vieira, João; Esteves da Silva, Joaquim C. G.

    2012-08-01

    This is the first report of a study employing a computational approach to study the binding of (D/L)-luciferyl-adenlyates and dehydroluciferyl-adenylate to firefly luciferase. A semi-empirical/molecular mechanics methodology was used to study the interaction between these ligands and active site molecules. All adenylates are complexed with the enzyme, mostly due to electrostatic interactions with cationic residues. Dehydroluciferyl-adenylate is expected to be a competitive inhibitor of luciferyl-adenylate, as their binding mechanism and affinity to luciferase are very similar. Both luciferyl-adenylates adopt the L-orientation in the active site of luciferase.

  15. Challenge of human Jurkat T-cells with the adenylate cyclase activator forskolin elicits major changes in cAMP phosphodiesterase (PDE) expression by up-regulating PDE3 and inducing PDE4D1 and PDE4D2 splice variants as well as down-regulating a novel PDE4A splice variant.

    PubMed Central

    Erdogan, S; Houslay, M D

    1997-01-01

    The cAMP phosphodiesterase (PDE) 3 and PDE4 isoforms provide the major cAMP-hydrolysing PDE activities in Jurkat T-cells, with additional contributions from the PDE1 and PDE2 isoforms. Challenge of cells with the adenylate cyclase activator forskolin led to a rapid, albeit transient, increase in PDE3 activity occurring over the first 45 min, followed by a sustained increase in PDE3 activity which began after approximately 3 h and continued for at least 24 h. Only this second phase of increase in PDE3 activity was blocked by the transcriptional inhibitor actinomycin D. After approximately 3 h of exposure to forskolin, PDE4 activity had increased, via a process that could be inhibited by actinomycin D, and it remained elevated for at least a 24 h period. Such actions of forskolin were mimicked by cholera toxin and 8-bromo-cAMP. Forskolin increased intracellular cAMP concentrations in a time-dependent fashion and its action was enhanced when PDE induction was blocked with actinomycin D. Reverse transcription (RT)-PCR analysis, using generic primers designed to detect transcripts representing enzymically active products of the four PDE4 genes, identified transcripts for PDE4A and PDE4D but not for PDE4B or PDE4C in untreated Jurkat T-cells. Forskolin treatment did not induce transcripts for either PDE4B or PDE4C; however, it reduced the RT-PCR signal for PDE4A transcripts and markedly enhanced that for PDE4D transcripts. Using RT-PCR primers for PDE4 splice variants, a weak signal for PDE4D1 was evident in control cells whereas, in forskolin-treated cells, clear signals for both PDE4D1 and PDE4D2 were detected. RT-PCR analysis of the PDE4A species indicated that it was not the PDE4A isoform PDE-46 (PDE4A4B). Immunoblotting of control cells for PDE4 forms identified a single PDE4A species of approximately 118 kDa, which migrated distinctly from the PDE4A4B isoform PDE-46, with immunoprecipitation analyses showing that it provided all of the PDE4 activity in control

  16. High-throughput screening using the differential radial capillary action of ligand assay identifies ebselen as an inhibitor of diguanylate cyclases.

    PubMed

    Lieberman, Ori J; Orr, Mona W; Wang, Yan; Lee, Vincent T

    2014-01-17

    The rise of bacterial resistance to traditional antibiotics has motivated recent efforts to identify new drug candidates that target virulence factors or their regulatory pathways. One such antivirulence target is the cyclic-di-GMP (cdiGMP) signaling pathway, which regulates biofilm formation, motility, and pathogenesis. Pseudomonas aeruginosa is an important opportunistic pathogen that utilizes cdiGMP-regulated polysaccharides, including alginate and pellicle polysaccharide (PEL), to mediate virulence and antibiotic resistance. CdiGMP activates PEL and alginate biosynthesis by binding to specific receptors including PelD and Alg44. Mutations that abrogate cdiGMP binding to these receptors prevent polysaccharide production. Identification of small molecules that can inhibit cdiGMP binding to the allosteric sites on these proteins could mimic binding defective mutants and potentially reduce biofilm formation or alginate secretion. Here, we report the development of a rapid and quantitative high-throughput screen for inhibitors of protein-cdiGMP interactions based on the differential radial capillary action of ligand assay (DRaCALA). Using this approach, we identified ebselen as an inhibitor of cdiGMP binding to receptors containing an RxxD domain including PelD and diguanylate cyclases (DGC). Ebselen reduces diguanylate cyclase activity by covalently modifying cysteine residues. Ebselen oxide, the selenone analogue of ebselen, also inhibits cdiGMP binding through the same covalent mechanism. Ebselen and ebselen oxide inhibit cdiGMP regulation of biofilm formation and flagella-mediated motility in P. aeruginosa through inhibition of diguanylate cyclases. The identification of ebselen provides a proof-of-principle that a DRaCALA high-throughput screening approach can be used to identify bioactive agents that reverse regulation of cdiGMP signaling by targeting cdiGMP-binding domains. PMID:24134695

  17. High-throughput screening using the differential radial capillary action of ligand assay identifies ebselen as an inhibitor of diguanylate cyclases

    PubMed Central

    Lieberman, Ori J.; Orr, Mona W.; Wang, Yan; Lee, Vincent T.

    2013-01-01

    The rise of bacterial resistance to traditional antibiotics has motivated recent efforts to identify new drug candidates that target virulence factors or their regulatory pathways. One such antivirulence target is the cyclic-di-GMP (cdiGMP) signaling pathway, which regulates biofilm formation, motility, and pathogenesis. Pseudomonas aeruginosa is an important opportunistic pathogen that utilizes cdiGMP-regulated polysaccharides, including alginate and pellicle polysaccharide (PEL), to mediate virulence and antibiotic resistance. CdiGMP activates PEL and alginate biosynthesis by binding to specific receptors including PelD and Alg44. Mutations that abrogate cdiGMP binding to these receptors prevent polysaccharide production. Identification of small molecules that can inhibit cdiGMP binding to the allosteric sites on these proteins could mimic binding defective mutants and potentially reduce biofilm formation or alginate secretion. Here, we report the development of a rapid and quantitative high-throughput screen for inhibitors of protein-cdiGMP interactions based on the differential radial capillary action of ligand assay (DRaCALA). Using this approach, we identified ebselen as an inhibitor of cdiGMP binding to receptors containing an RxxD domain including PelD and diguanylate cyclases (DGC). Ebselen reduces diguanylate cyclase activity by covalently modifying cysteine residues. Ebselen oxide, the selenone analogue of ebselen, also inhibits cdiGMP binding through the same covalent mechanism. Ebselen and ebselen oxide inhibit cdiGMP regulation of biofilm formation and flagella-mediated motility in P. aeruginosa through inhibition of diguanylate cyclases. The identification of ebselen provides a proof-of-principle that a DRaCALA high-throughput screening approach can be used to identify bioactive agents that reverse regulation of cdiGMP signaling by targeting cdiGMP-binding domains. PMID:24134695

  18. Biochemical and Structural Characterization of Bisubstrate Inhibitors of BasE, the Self-standing Non-Ribosomal Peptide Synthetase Adenylate-Forming Enzyme of Acinetobactin Synthesis†,‡

    PubMed Central

    Drake, Eric J.; Duckworth, Benjamin P.; Neres, João; Aldrich, Courtney C.; Gulick, Andrew M.

    2010-01-01

    The human pathogen Acinetobacter baumannii produces a siderophore called acinetobactin that is derived from one molecule each of threonine, histidine, and 2,3-dihydroxybenzoic acid (DHB). The activity of several non-ribosomal peptide synthetase (NRPS) enzymes is used to combine the building blocks into the final molecule. The acinetobactin synthesis pathway initiates with a self-standing adenylation enzyme, BasE, that activates the DHB molecule and covalently transfers it to the pantetheine cofactor of an aryl-carrier protein of BasF, a strategy that is shared with many siderophore-producing NRPS clusters. In this reaction, DHB reacts with ATP to form the aryl adenylate and pyrophosphate. In a second partial reaction, the DHB is transferred to the carrier protein. Inhibitors of BasE and related enzymes have been identified that prevent growth of bacteria on iron-limiting media. Recently, a new inhibitor of BasE has been identified via high-throughput screening using a fluorescence polarization displacement assay. We present here biochemical and structural studies to examine the binding mode of this inhibitor. The kinetics of the wild-type BasE enzyme is shown and inhibition studies demonstrate that the new compound exhibits competitive inhibition against both ATP and 2,3-dihydroxybenzoate. Structural examination of BasE bound to this inhibitor illustrates a novel binding mode in which the phenyl moiety partially fills the enzyme pantetheine binding tunnel. Structures of rationally designed bisubstrate inhibitors are also presented. PMID:20853905

  19. A Diguanylate Cyclase Acts as a Cell Division Inhibitor in a Two-Step Response to Reductive and Envelope Stresses

    PubMed Central

    Kim, Hyo Kyung

    2016-01-01

    ABSTRACT Cell division arrest is a universal checkpoint in response to environmental assaults that generate cellular stress. In bacteria, the cyclic di-GMP (c-di-GMP) signaling network is one of several signal transduction systems that regulate key processes in response to extra-/intracellular stimuli. Here, we find that the diguanylate cyclase YfiN acts as a bifunctional protein that produces c-di-GMP in response to reductive stress and then dynamically relocates to the division site to arrest cell division in response to envelope stress in Escherichia coli. YfiN localizes to the Z ring by interacting with early division proteins and stalls cell division by preventing the initiation of septal peptidoglycan synthesis. These studies reveal a new role for a diguanylate cyclase in responding to environmental change, as well as a novel mechanism for arresting cell division. PMID:27507823

  20. Synthesis of Chromone, Quinolone, and Benzoxazinone Sulfonamide Nucleosides as Conformationally Constrained Inhibitors of Adenylating Enzymes Required for Siderophore Biosynthesis

    PubMed Central

    Engelhart, Curtis A.; Aldrich, Courtney C.

    2013-01-01

    MbtA catalyzes the first committed step of mycobactin biosynthesis in Mycobacterium tuberculosis (Mtb) and is responsible for the incorporation of salicylic acid into the mycobactin siderophores. 5′-O-[N-(Salicyl)sulfamoyl]adenosine (Sal-AMS) is an extremely potent nucleoside inhibitor of MbtA that possesses excellent activity against whole-cell Mtb, but suffers from poor bioavailability. In an effort to improve the bioavailability, we have designed four conformationally constrained analogues of Sal-AMS that remove two rotatable bonds and the ionized sulfamate group based on computational and structural studies. Herein we describe the synthesis, biochemical, and microbiological evaluation of chromone-, quinolone-, and benzoxazinone-3-sulfonamide derivatives of Sal-AMS. We developed new chemistry to assemble these three heterocycles from common β-ketosulfonamide intermediates. The synthesis of the chromone- and quinolone-3-sulfonamide intermediates features formylation of a β-ketosulfonamide employing dimethylformamide dimethyl acetal to afford an enaminone that can react intramolecularly with a phenol or intermolecularly with a primary amine via addition-elimination reaction(s). The benzoxazinone-3-sulfonamide was prepared by nitrosation of a β-ketosulfonamide followed by intramolecular nucleophilic aromatic substitution. Mitsunobu coupling of these bicyclic sulfonamides with a protected adenosine derivative followed by global deprotection provides a concise synthesis of the respective inhibitors. PMID:23805993

  1. Adenylate-forming enzymes.

    PubMed

    Schmelz, Stefan; Naismith, James H

    2009-12-01

    Thioesters, amides, and esters are common chemical building blocks in a wide array of natural products. The formation of these bonds can be catalyzed in a variety of ways. For chemists, the use of an activating group is a common strategy and adenylate enzymes are exemplars of this approach. Adenylating enzymes activate the otherwise unreactive carboxylic acid by transforming the normal hydroxyl leaving group into adenosine monophosphate. Recently there have been a number of studies of such enzymes and in this review we suggest a new classification scheme. The review highlights the diversity in enzyme fold, active site architecture, and metal coordination that has evolved to catalyze this particular reaction. PMID:19836944

  2. Phosphonate analogues of aminoacyl adenylates.

    PubMed Central

    Southgate, C C; Dixon, H B

    1978-01-01

    Phosphonomethyl analogues of glycyl phosphate and valyl phosphate, i.e. NH2-CHR-CO-CH2-PO(OH)2, were synthesized and esterified with adenosine to give analogues of aminoacyl adenylates. The interaction of these adenylate analogues with valyl-tRNA synthetase from Escherichia coli was studied by fluorescence titration. The analogue of valyl phosphate has an affinity for the enzyme comparable with that of valine, but that of valyl adenylate is bound much less tightly than either valyl adenylate or corresponding derivative of valinol. The affinity of the analogue of glycyl adenylate was too low to be measured. We conclude that this enzyme interacts specifically with both the side chain and the anhydride linkage of the adenylate intermediate. PMID:743207

  3. Pharmacophore based approach to design inhibitors in crustaceans: an insight into the molt inhibition response to the receptor guanylyl cyclase.

    PubMed

    Shrivastava, Sajal; Princy, S Adline

    2014-04-01

    The first set of competitive inhibitors of molt inhibiting hormone (MIH) has been developed using the effective approaches such as Hip-Hop, virtual screening and manual alterations. Moreover, the conserved residues at 71 and 72 positions in the molt inhibiting hormone is known to be significant for selective inhibition of ecdysteroidogenesis; thus, the information from mutation and solution structure were used to generate common pharmacophore features. The geometry of the final six-feature pharmacophore was also found to be consistent with the homology-modeled MIH structures from various other decapod crustaceans. The Hypo-1, comprising six features hypothesis was carefully selected as a best pharmacophore model for virtual screening created on the basis of rank score and cluster processes. The hypothesis was validated and the database was virtually screened using this 3D query and the compounds were then manually altered to enhance the fit value. The hits obtained were further filtered for drug-likeness, which is expressed as physicochemical properties that contribute to favorable ADME/Tox profiles to eliminate the molecules exhibit toxicity and poor pharmacokinetics. In conclusion, the higher fit values of CI-1 (4.6), CI-4 (4.9) and CI-7 (4.2) in conjunction with better pharmacokinetic profile made these molecules practically helpful tool to increase production by accelerating molt in crustaceans. The use of feeding sub-therapeutic dosages of these growth enhancers can be very effectively implemented and certainly turn out to be a vital part of emerging nutritional strategies for economically important crustacean livestock. PMID:24772941

  4. MEK Inhibitors Reverse cAMP-Mediated Anxiety in Zebrafish.

    PubMed

    Lundegaard, Pia R; Anastasaki, Corina; Grant, Nicola J; Sillito, Rowland R; Zich, Judith; Zeng, Zhiqiang; Paranthaman, Karthika; Larsen, Anders Peter; Armstrong, J Douglas; Porteous, David J; Patton, E Elizabeth

    2015-10-22

    Altered phosphodiesterase (PDE)-cyclic AMP (cAMP) activity is frequently associated with anxiety disorders, but current therapies act by reducing neuronal excitability rather than targeting PDE-cAMP-mediated signaling pathways. Here, we report the novel repositioning of anti-cancer MEK inhibitors as anxiolytics in a zebrafish model of anxiety-like behaviors. PDE inhibitors or activators of adenylate cyclase cause behaviors consistent with anxiety in larvae and adult zebrafish. Small-molecule screening identifies MEK inhibitors as potent suppressors of cAMP anxiety behaviors in both larvae and adult zebrafish, while causing no anxiolytic behavioral effects on their own. The mechanism underlying cAMP-induced anxiety is via crosstalk to activation of the RAS-MAPK signaling pathway. We propose that targeting crosstalk signaling pathways can be an effective strategy for mental health disorders, and advance the repositioning of MEK inhibitors as behavior stabilizers in the context of increased cAMP. PMID:26388333

  5. Primary structure of maize chloroplast adenylate kinase.

    PubMed

    Schiltz, E; Burger, S; Grafmüller, R; Deppert, W R; Haehnel, W; Wagner, E

    1994-06-15

    This paper describes the sequence of adenylate kinase (Mg-ATP+AMP<-->Mg-ADP+ADP) from maize chloroplasts. This light-inducible enzyme is important for efficient CO2 fixation in the C4 cycle, by removing and recycling AMP produced in the reversible pyruvate phosphate dikinase reaction. The complete sequence was determined by analyzing peptides from cleavages with trypsin, AspN protease and CNBr and subcleavage of a major CNBr peptide with chymotrypsin. N-terminal Edman degradation and carboxypeptidase digestion established the terminal residues. Electrospray mass spectrometry confirmed the final sequence of 222 residues (M(r) = 24867) including one cysteine and one tryptophan. The sequence shows this enzyme to be a long-variant-type adenylate kinase, the nearest relatives being adenylate kinases from Enterobacteriaceae. Alignment of the sequence with the adenylate kinase from Escherichia coli reveals 44% identical residues. Since the E. coli structure has been published recently at 0.19-nm resolution with the inhibitor adenosine(5')pentaphospho(5')adenosine (Ap5A) [Müller, C. W. & Schulz, G. E. (1992) J. Mol. Biol. 224, 159-177], catalytically essential residues could be compared and were found to be mostly conserved. Surprisingly, in the nucleotide-binding Gly-rich loop Gly-Xaa-Pro-Gly-Xaa-Gly-Lys the middle Gly is replaced by Ala. This is, however, compensated by an Ile-->Val exchange in the nearest spatial neighborhood. A Thr-->Ala exchange explains the unusual tolerance of the enzyme for pyrimidine nucleotides in the acceptor site. PMID:8026505

  6. Antitumor/Antifungal Celecoxib Derivative AR-12 is a Non-Nucleoside Inhibitor of the ANL-Family Adenylating Enzyme Acetyl CoA Synthetase

    PubMed Central

    2016-01-01

    AR-12/OSU-03012 is an antitumor celecoxib-derivative that has progressed to Phase I clinical trial as an anticancer agent and has activity against a number of infectious agents including fungi, bacteria and viruses. However, the mechanism of these activities has remained unclear. Based on a chemical-genetic profiling approach in yeast, we have found that AR-12 is an ATP-competitive, time-dependent inhibitor of yeast acetyl coenzyme A synthetase. AR-12-treated fungal cells show phenotypes consistent with the genetic reduction of acetyl CoA synthetase activity, including induction of autophagy, decreased histone acetylation, and loss of cellular integrity. In addition, AR-12 is a weak inhibitor of human acetyl CoA synthetase ACCS2. Acetyl CoA synthetase activity is essential in many fungi and parasites. In contrast, acetyl CoA is primarily synthesized by an alternate enzyme, ATP-citrate lyase, in mammalian cells. Taken together, our results indicate that AR-12 is a non-nucleoside acetyl CoA synthetase inhibitor and that acetyl CoA synthetase may be a feasible antifungal drug target. PMID:27088128

  7. Non-nucleoside Inhibitors of BasE, An Adenylating Enzyme in the Siderophore Biosynthetic Pathway of the Opportunistic Pathogen Acinetobacter baumannii

    PubMed Central

    Neres, João; Engelhart, Curtis A.; Drake, Eric J.; Wilson, Daniel J.; Fu, Peng; Boshoff, Helena I.; Barry, Clifton E.; Gulick, Andrew M.; Aldrich, Courtney C.

    2013-01-01

    Siderophores are small-molecule iron chelators produced by bacteria and other microorganisms for survival under iron limiting conditions, such as found in a mammalian host. Siderophore biosynthesis is essential for the virulence of many important Gram-negative pathogens including Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli. We performed high-throughput screening of against BasE, which is involved in siderophore biosynthesis in A. baumannii and identified 6-phenyl-1-(pyridin-4-ylmethyl)-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid 15. Herein we report the synthesis, biochemical, and microbiological evaluation of a systematic series of analogues of the HTS hit 15. Analogue 67 is the most potent analogue with a KD of 2 nM against BasE. Structural characterization of the inhibitors with BasE reveal they bind in a unique orientation in the active site occupying all three substrate binding sites, and thus can be considered multisubstrate inhibitors. These results provide a foundation for future studies aimed at both increasing enzyme potency and antibacterial activity. PMID:23437866

  8. Facilitation of corticostriatal transmission following pharmacological inhibition of striatal phosphodiesterase 10A: role of nitric oxide-soluble guanylyl cyclase-cGMP signaling pathways.

    PubMed

    Padovan-Neto, Fernando E; Sammut, Stephen; Chakroborty, Shreaya; Dec, Alexander M; Threlfell, Sarah; Campbell, Peter W; Mudrakola, Vishnu; Harms, John F; Schmidt, Christopher J; West, Anthony R

    2015-04-01

    The striatum contains a rich variety of cyclic nucleotide phosphodiesterases (PDEs), which play a critical role in the regulation of cAMP and cGMP signaling. The dual-substrate enzyme PDE10A is the most highly expressed PDE in striatal medium-sized spiny neurons (MSNs) with low micromolar affinity for both cyclic nucleotides. Previously, we have shown that systemic and local administration of the selective PDE10A inhibitor TP-10 potently increased the responsiveness of MSNs to cortical stimulation. However, the signaling mechanisms underlying PDE10A inhibitor-induced changes in corticostriatal transmission are only partially understood. The current studies assessed the respective roles of cAMP and cGMP in the above effects using soluble guanylyl cyclase (sGC) or adenylate cyclase (AC) specific inhibitors. Cortically evoked spike activity was monitored in urethane-anesthetized rats using in vivo extracellular recordings performed proximal to a microdialysis probe during local infusion of vehicle, the selective sGC inhibitor ODQ, or the selective AC inhibitor SQ 22536. Systemic administration of TP-10 (3.2 mg/kg) robustly increased cortically evoked spike activity in a manner that was blocked following intrastriatal infusion of ODQ (50 μm). The effects of TP-10 on evoked activity were due to accumulation of cGMP, rather than cAMP, as the AC inhibitor SQ was without effect. Consistent with these observations, studies in neuronal NO synthase (nNOS) knock-out (KO) mice confirmed that PDE10A operates downstream of nNOS to limit cGMP production and excitatory corticostriatal transmission. Thus, stimulation of PDE10A acts to attenuate corticostriatal transmission in a manner largely dependent on effects directed at the NO-sGC-cGMP signaling cascade. PMID:25855188

  9. Bacterial terpene cyclases.

    PubMed

    Dickschat, Jeroen S

    2016-01-01

    Covering: up to 2015. This review summarises the accumulated knowledge about characterised bacterial terpene cyclases. The structures of identified products and of crystallised enzymes are included, and the obtained insights into enzyme mechanisms are discussed. After a summary of mono-, sesqui- and diterpene cyclases the special cases of the geosmin and 2-methylisoborneol synthases that are both particularly widespread in bacteria will be presented. A total number of 63 enzymes that have been characterised so far is presented, with 132 cited references. PMID:26563452

  10. Carnosine as a regulator of soluble guanylate cyclase.

    PubMed

    Severina, I S; Bussygina, O G; Pyatakova, N V

    2000-07-01

    The molecular mechanism of the participation of carnosine in the functioning of soluble guanylate cyclase is discussed. It is shown that carnosine inhibits the activation of soluble guanylate cyclase by sodium nitroprusside and a derivative of furoxan--1,2,5-oxadiazolo-trioxide (an NO donor). However, carnosine has no effect on stimulation of the enzyme by a structural analog of the latter compound, a furazan derivative (1,2,5-oxadiazolo-dioxide) that is not an NO donor; nor was carnosine involved in the enzyme activation by protoporphyrin IX, whose stimulatory effect is not associated with the guanylate cyclase heme. The inhibition by carnosine of guanylate cyclase activation by an NO donor is due to the interaction of carnosine with heme iron with subsequent formation of a chelate complex. It was first demonstrated that carnosine is a selective inhibitor of NO-dependent activation of guanylate cyclase and may be used for suppression of activity of the intracellular signaling system NO-soluble guanylate cyclase-cGMP, whose sharp increase is observed in malignant tumors, sepsis, septic shock, asthma, and migraine. PMID:10951096

  11. Sustained and selective suppression of intestinal cholesterol synthesis by Ro 48-8071, an inhibitor of 2,3-oxidosqualene:lanosterol cyclase, in the BALB/c mouse.

    PubMed

    Chuang, Jen-Chieh; Valasek, Mark A; Lopez, Adam M; Posey, Kenneth S; Repa, Joyce J; Turley, Stephen D

    2014-04-01

    The small intestine plays a fundamentally important role in regulating whole body cholesterol balance and plasma lipoprotein composition. This is articulated through the interplay of a constellation of genes that ultimately determines the net amount of chylomicron cholesterol delivered to the liver. Major advances in our insights into regulation of the cholesterol absorption pathway have been made using genetically manipulated mouse models and agents such as ezetimibe. One unresolved question is how a sustained pharmacological inhibition of intestinal cholesterol synthesis in vivo may affect cholesterol handling by the absorptive cells. Here we show that the lanosterol cyclase inhibitor, Ro 48-8071, when fed to BALB/c mice in a chow diet (20 mg/day/kg body weight), leads to a rapid and sustained inhibition (>50%) of cholesterol synthesis in the whole small intestine. Sterol synthesis was also reduced in the large intestine and stomach. In contrast, hepatic cholesterol synthesis, while markedly suppressed initially, rebounded to higher than baseline rates within 7 days. Whole body cholesterol synthesis, fractional cholesterol absorption, and fecal neutral and acidic sterol excretion were not consistently changed with Ro 48-8071 treatment. There were no discernible effects of this agent on intestinal histology as determined by H&E staining and the level of Ki67, an index of proliferation. The mRNA expression for multiple genes involved in intestinal cholesterol regulation including NPC1L1 was mostly unchanged although there was a marked rise in the mRNA level for the PXR target genes CYP3A11 and CES2A. PMID:24486573

  12. Uridylation and adenylation of RNAs.

    PubMed

    Song, JianBo; Song, Jun; Mo, BeiXin; Chen, XueMei

    2015-11-01

    The posttranscriptional addition of nontemplated nucleotides to the 3' ends of RNA molecules can have a significant impact on their stability and biological function. It has been recently discovered that nontemplated addition of uridine or adenosine to the 3' ends of RNAs occurs in different organisms ranging from algae to humans, and on different kinds of RNAs, such as histone mRNAs, mRNA fragments, U6 snRNA, mature small RNAs and their precursors etc. These modifications may lead to different outcomes, such as increasing RNA decay, promoting or inhibiting RNA processing, or changing RNA activity. Growing pieces of evidence have revealed that such modifications can be RNA sequence-specific and subjected to temporal or spatial regulation in development. RNA tailing and its outcomes have been associated with human diseases such as cancer. Here, we review recent developments in RNA uridylation and adenylation and discuss the future prospects in this research area. PMID:26563174

  13. Inhibition of vaccinia mRNA methylation by 2',5'-linked oligo(adenylic acid) triphosphate

    SciTech Connect

    Sharma, O.K.; Goswami, B.B.

    1981-04-01

    Extracts of interferon-treated cells synthesize unique 2',5'-linked oligo(adenylic acid) 5'-phosphates in the presence of ATP and double-stranded RNA. 2',5'-linked oligo(adenylic acid) 5'-triphosphate inhibits protein synthesis at nanomolar concentrations by activating RNase. We have observed that oligo(adenylic acid) 5'-monophosphate and 5'-triphosphate are potent inhibitors of vaccinia mRNA methylation in vitro. Both the methylation of the 5'-terminal guanine at the 7 position and the 2'-O-ribose methylation of the penultimate nucleoside are inhibited. Such inhibition of mRNA methylation is not due to degradation of the mRNA. Inhibition of the requisite modification of the 5' terminus of mRNA by 2',5'-linked oligo(adenylic acids) may be a mechanism of interferon action against both DNA and RNA viruses in which mRNAs derived from them are capped.

  14. A Novel Mechanism for Adenylyl Cyclase Inhibition from the Crystal Structure of its Complex with Catechol Estrogen

    SciTech Connect

    Steegborn,C.; Litvin, T.; Hess, K.; Capper, A.; Taussig, R.; Buck, J.; Levin, L.; Wu, H.

    2005-01-01

    Catechol estrogens are steroid metabolites that elicit physiological responses through binding to a variety of cellular targets. We show here that catechol estrogens directly inhibit soluble adenylyl cyclases and the abundant trans-membrane adenylyl cyclases. Catechol estrogen inhibition is non-competitive with respect to the substrate ATP, and we solved the crystal structure of a catechol estrogen bound to a soluble adenylyl cyclase from Spirulina platensis in complex with a substrate analog. The catechol estrogen is bound to a newly identified, conserved hydrophobic patch near the active center but distinct from the ATP-binding cleft. Inhibitor binding leads to a chelating interaction between the catechol estrogen hydroxyl groups and the catalytic magnesium ion, distorting the active site and trapping the enzyme substrate complex in a non-productive conformation. This novel inhibition mechanism likely applies to other adenylyl cyclase inhibitors, and the identified ligand-binding site has important implications for the development of specific adenylyl cyclase inhibitors.

  15. MEK Inhibitors Reverse cAMP-Mediated Anxiety in Zebrafish

    PubMed Central

    Lundegaard, Pia R.; Anastasaki, Corina; Grant, Nicola J.; Sillito, Rowland R.; Zich, Judith; Zeng, Zhiqiang; Paranthaman, Karthika; Larsen, Anders Peter; Armstrong, J. Douglas; Porteous, David J.; Patton, E. Elizabeth

    2015-01-01

    Summary Altered phosphodiesterase (PDE)-cyclic AMP (cAMP) activity is frequently associated with anxiety disorders, but current therapies act by reducing neuronal excitability rather than targeting PDE-cAMP-mediated signaling pathways. Here, we report the novel repositioning of anti-cancer MEK inhibitors as anxiolytics in a zebrafish model of anxiety-like behaviors. PDE inhibitors or activators of adenylate cyclase cause behaviors consistent with anxiety in larvae and adult zebrafish. Small-molecule screening identifies MEK inhibitors as potent suppressors of cAMP anxiety behaviors in both larvae and adult zebrafish, while causing no anxiolytic behavioral effects on their own. The mechanism underlying cAMP-induced anxiety is via crosstalk to activation of the RAS-MAPK signaling pathway. We propose that targeting crosstalk signaling pathways can be an effective strategy for mental health disorders, and advance the repositioning of MEK inhibitors as behavior stabilizers in the context of increased cAMP. PMID:26388333

  16. Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia.

    PubMed

    Dong, Qian; Ernst, Sarah E; Ostedgaard, Lynda S; Shah, Viral S; Ver Heul, Amanda R; Welsh, Michael J; Randak, Christoph O

    2015-05-29

    The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. PMID:25887396

  17. Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia*

    PubMed Central

    Dong, Qian; Ernst, Sarah E.; Ostedgaard, Lynda S.; Shah, Viral S.; Ver Heul, Amanda R.; Welsh, Michael J.; Randak, Christoph O.

    2015-01-01

    The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P1,P5-di(adenosine-5′) pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5′-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5′-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl− channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. PMID:25887396

  18. Nerve growth factor-induced differentiation of PC12 cells is accompanied by elevated adenylyl cyclase activity.

    PubMed

    Yung, H S; Lai, K H; Chow, K B S; Ip, N Y; Tsim, K W K; Wong, Y H; Wu, Z; Wise, H

    2010-01-01

    Rat pheochromocytoma (PC12) cells characteristically undergo differentiation when cultured with nerve growth factor (NGF). Here we show that NGF dramatically increased the adenylyl cyclase-activating property of forskolin in PC12 cells. This effect of NGF was well maintained even when NGF was removed after 4 days, even though the morphological features of neuronal differentiation were rapidly lost on removal of NGF. The enhanced cAMP production in response to forskolin could be due to a synergistic interaction between forskolin and endogenously released agonists acting on G(s)-coupled receptors. However, responses to forskolin were not attenuated by antagonists of adenosine A2 receptors or pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, suggesting that adenosine and PACAP were not involved. Adenylyl cyclases 3, 6 and 9 were the predominant isoforms expressed in PC12 cells, but we found no evidence for NGF-induced changes in expression levels of any of the 9 adenylyl cyclase isoforms, nor in the expression of Gα(s). These findings highlight that NGF has a subtle influence on adenylyl cyclase activity in PC12 cells which may influence more than the neurite extension process classically associated with neuronal differentiation. PMID:20389133

  19. Loss of Adenylate Cyclase Tonxin among closely related B. bronchiseptica strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bordetella bronchiseptica is a multi-host, gram-negative respiratory pathogen that causes everything from asymptomatic infection to fatal pneumonia. We have identified a strain of B. bronchiseptica, 253, that is inefficient at persisting in the lower respiratory tract of mice compared to the typica...

  20. Renal adenylate cyclase assay for biologically active parathyroid hormone: clinical utility and physiological significance.

    PubMed

    Auf'mkolk, B; Hesch, R D

    1986-01-01

    The stimulation of cyclic AMP production by human renal cortical membranes in the presence of the GTP analogue 5'-guanylimidodiphosphate and a calcium chelator represents a homologous assay system for the evaluation of biologically active parathyroid hormone (bioPTH) in human serum. Bioactive PTH was raised above normal (normal range: undetectable to 4.6 pmol human PTH(1-34) per 1) in 13/17 (76%) patients with primary hyperparathyroidism, in 5/6 (83%) patients with surgically proven hyperparathyroidism secondary to chronic renal failure, in 4/5 (80%) patients with hyperparathyroidism secondary to hypocalcaemia, in all three patients with pseudohypoparathyroidism, in 5/17 (29%) patients with osteoporosis and in 1/9 (11%) patients with renal stones and/or hypercalciuria. Bioactive PTH correlated positively with immunoreactive PTH (iPTH) measured with a radioimmunoassay predominantly recognizing the middle- and carboxyl-terminal region of the PTH molecule (r = 0.503, P less than 0.001). A positive correlation (r = 0.572, P less than 0.05) was found between values of serum calcium and bioPTH in the group with primary hyperparathyroidism. Immunoreactive PTH did not correlate significantly with calcium in this group. In the other patients except those who had chronic renal failure, a negative correlation between serum calcium and both bioPTH and iPTH was observed (P less than 0.01). When alkaline phosphatase was compared with bioPTH in all patients, the correlation was positive (r = 0.390, P less than 0.01); no significant correlation existed between iPTH and alkaline phosphatase in the patients studied.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3944539

  1. Gustatory Habituation in "Drosophila" Relies on "Rutabaga" (Adenylate Cyclase)-Dependent Plasticity of GABAergic Inhibitory Neurons

    ERIC Educational Resources Information Center

    Paranjpe, Pushkar; Rodrigues, Veronica; VijayRaghavan, K.; Ramaswami, Mani

    2012-01-01

    In some situations, animals seem to ignore stimuli which in other contexts elicit a robust response. This attenuation in behavior, which enables animals to ignore a familiar, unreinforced stimulus, is called habituation. Despite the ubiquity of this phenomenon, it is generally poorly understood in terms of the underlying neural circuitry. Hungry…

  2. Identification of a D1 dopamine receptor, not linked to adenylate cyclase, on lactotroph cells.

    PubMed Central

    Schoors, D. F.; Vauquelin, G. P.; De Vos, H.; Smets, G.; Velkeniers, B.; Vanhaelst, L.; Dupont, A. G.

    1991-01-01

    1. We studied the lactotroph cells of the rat by both in vivo and in vitro pharmacological techniques for the presence of D1-receptors. Both approaches revealed the presence of D2-receptor, stimulated by quinpirole (resulting in an inhibition of prolactin secretion) and blocked by domperidone. 2. Administration of fenoldopam, the most selective D1-receptor agonist currently available, resulted in a dose-dependent decrease of prolactin secretion in vivo (after pretreatment with alpha-methyl-p-tyrosine) and in vitro (cultured pituitary cells). This increase was dose-dependently blocked by the selective D1-receptor antagonist, SCH 23390, and although the effect of fenoldopam was less than that obtained by D2-receptor stimulation, these data suggest that a D1-receptor also controls prolactin secretion. 3. In order to detect the location of these dopamine receptors, autoradiographic studies were performed by use of [3H]-SCH 23390 and [3H]-spiperone as markers for D1- and D2-receptors, respectively. Specific binding sites for [3H]-SCH 23390 were demonstrated. Fenoldopam dose-dependently reduced [3H]-SCH 23390 binding, but had no effect on [3H]-spiperone binding. Immunocytochemical labelling of prolactin cells after incubation with [3H]-SCH 23390 revealed that the granulae and hence, D1 binding sites were present on the lactotroph cells. 4. Radioligand binding studies performed on membranes from anterior pituitary cells revealed the presence of the D2-receptor (54 fmol mg-1 protein) with a Kd of 0.58 nM for [3H]-spiperone, but failed to detect D1-receptors. 5. Finally, we studied the effect of dopamine and of fenoldopam on the adenosine 3':5'-cyclic monophosphate (cyclic AMP) content of anterior pituitary cells.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 4 Figure 5 PMID:1833020

  3. Asymmetrically acting lycopene beta-cyclases (CrtLm) from non-photosynthetic bacteria.

    PubMed

    Tao, L; Picataggio, S; Rouvière, P E; Cheng, Q

    2004-03-01

    Carotenoids have important functions in photosynthesis, nutrition, and protection against oxidative damage. Some natural carotenoids are asymmetrical molecules that are difficult to produce chemically. Biological production of carotenoids using specific enzymes is a potential alternative to extraction from natural sources. Here we report the isolation of lycopene beta-cyclases that selectively cyclize only one end of lycopene or neurosporene. The crtLm genes encoding the asymmetrically acting lycopene beta-cyclases were isolated from non-photosynthetic bacteria that produced monocyclic carotenoids. Co-expression of these crtLm genes with the crtEIB genes from Pantoea stewartii (responsible for lycopene synthesis) resulted in the production of monocyclic gamma-carotene in Escherichia coli. The asymmetric cyclization activity of CrtLm could be inhibited by the lycopene beta-cyclase inhibitor 2-(4-chlorophenylthio)-triethylamine (CPTA). Phylogenetic analysis suggested that bacterial CrtL-type lycopene beta-cyclases might represent an evolutionary link between the common bacterial CrtY-type of lycopene beta-cyclases and plant lycopene beta- and epsilon-cyclases. These lycopene beta-cyclases may be used for efficient production of high-value asymmetrically cyclized carotenoids. PMID:14740205

  4. Endothelin-1, superoxide and adeninediphosphate ribose cyclase in shark vascular smooth muscle.

    PubMed

    Fellner, Susan K; Parker, Laurel

    2005-03-01

    In vascular smooth muscle (VSM) of Squalus acanthias, endothelin-1 (ET-1) signals via the ET(B) receptor. In both shark and mammalian VSM, ET-1 induces a rise in cytosolic Ca(2+) concentration ([Ca(2+)](i)) via activation of the inositol trisphosphate (IP(3)) receptor (IP(3)R) and subsequent release of Ca(2+) from the sarcoplasmic reticulum (SR). IP(3)R-mediated release of SR Ca(2+) causes calcium-induced calcium release (CICR) via the ryanodine receptor (RyR), which can be sensitized by cyclic adeninediphosphate ribose (cADPR). cADPR is synthesized from NAD(+) by a membrane-bound bifunctional enzyme, ADPR cyclase. We have previously shown that the antagonists of the RyR, Ruthenium Red, high concentrations of ryanodine and 8-Br cADPR, diminish the [Ca(2+)](i) response to ET-1 in shark VSM. To investigate how ET-1 might influence the activity of the ADPR cyclase, we employed inhibitors of the cyclase. To explore the possibility that ET-1-induced production of superoxide (O(2)*-) might activate the cyclase, we used an inhibitor of NAD(P)H oxidase (NOX), DPI and a scavenger of O(2)*-, TEMPOL. Anterior mesenteric artery VSM was loaded with fura-2AM to measure [Ca(2+)](i). In Ca(2+)-free shark Ringers, ET-1 increased [Ca(2+)](i) by 104+/-8 nmol l(-1). The VSM ADPR cyclase inhibitors, nicotinamide and Zn(2+), diminished the response by 62% and 72%, respectively. Both DPI and TEMPOL reduced the response by 63%. The combination of the IP(3)R antagonists, 2-APB or TMB-8, with DPI or TEMPOL further reduced the response by 83%. We show for the first time that in shark VSM, inhibition of the ADPR cyclase reduces the [Ca(2+)](i) response to ET-1 and that superoxide may be involved in the activation of the cyclase. PMID:15767306

  5. Upregulation of cAMP-specific PDE-4 activity following ligation of the TCR complex on thymocytes is blocked by selective inhibitors of protein kinase C and tyrosyl kinases.

    PubMed

    Michie, A M; Rena, G; Harnett, M M; Houslay, M D

    1998-01-01

    We have previously shown that the major cAMP phosphodiesterase (PDE) isoforms present in murine thymocytes are the cGMP-stimulated PDE activity (PDE-2) and the cAMP-specific PDE activity (PDE-4), and that these isoforms are differentially regulated following ligation of the TCR (Michie, A.M., Lobban, M. D., Mueller, T., Harnett, M. M., and Houslay, M.D. [1996] Cell. Signalling 8, 97-110). We show here that the anti-CD3-stimulated elevation in PDE-4 activity in murine thymocytes is dependent on protein tyrosine kinase and protein kinase C (PKC)-mediated signals as the TCR-coupled increase in PDE-4 activity can be abrogated by both the tyrosine kinase inhibitor, genistein, and the PKC selective inhibitors chelerythrine and staurosporine. Moreover, the PKC-activating phorbol ester, phorbol-12-myristate, 13-acetate (PMA) caused an increase in PDE-4 activity, similar to that observed in cells challenged with anti-CD3 monoclonal antibodies and which was not additive with cochallenge using anti-CD3 antibodies. Both the PMA- and the anti-CD3 antibody-mediated increases in PDE-4 activity were blocked by treatment with either cycloheximide or actinomycin D. Despite the upregulation of PDE-4 activity consequent to TCR ligation, intracellular cAMP levels increased on challenge of thymocytes with anti-CD3 antibody, indicating that adenylate cyclase activity was also increased by TCR ligation. It is suggested that the anti-CD3-mediated increase in PDE-4 activity was owing to a rapid PKC-dependent induction of PDE-4 activity following crosslinking of the TCR complex. This identifies "crosstalk" occurring between the PKA and PKC signaling pathways initiated by ligation of the antigen receptor in murine thymocytes. That both adenylate cyclase and PDE-4 activities were increased may indicate the presence of compartmentalized cAMP responses present in these cells. PMID:9515165

  6. Inhibitors

    MedlinePlus

    ... Community Counts Blood Safety Inhibitors Articles & Key Findings Free Materials Videos Starting the Conversation Playing it Safe A Look at Hemophilia Joint Range of Motion My Story Links to Other Websites ...

  7. Guanylyl cyclase structure, function and regulation

    PubMed Central

    Potter, Lincoln R.

    2016-01-01

    Nitric oxide, bicarbonate, natriuretic peptides (ANP, BNP and CNP), guanylins, uroguanylins and guanylyl cyclase activating proteins (GCAPs) activate a family of enzymes variously called guanyl, guanylyl or guanylate cyclases that catalyze the conversion of guanosine triphosphate to cyclic guanosine monophosphate (cGMP) and pyrophosphate. Intracellular cyclic GMP is a second messenger that modulates: platelet aggregation, neurotransmission, sexual arousal, gut peristalsis, blood pressure, long bone growth, intestinal fluid secretion, lipolysis, phototransduction, cardiac hypertrophy and oocyte maturation. This review briefly discusses the discovery of cGMP and guanylyl cyclases, then nitric oxide, nitric oxide synthase and soluble guanylyl cyclase are described in slightly greater detail. Finally, the structure, function, and regulation of the individual mammalian single membrane-spanning guanylyl cyclases GC-A, GC-B, GC-C, GC-D, GC-E, GC-F and GC-G are described in greatest detail as determined by biochemical, cell biological and gene-deletion studies. PMID:21914472

  8. Attenuation by phosphodiesterase inhibitors of lipopolysaccharide-induced thromboxane release and bronchoconstriction in rat lungs.

    PubMed

    Uhlig, S; Featherstone, R L; Held, H D; Nüsing, R; Schudt, C; Wendel, A

    1997-12-01

    Exposure of perfused rat lungs to lipopolysaccharides (LPS) causes induction of cyclooxygenase-2 followed by thromboxane (TX)-mediated bronchoconstriction (BC). Recently, phosphodiesterase (PDE) inhibitors have received much interest because they not only are bronchodilators but also can suppress release of proinflammatory mediators. In the present study, we investigated the effect of three different PDE inhibitors on TX release and BC in LPS-exposed perfused rat lungs. The PDE inhibitors used were motapizone (PDE III specific), rolipram (PDE IV specific), and zardaverine (mixed PDE III and IV specific). At 5 microM, a concentration at which all three compounds selectively block their respective PDE isoenzyme, rolipram (IC50 = 0.04 microM) and zardaverine (IC50 = 1.8 microM) largely attenuated the LPS-induced BC, whereas motapizone was almost ineffective (IC50 = 40 microM). In contrast to LPS, BC induced by the TX-mimetic U46619 was prevented with comparable strength by motapizone and rolipram. In LPS-treated lungs, the TX release was reduced to 50% of controls by rolipram and zardaverine but was unaltered in the presence of 5 microM motapizone. Increasing intracellular cAMP through perfusion of db-cAMP or forskolin (activates adenylate cyclase) also reduced TX release and BC. We conclude that PDE inhibitors act via elevation of intracellular cAMP. Although both PDE III and PDE IV inhibitors can relax airway smooth muscle, in the model of LPS-induced BC, PDE IV inhibitors are more effective because (in contrast to PDE III inhibitors) they also attenuate TX release. PMID:9400021

  9. Transmembrane adenylyl cyclase regulates amphibian sperm motility through Protein Kinase A activation

    PubMed Central

    O’Brien, Emma D.; Krapf, Darío; Cabada, Marcelo O.; Visconti, Pablo E.; Arranz, Silvia E.

    2014-01-01

    Sperm motility is essential for achieving fertilization. In animals with external fertilization as amphibians, spermatozoa are stored in a quiescent state in the testis. Spermiation to hypotonic fertilization media triggers activation of sperm motility. Bufo arenarum sperm are immotile in artificial seminal plasma (ASP) but acquire in situ flagellar beating upon dilution. In addition to the effect of low osmolarity on sperm motility activation, we report that diffusible factors of the egg jelly coat (EW) regulate motility patterns, switching from in situ to progressive movement. The signal transduction pathway involved in amphibian sperm motility activation is mostly unknown. In the present study, we show a correlation between motility activation triggered by low osmotic pressure and activation of protein kinase A (PKA). Moreover, this is the first study to present strong evidences that point toward a role of a transmembrane adenyl-cyclase (tmAC) in the regulation of amphibian sperm motility through PKA activation. PMID:21126515

  10. Adenylation of maternally inherited microRNAs by Wispy.

    PubMed

    Lee, Mihye; Choi, Yeon; Kim, Kijun; Jin, Hua; Lim, Jaechul; Nguyen, Tuan Anh; Yang, Jihye; Jeong, Minsun; Giraldez, Antonio J; Yang, Hui; Patel, Dinshaw J; Kim, V Narry

    2014-12-01

    Early development depends heavily on accurate control of maternally inherited mRNAs, and yet it remains unknown how maternal microRNAs are regulated during maternal-to-zygotic transition (MZT). We here find that maternal microRNAs are highly adenylated at their 3' ends in mature oocytes and early embryos. Maternal microRNA adenylation is widely conserved in fly, sea urchin, and mouse. We identify Wispy, a noncanonical poly(A) polymerase, as the enzyme responsible for microRNA adenylation in flies. Knockout of wispy abrogates adenylation and results in microRNA accumulation in eggs, whereas overexpression of Wispy increases adenylation and reduces microRNA levels in S2 cells. Wispy interacts with Ago1 through protein-protein interaction, which may allow the effective and selective adenylation of microRNAs. Thus, adenylation may contribute to the clearance of maternally deposited microRNAs during MZT. Our work provides mechanistic insights into the regulation of maternal microRNAs and illustrates the importance of RNA tailing in development. PMID:25454948

  11. Dipeptidyl peptidase inhibitors as new drugs for the treatment of type 2 diabetes.

    PubMed

    Mest, H-J; Mentlein, R

    2005-04-01

    Inhibitors of the regulatory protease dipeptidyl peptidase-IV (DPP-IV) are currently under development in preclinical and clinical studies (several pharmaceutical companies, now in Phase III) as potential drugs for the treatment of type 2 diabetes. Their development is based on the observation that DPP-IV rapidly inactivates the incretin hormone glucagon-like peptide-1 (GLP-1), which is released postprandially from the gut and increases insulin secretion. DPP-IV inhibitors stabilise endogenous GLP-1 at physiological concentrations, and induce insulin secretion in a glucose-dependent manner; therefore, they do not demonstrate any hypoglycaemic effects. Furthermore, they are orally bioavailable. In addition to their ability to protect GLP-1 against degradation, DPP-IV inhibitors also stabilise other incretins, including gastric inhibitory peptide and pituitary adenylate cyclase-activating peptide. They also reduce the antagonistic and desensitising effects of the fragments formed by truncation of the incretins. In clinical studies, when used for the treatment of diabetes over a 1-year period, DPP-IV inhibitors show improved efficacy over time. This finding can be explained by a GLP-1-induced increase in the number of beta cells. Potential risks associated with DPP-IV inhibitors include the prolongation of the action of other peptide hormones, neuropeptides and chemokines cleaved by the protease, and their interaction with DPP-IV-related proteases. Based on their mode of action, DPP-IV inhibitors seem to be of particular value in early forms of type 2 diabetes, either alone or in combination with other types of oral agents. PMID:15770466

  12. Differential effects of ceramides upon adenylyl cyclase subtypes.

    PubMed

    Bösel, A; Pfeuffer, T

    1998-01-30

    Ceramides are reported to stimulate different effector systems, among them atypical protein kinases C (PKCs). When HEK 293 cells, stably expressing adenylyl cyclase type II (AC II), were treated with various ceramide derivatives, adenylyl cyclase activity was enhanced 8-15-fold. The stimulation by the most potent analog, C18/C24 ceramide, was comparable to that by the phorbolester TPA. The stimulatory effect of ceramide was not restricted to AC II, although the type I and type V enzymes were affected less dramatically. Unexpectedly, the dihydro derivatives of ceramides, generally serving as non-activating controls, exhibited only slightly lower stimulation than ceramides, whereas short-chain ceramides (e.g. C2) were without effect. The action of ceramides was at least partially inhibited by okadaic acid, suggesting involvement of a phosphatase. Furthermore, ceramides and TPA operated synergistically. While the PKC inhibitor staurosporine counteracted the action of phorbol-esters, it significantly (2.5x) enhanced the effect of ceramides. PMID:9490008

  13. Affinity Purification Method for the Identification of Nonribosomal Peptide Biosynthetic Enzymes Using a Synthetic Probe for Adenylation Domains.

    PubMed

    Ishikawa, Fumihiro; Kakeya, Hideaki

    2016-01-01

    A series of inhibitors have been designed based on 5'-O-sulfamoyl adenosine (AMS) that display tight binding characteristics towards the inhibition of adenylation (A) domains in nonribosomal peptide synthetases (NRPSs). We recently developed an affinity probe for A domains that could be used to facilitate the specific isolation and identification of NRPS modules. Our synthetic probe, which is a biotinylated variant of L-Phe-AMS (L-Phe-AMS-biotin), selectively targets the A domains in NRPS modules that recognize and convert L-Phe to an aminoacyl adenylate in whole proteomes. In this chapter, we describe the design and synthesis of L-Phe-AMS-biotin and provide a summary of our work towards the development of a series of protocols for the specific enrichment of NRPS modules using this probe. PMID:26831701

  14. The Cyclase-Associated Protein Cap1 Is Important for Proper Regulation of Infection-Related Morphogenesis in Magnaporthe oryzae

    PubMed Central

    Zhou, Xiaoying; Zhang, Haifeng; Li, Guotian; Shaw, Brian; Xu, Jin-Rong

    2012-01-01

    Surface recognition and penetration are critical steps in the infection cycle of many plant pathogenic fungi. In Magnaporthe oryzae, cAMP signaling is involved in surface recognition and pathogenesis. Deletion of the MAC1 adenylate cyclase gene affected appressorium formation and plant infection. In this study, we used the affinity purification approach to identify proteins that are associated with Mac1 in vivo. One of the Mac1-interacting proteins is the adenylate cyclase-associated protein named Cap1. CAP genes are well-conserved in phytopathogenic fungi but none of them have been functionally characterized. Deletion of CAP1 blocked the effects of a dominant RAS2 allele and resulted in defects in invasive growth and a reduced intracellular cAMP level. The Δcap1 mutant was defective in germ tube growth, appressorium formation, and formation of typical blast lesions. Cap1-GFP had an actin-like localization pattern, localizing to the apical regions in vegetative hyphae, at the periphery of developing appressoria, and in circular structures at the base of mature appressoria. Interestingly, Cap1, similar to LifeAct, did not localize to the apical regions in invasive hyphae, suggesting that the apical actin cytoskeleton differs between vegetative and invasive hyphae. Domain deletion analysis indicated that the proline-rich region P2 but not the actin-binding domain (AB) of Cap1 was responsible for its subcellular localization. Nevertheless, the AB domain of Cap1 must be important for its function because CAP1ΔAB only partially rescued the Δcap1 mutant. Furthermore, exogenous cAMP induced the formation of appressorium-like structures in non-germinated conidia in CAP1ΔAB transformants. This novel observation suggested that AB domain deletion may result in overstimulation of appressorium formation by cAMP treatment. Overall, our results indicated that CAP1 is important for the activation of adenylate cyclase, appressorium morphogenesis, and plant infection in M

  15. Control of gastric acid secretion. Histamine H2-receptor antagonists and H+K(+)-ATPase inhibitors.

    PubMed

    Shamburek, R D; Schubert, M L

    1992-09-01

    Gastric acid secretion is regulated by an intricate interplay of neural (acetylcholine), hormonal (gastrin), and paracrine (histamine, somatostatin) mechanisms. Receptors for each of these agents and the signal transduction pathways to which these receptors are coupled have been identified on the parietal cell. The stimulatory effect of acetylcholine and gastrin is mediated by an increase in cytosolic calcium, whereas that of histamine is mediated by activation of adenylate cyclase and generation of cAMP. Strong potentiation between histamine and either gastrin or acetylcholine reflects postreceptor interaction between the distinct pathways as well as the ability of acetylcholine and gastrin to release histamine from mucosal ECL cells. The inhibitory effects of somatostatin on acid secretion are mediated by receptors coupled by guanine nucleotide-binding proteins to inhibition of adenylate cyclase activity. All the pathways converge on and modulate the activity of the luminal enzyme, H+K(+)-ATPase, the proton pump of the parietal cell. Precise information on the mechanisms involved in gastric acid secretion has led to the development of potent drugs capable of inhibiting acid secretion. These include competitive antagonists that interact with stimulatory receptors (e.g., histamine H2-receptor antagonists) as well as noncompetitive inhibitors of H+K(+)-ATPase (e.g., omeprazole). The histamine H2-receptor antagonists (cimetidine, ranitidine, famotidine, and nizatidine) continue as first-line therapy for peptic ulcer disease and are effective in preventing relapse. Although they are generally well tolerated, histamine H2-receptor antagonists may cause untoward CNS, cardiac, and endocrine effects as well as interference with the absorption, metabolism, and elimination of various drugs. Omeprazole is a weak base that reaches the parietal cell through the bloodstream, diffuses through the cytoplasm, and becomes activated and trapped as a sulfenamide in the acidic

  16. Sustained and Selective Suppression of Intestinal Cholesterol Synthesis by Ro 48-8071, an Inhibitor of 2,3-Oxidosqualene:Lanosterol Cyclase, in the BALB/c Mouse

    PubMed Central

    Chuang, Jen-Chieh; Valasek, Mark A.; Lopez, Adam M.; Posey, Kenneth S.; Repa, Joyce J.; Turley, Stephen D.

    2014-01-01

    The small intestine plays a fundamentally important role in regulating whole body cholesterol balance and plasma lipoprotein composition. This is articulated through the interplay of a constellation of genes that ultimately determines the net amount of chylomicron cholesterol delivered to the liver. Major advances in our insights into regulation of the cholesterol absorption pathway have been made using genetically manipulated mouse models and agents such as ezetimibe. One unresolved question is how a sustained pharmacological inhibition of intestinal cholesterol synthesis in vivo may affect cholesterol handling by the absorptive cells. Here we show that the lanosterolcyclase inhibitor, Ro 48-8071, when fed to BALB/c mice in a chow diet (20 mg/day/kg body weight), leads to a rapid and sustained inhibition (>50%) of cholesterol synthesis in the whole small intestine. Sterol synthesis was also reduced in the large intestine and stomach. In contrast, hepatic cholesterol synthesis, while markedly suppressed initially, rebounded to higher than baseline rates within 7 days. Whole body cholesterol synthesis, fractional cholesterol absorption, and fecal neutral and acidic sterol excretion were not consistently changed with Ro 48-8071 treatment. There were no discernible effects of this agent on intestinal histology as determined by H&E staining and the level of Ki67, an index of proliferation. The mRNA expression for multiple genes involved in intestinal cholesterol regulation including NPC1L1 was mostly unchanged although there was a marked rise in the mRNA level for the PXR target genes CYP3A11 and CES2A. PMID:24486573

  17. Structure of the adenylation domain of NAD[superscript +]-dependent DNA ligase from Staphylococcus aureus

    SciTech Connect

    Han, Seungil; Chang, Jeanne S.; Griffor, Matt; Pfizer

    2010-09-17

    DNA ligase catalyzes phosphodiester-bond formation between immediately adjacent 5'-phosphate and 3''-hydroxyl groups in double-stranded DNA and plays a central role in many cellular and biochemical processes, including DNA replication, repair and recombination. Bacterial NAD{sup +}-dependent DNA ligases have been extensively characterized as potential antibacterial targets because of their essentiality and their structural distinction from human ATP-dependent DNA ligases. The high-resolution structure of the adenylation domain of Staphylococcus aureus NAD{sup +}-dependent DNA ligase establishes the conserved domain architecture with other bacterial adenylation domains. Two apo crystal structures revealed that the active site possesses the preformed NAD{sup +}-binding pocket and the 'C2 tunnel' lined with hydrophobic residues: Leu80, Phe224, Leu287, Phe295 and Trp302. The C2 tunnel is unique to bacterial DNA ligases and the Leu80 side chain at the mouth of the tunnel points inside the tunnel and forms a narrow funnel in the S. aureus DNA ligase structure. Taken together with other DNA ligase structures, the S. aureus DNA ligase structure provides a basis for a more integrated understanding of substrate recognition and catalysis and will be also be of help in the development of small-molecule inhibitors.

  18. Regulation of Plant Acetyl-CoA Carboxylase by Adenylate Nucleotides 1

    PubMed Central

    Eastwell, Kenneth C.; Stumpf, Paul K.

    1983-01-01

    The assay of acetyl-CoA carboxylase (EC 6.4.1.2) does not follow ideal zero-order kinetics when assayed in a crude extract from wheat (Triticum aestivum L.) germ. Our results show that the lack of ideality is the consequence of contamination by ATPase and adenylate kinase. These enzyme activities generate significant amounts of ADP and AMP in the assay mixture, thus limiting the availability of ATP for the carboxylase reaction. Moreover, ADP and AMP are competitive inhibitors, with respect to ATP, of acetyl-CoA carboxylase. Similar relationships between adenylate nucleotides and acetyl-CoA carboxylase are found in isolated chloroplasts. There is no evidence that acetyl-CoA carboxylase activity in the extracts of the plant systems examined is altered by covalent modification, such as a phosphorylation-dephosphorylation cycle. A scheme is presented that illustrates the dependency of acetyl-CoA carboxylase and fatty acid synthesis on the energy demands of the chloroplasts in vivo. PMID:16662980

  19. Pituitary adenylate cyclase-activating polypeptide (PACAP): a regulator of the innate and acquired immune functions in juvenile fish.

    PubMed

    Lugo, Juana Maria; Carpio, Yamila; Oliva, Aymé; Morales, Antonio; Estrada, Mario Pablo

    2010-09-01

    To date, published in-vivo studies on the action of the PACAP in fish are few and these are concerned with reproduction, brain development and feeding behavior. Recently, we demonstrated for the first time that PACAP, apart from its neuroendocrine role, influences immune functions in fish larvae. In this work, we have evaluated the effects of recombinant Clarias gariepinus PACAP administration by intraperitoneal injection on important immune parameters in juvenile fish. We observed that a single injection of the recombinant peptide (0.1 microg per g of body weight) was able to increase the nitric oxide synthase-derived metabolites (NOS) and total immunoglobulin M (IgM) concentration in serum of juvenile catfish C. gariepinus and tilapia Orechromis niloticus respectively, after 24 h of its administration. In addition, our results showed that recombinant PACAP increases IgM, NOS and lysozyme in serum correlated with its ability to enhance growth performance in juvenile fish. Finally, the PACAP mRNA expression and PACAP immunoreactivity detected in peripheral blood leucocytes from juvenile catfish suggest a direct autocrine or/and paracrine mechanism of regulation of this peptide to mediate immune functions in fish. PMID:20510368

  20. Mutations That Affect Transcription and Cyclic Amp-Crp Regulation of the Adenylate Cyclase Gene (Cya) of Salmonella Typhimurium

    PubMed Central

    Fandl, J. P.; Thorner, L. K.; Artz, S. W.

    1990-01-01

    We studied the expression of the cya promoter(s) in cya-lac fusion strains of Salmonella typhimurium and demonstrated cAMP receptor protein (CRP)-dependent repression by cAMP. Expression of cya was reduced about fourfold in cultures grown in acetate minimal medium as compared to cultures grown in glucose-6-phosphate minimal medium. Expression of cya was also reduced about fourfold by addition of 5 mM cAMP to cultures grown in glucose minimal medium. We constructed in vitro deletion and insertion mutations altering a major cya promoter (P2) and a putative CRP binding site overlapping P2. These mutations were recombined into the chromosome by allele replacement with M13mp::cya recombinant phages and the regulation of the mutant promoters was analyzed. A 4-bp deletion of the CRP binding site and a 4-bp insertion in this site nearly eliminated repression by cAMP. A mutant with the P2 promoter and the CRP binding site both deleted exhibited an 80% reduction in cya expression; the 20% residual expression was insensitive to cAMP repression. This mutant retained a Cya(+) phenotype. Taken together, the results establish that the cya gene is transcribed from multiple promoters one of which, P2, is negatively regulated by the cAMP-CRP complex. Correction for the contribution to transcription by the cAMP-CRP nonregulated cya promoters indicates that the P2 promoter is repressed at least eightfold by cAMP-CRP. PMID:2168849

  1. Correlation between Activity and Domain Complementation in Adenylyl Cyclase Demonstrated with a Novel Fluorescence Resonance Energy Transfer Sensor.

    PubMed

    Ritt, Michael; Sivaramakrishnan, Sivaraj

    2016-04-01

    Adenylyl cyclase (AC) activity relies on multiple effectors acting through distinct binding sites. Crystal structures have revealed the location of these sites, and biochemical studies have explored the kinetics of ACs, but the interplay between conformation and activity remains incompletely understood. Here, we describe a novel fluorescence resonance energy transfer (FRET) sensor that functions both as a soluble cyclase and a reporter of complementation within the catalytic domain. We report a strong linear correlation between catalytic domain complementation and cyclase activity upon stimulation with forskolin and Gαs. Exploiting this, we dissect the mechanism of action of a series of forskolin analogs and a P-site inhibitor, 2'-d3'-AMP. Finally, we demonstrate that this sensor is functional in live cells, wherein it reports forskolin-stimulated activity of AC. PMID:26801393

  2. The isoenzyme of glutaminyl cyclase is an important regulator of monocyte infiltration under inflammatory conditions

    PubMed Central

    Cynis, Holger; Hoffmann, Torsten; Friedrich, Daniel; Kehlen, Astrid; Gans, Kathrin; Kleinschmidt, Martin; Rahfeld, Jens-Ulrich; Wolf, Raik; Wermann, Michael; Stephan, Anett; Haegele, Monique; Sedlmeier, Reinhard; Graubner, Sigrid; Jagla, Wolfgang; Müller, Anke; Eichentopf, Rico; Heiser, Ulrich; Seifert, Franziska; Quax, Paul H A; de Vries, Margreet R; Hesse, Isabel; Trautwein, Daniela; Wollert, Ulrich; Berg, Sabine; Freyse, Ernst-Joachim; Schilling, Stephan; Demuth, Hans-Ulrich

    2011-01-01

    Acute and chronic inflammatory disorders are characterized by detrimental cytokine and chemokine expression. Frequently, the chemotactic activity of cytokines depends on a modified N-terminus of the polypeptide. Among those, the N-terminus of monocyte chemoattractant protein 1 (CCL2 and MCP-1) is modified to a pyroglutamate (pE-) residue protecting against degradation in vivo. Here, we show that the N-terminal pE-formation depends on glutaminyl cyclase activity. The pE-residue increases stability against N-terminal degradation by aminopeptidases and improves receptor activation and signal transduction in vitro. Genetic ablation of the glutaminyl cyclase iso-enzymes QC (QPCT) or isoQC (QPCTL) revealed a major role of isoQC for pE1-CCL2 formation and monocyte infiltration. Consistently, administration of QC-inhibitors in inflammatory models, such as thioglycollate-induced peritonitis reduced monocyte infiltration. The pharmacologic efficacy of QC/isoQC-inhibition was assessed in accelerated atherosclerosis in ApoE3*Leiden mice, showing attenuated atherosclerotic pathology following chronic oral treatment. Current strategies targeting CCL2 are mainly based on antibodies or spiegelmers. The application of small, orally available inhibitors of glutaminyl cyclases represents an alternative therapeutic strategy to treat CCL2-driven disorders such as atherosclerosis/restenosis and fibrosis. PMID:21774078

  3. A cAMP Biosensor-Based High-Throughput Screening Assay for Identification of Gs-Coupled GPCR Ligands and Phosphodiesterase Inhibitors.

    PubMed

    Vedel, Line; Bräuner-Osborne, Hans; Mathiesen, Jesper Mosolff

    2015-08-01

    Cyclic adenosine 3',5'-monophosphate (cAMP) is an important second messenger, and quantification of intracellular cAMP levels is essential in studies of G protein-coupled receptors (GPCRs). The intracellular cAMP levels are regulated by the adenylate cyclase (AC) upon activation of either Gs- or Gi-coupled GPCRs, which leads to increased or decreased cAMP levels, respectively. Here we describe a real-time Förster resonance energy transfer (FRET)-based cAMP high-throughput screening (HTS) assay for identification and characterization of Gs-coupled GPCR ligands and phosphodiesterase (PDE) inhibitors in living cells. We used the β2-adrenergic receptor (β(2)AR) as a representative Gs-coupled receptor and characterized two cell lines with different expression levels. Low receptor expression allowed detection of desensitization kinetics and delineation of partial agonism, whereas high receptor expression resulted in prolonged signaling and enabled detection of weak partial agonists and/or ligands with low potency, which is highly advantageous in large HTS settings and hit identification. In addition, the assay enabled detection of β(2)AR inverse agonists and PDE inhibitors. High signal-to-noise ratios were also observed for the other representative Gs-coupled GPCRs tested, GLP-1R and GlucagonR. The FRET-based cAMP biosensor assay is robust, reproducible, and inexpensive with good Z factors and is highly applicable for HTS. PMID:25851033

  4. Energetics and Structural Characterization of the large-scale Functional Motion of Adenylate Kinase

    NASA Astrophysics Data System (ADS)

    Formoso, Elena; Limongelli, Vittorio; Parrinello, Michele

    2015-02-01

    Adenylate Kinase (AK) is a signal transducing protein that regulates cellular energy homeostasis balancing between different conformations. An alteration of its activity can lead to severe pathologies such as heart failure, cancer and neurodegenerative diseases. A comprehensive elucidation of the large-scale conformational motions that rule the functional mechanism of this enzyme is of great value to guide rationally the development of new medications. Here using a metadynamics-based computational protocol we elucidate the thermodynamics and structural properties underlying the AK functional transitions. The free energy estimation of the conformational motions of the enzyme allows characterizing the sequence of events that regulate its action. We reveal the atomistic details of the most relevant enzyme states, identifying residues such as Arg119 and Lys13, which play a key role during the conformational transitions and represent druggable spots to design enzyme inhibitors. Our study offers tools that open new areas of investigation on large-scale motion in proteins.

  5. Diterpene Cyclases and the Nature of the Isoprene Fold

    PubMed Central

    Cao, Rong; Zhang, Yonghui; Mann, Francis M.; Huang, Cancan; Mukkamala, Dushyant; Hudock, Michael P.; Mead, Matthew; Prisic, Sladjana; Wang, Ke; Lin, Fu-Yang; Chang, Ting-Kai; Peters, Reuben; Oldfield, Eric

    2013-01-01

    The structures and mechanism of action of many terpene cyclases are known, but there are no structures of diterpene cyclases. Here, we propose structural models based on bioinformatics, site-directed mutagenesis, domain swapping, enzyme inhibition and spectroscopy that help explain the nature of diterpene cyclase structure, function, and evolution. Bacterial diterpene cyclases contain ∼20 α-helices and the same conserved “QW” and DxDD motifs as in triterpene cyclases, indicating the presence of a βγ barrel structure. Plant diterpene cyclases have a similar catalytic motif and βγ-domain structure together with a third, α-domain, forming an αβγ structure, and in H+-initiated cyclases, there is an EDxxD-like Mg2+/diphosphate binding motif located in the γ-domain. The results support a new view of terpene cyclase structure and function and suggest evolution from ancient (βγ) bacterial triterpene cyclases to (βγ) bacterial and thence to (αβγ) plant diterpene cyclases. PMID:20602361

  6. Structure, signaling mechanism and regulation of natriuretic peptide receptor-guanylate cyclase

    PubMed Central

    Misono, Kunio S.; Philo, John S.; Arakawa, Tsutomu; Ogata, Craig M.; Qiu, Yue; Ogawa, Haruo; Young, Howard S.

    2011-01-01

    Summary Atrial natriuretic peptide (ANP) and homologous B-type natriuretic peptide (BNP) are cardiac hormones that dilate blood vessels and stimulate natriuresis and diuresis, thereby lowering blood pressure and blood volume. ANP and BNP counterbalance the actions of the renin-angiotensin-aldosterone and neurohormonal systems, and play a central role in cardiovascular regulation. These activities are mediated by the A-type natriuretic peptide receptor (NPRA), a single transmembrane segment, guanylate cyclase (GC) linked receptor that occurs as a homodimer. Here we present an overview of the structure, possible chloride-mediated regulation, and signaling mechanism of the NPRA and other receptor-GCs. Earlier, we determined the crystal structures of the NPRA extracellular domain with and without bound ANP. Their structural comparison has revealed a novel ANP-induced rotation mechanism occurring in the juxtamembrane region that apparently triggers transmembrane signal transduction. More recently, the crystal structures of the dimerized catalytic domain of green algae GC Cyg12 and that of cyanobacter GC Cya2 have been reported. These structures closely resemble that of the adenylate cyclase catalytic domain consisting of C1 and C2 subdomain heterodimer. AC is activated by binding of Gsα to C2 and ensuing 7° rotation of C1 around an axis parallel to the central cleft, thereby inducing the heterodimer into a catalytically active conformation. We speculate that, in the NPRA, the ANP-induced rotation of the juxtamembrane domains, transmitted across the transmembrane helices, may induce a similar rotation in each of the dimerized GC catalytic domains, leading to the stimulation of the GC catalytic activity. PMID:21375693

  7. Soluble Adenylyl Cyclase in Health and Disease

    PubMed Central

    Schmid, Andreas; Meili, Dimirela; Salathe, Matthias

    2014-01-01

    The second messenger cAMP is integral for many physiological processes. Soluble adenylyl cyclase (sAC) was recently identified as a widely expressed intracellular source of cAMP in mammalian cells. sAC is evolutionary, structurally, and biochemically distinct from the G-protein-responsive transmembranous adenylyl cyclases (tmAC). The structure of the catalytic unit of sAC is similar to tmAC, but sAC does not contain transmembranous domains, allowing localizations independent of the membranous compartment. sAC activity is stimulated by HCO3-, Ca2+ and is sensitive to physiologically relevant ATP fluctuations. sAC functions as a physiological sensor for carbon dioxide and bicarbonate, and therefore indirectly for pH. Here we review the physiological role of sAC in different human tissues with a major focus on the lung. PMID:25064591

  8. Membrane guanylyl cyclase receptors: an update

    PubMed Central

    Garbers, David L.; Chrisman, Ted D.; Wiegn, Phi; Katafuchi, Takeshi; Albanesi, Joseph P.; Bielinski, Vincent; Barylko, Barbara; Redfield, Margaret M.; Burnett, John C.

    2007-01-01

    Recent studies have demonstrated key roles for several membrane guanylyl cyclase receptors in the regulation of cell hyperplasia, hypertrophy, migration and extracellular matrix production, all of which having an impact on clinically relevant diseases, including tissue remodeling after injury. Additionally, cell differentiation, and even tumor progression, can be profoundly influenced by one or more of these receptors. Some of these receptors also mediate important communication between the heart and intestine, and the kidney to regulate blood volume and Na+ balance. PMID:16815030

  9. An old friend with new skills: Imiquimod as novel inhibitor of Hedgehog signaling in basal cell carcinoma

    PubMed Central

    Gruber, Wolfgang; Frischauf, Anna-Maria; Aberger, Fritz

    2014-01-01

    Deregulated Hedgehog (HH)/GLI signaling plays an etiologic role in the initiation, progression and maintenance of many cancers. Small molecule targeting of HH signaling by inhibiting the essential pathway effector Smoothened (SMO) has proven exceptionally efficient for the treatment of advanced and metastatic basal cell carcinoma. That said, severe side effects, limited response rates, SMO-independent GLI signaling and rapid development of drug resistance limit the therapeutic success of SMO antagonists, urgently calling for the identification of alternative and additional strategies repressing oncogenic HH signaling. In this perspective article we highlight recent findings showing that the Toll-like receptor-7/8 (TLR7/8) agonist imiquimod (IMQ), an immune modulator approved for the treatment of basal cell carcinoma, can also act as a potent cell autonomous inhibitor of oncogenic HH signaling. Surprisingly, IMQ reduces HH signal strength independent of TLR signaling, via adenosine receptor (ADORA)/Adenylate cyclase (AC)/Protein kinase A (PKA) activation. We here highlight the molecular mechanisms of IMQ-mediated repression of HH/GLI and discuss the possible benefits as well as challenges of using ADORA agonists for the treatment of HH-associated cancer. PMID:25594066

  10. Soluble adenylyl cyclase is not required for axon guidance to netrin-1.

    PubMed

    Moore, Simon W; Lai Wing Sun, Karen; Xie, Fang; Barker, Philip A; Conti, Marco; Kennedy, Timothy E

    2008-04-01

    During development, axons are directed to their targets by extracellular guidance cues. The axonal response to the guidance cue netrin-1 is profoundly influenced by the concentration of cAMP within the growth cone. In some cases, cAMP affects the sensitivity of the growth cone to netrin-1, whereas in others it changes the response to netrin-1 from attraction to repulsion. The effects of cAMP on netrin-1 action are well accepted, but the critical issue of whether cAMP production is activated by a netrin-1 induced signaling cascade remains uncertain. A previous report has suggested that axon guidance in response to netrin-1 requires cAMP production mediated by soluble adenyl cyclase (sAC). We have used genetic, molecular and biochemical strategies to assess this issue. Surprisingly, we found only extremely weak expression of sAC in embryonic neurons and determined that, under conditions where netrin-1 directs axonal pathfinding, exposure to netrin-1 does not alter cAMP levels. Furthermore, although netrin-1-deficient mice exhibit major axon guidance defects, we show that pathfinding is normal in sAC-null mice. Therefore, although cAMP can alter the response of axons to netrin-1, we conclude that netrin-1 does not alter cAMP levels in axons attracted by this cue, and that sAC is not required for axon attraction to netrin-1. PMID:18400890

  11. Guanylyl cyclase and cGMP-specific phosphodiesterase participate in the acrosome reaction of starfish sperm.

    PubMed

    Kawase, Osamu; Ueno, Seiichi; Minakata, Hiroyuki; Hoshi, Motonori; Matsumoto, Midori

    2004-11-01

    In the starfish, Asterias amurensis, the cooperation of three components of the egg jelly, i.e. ARIS (acrosome reaction-inducing substance), Co-ARIS and asterosap, is responsible for inducing the acrosome reaction. Experimentally, ARIS and asterosap are sufficient for the induction. However, when sperm are treated only with asterosap, they become unresponsive to the egg jelly to undergo the reaction. In this study, we analysed the mechanism of the acrosome reaction, using sperm inactivation by asterosap as a clue. Asterosap causes a rapid and transient increase in intracellular cGMP through the activation of the asterosap receptor, a guanylyl cyclase, and causes an increase in intracellular Ca(2+). When sperm were pretreated with asterosap, the guanylyl cyclase seemed to be inactivated irreversibly by dephosphorylation. They were still responsive to ARIS but no longer to asterosap. However, in the presence of IBMX or zaprinast, inhibitors against phosphodiesterases (PDEs), they retained their capacity to undergo the acrosome reaction in response to the egg jelly or ARIS alone. IBMX and zaprinast suppressed the intracellular catabolism of cGMP, but not of cAMP. These results suggest that guanylyl cyclase and cGMP-specific, IBMX- and zaprinast-susceptible PDEs are involved in the regulation of the acrosome reaction. PMID:15751545

  12. H2S induces vasoconstriction of rat cerebral arteries via cAMP/adenylyl cyclase pathway.

    PubMed

    Li, Sen; Ping, Na-Na; Cao, Lei; Mi, Yan-Ni; Cao, Yong-Xiao

    2015-12-15

    Hydrogen sulfide (H2S), traditionally known for its toxic effects, is now involved in regulating vascular tone. Here we investigated the vasoconstrictive effect of H2S on cerebral artery and the underlying mechanism. Sodium hydrosulfide (NaHS), a donor of H2S, concentration-dependently induced vasoconstriction on basilar artery, which was enhanced in the presence of isoprenaline, a β-adrenoceptor agonist or forskolin, an adenylyl cyclase activator. Administration of NaHS attenuated the vasorelaxant effects of isoprenaline or forskolin. Meanwhile, the NaHS-induced vasoconstriction was diminished in the presence of 8B-cAMP, an analog of cAMP, but was not affected by Bay K-8644, a selective L-type Ca(2+) channel agonist. These results could be explained by the revised effects of NaHS on isoprenaline-induced cAMP elevation and forskolin-stimulated adenylyl cyclase activity. Additionally, NaHS-induced vasoconstriction was enhanced by removing the endothelium or in the presence of L-NAME, an inhibitor of nitric oxide synthase. L-NAME only partially attenuated the effect of NaHS which was given together with forskolin on the pre-contracted artery. In conclusion, H2S induces vasoconstriction of cerebral artery via, at least in part, cAMP/adenylyl cyclase pathway. PMID:26524654

  13. The energy landscape of adenylate kinase during catalysis

    PubMed Central

    Kerns, S. Jordan; Agafonov, Roman V.; Cho, Young-Jin; Pontiggia, Francesco; Otten, Renee; Pachov, Dimitar V.; Kutter, Steffen; Phung, Lien A.; Murphy, Padraig N.; Thai, Vu; Alber, Tom; Hagan, Michael F.; Kern, Dorothee

    2014-01-01

    Kinases perform phosphoryl-transfer reactions in milliseconds; without enzymes, these reactions would take about 8000 years under physiological conditions. Despite extensive studies, a comprehensive understanding of kinase energy landscapes, including both chemical and conformational steps, is lacking. Here we scrutinize the microscopic steps in the catalytic cycle of adenylate kinase, through a combination of NMR measurements during catalysis, pre-steady-state kinetics, MD simulations, and crystallography of active complexes. We find that the Mg2+ cofactor activates two distinct molecular events, phosphoryl transfer (>105-fold) and lid-opening (103-fold). In contrast, mutation of an essential active-site arginine decelerates phosphoryl transfer 103-fold without substantially affecting lid-opening. Our results highlight the importance of the entire energy landscape in catalysis and suggest that adenylate kinases have evolved to activate key processes simultaneously by precise placement of a single, charged and very abundant cofactor in a pre-organized active site. PMID:25580578

  14. Aminoacyl transfer from an adenylate anhydride to polyribonucleotides

    NASA Technical Reports Server (NTRS)

    Weber, A. L.; Lacey, J. C., Jr.

    1975-01-01

    Imidazole catalysis of phenylalanyl transfer from phenylalanine adenylate to hydroxyl groups of homopolyribonucleotides is studied as a possible chemical model of biochemical aminoacylation of transfer RNA (tRNA). The effect of pH on imidazole-catalyzed transfer of phenylalanyl residues to poly(U) and poly(A) double helix strands, the number of peptide linkages and their lability to base and neutral hydroxylamine, and the nature of adenylate condensation products are investigated. The chemical model entertained exhibits a constraint by not acylating the hydroxyl groups of polyribonucleotides in a double helix. The constraint is consistent with selective biochemical aminoacylation at the tRNA terminus. Interest in imidazole as a model of histidine residue in protoenzymes participating in prebiotic aminoacyl transfer to polyribonucleotides, and in rendering the tRNA a more efficient adaptor, is indicated.

  15. Subcellular localization of adenylate kinases in Plasmodium falciparum.

    PubMed

    Ma, Jipeng; Rahlfs, Stefan; Jortzik, Esther; Schirmer, R Heiner; Przyborski, Jude M; Becker, Katja

    2012-09-21

    Adenylate kinases (AK) play a key role in nucleotide signaling processes and energy metabolism by catalyzing the reversible conversion of ATP and AMP to 2 ADP. In the malaria parasite Plasmodium falciparum this reaction is mediated by AK1, AK2, and a GTP:AMP phosphotransferase (GAK). Here, we describe two additional adenylate kinase-like proteins: PfAKLP1, which is homologous to human AK6, and PfAKLP2. Using GFP-fusion proteins and life cell imaging, we demonstrate a cytosolic localization for PfAK1, PfAKLP1, and PfAKLP2, whereas PfGAK is located in the mitochondrion. PfAK2 is located at the parasitophorous vacuole membrane, and this localization is driven by N-myristoylation. PMID:22819813

  16. Comparative analysis of plant lycopene cyclases.

    PubMed

    Koc, Ibrahim; Filiz, Ertugrul; Tombuloglu, Huseyin

    2015-10-01

    Carotenoids are essential isoprenoid pigments produced by plants, algae, fungi and bacteria. Lycopene cyclase (LYC) commonly cyclize carotenoids, which is an important branching step in the carotenogenesis, at one or both end of the backbone. Plants have two types of LYC (β-LCY and ϵ-LCY). In this study, plant LYCs were analyzed. Based on domain analysis, all LYCs accommodate lycopene cyclase domain (Pf05834). Furthermore, motif analysis indicated that motifs were conserved among the plants. On the basis of phylogenetic analysis, β-LCYs and ϵ-LCYs were classified in β and ϵ groups. Monocot and dicot plants separated from each other in the phylogenetic tree. Subsequently, Oryza sativa Japonica Group and Zea mays of LYCs as monocot plants and Vitis vinifera and Solanum lycopersicum of LYCs as dicot plants were analyzed. According to nucleotide diversity analysis of β-LCY and ϵ-LCY genes, nucleotide diversities were found to be π: 0.30 and π: 0.25, respectively. The result highlighted β-LCY genes showed higher nucleotide diversity than ϵ-LCY genes. LYCs interacting genes and their co-expression partners were also predicted using String server. The obtained data suggested the importance of LYCs in carotenoid metabolism. 3D modeling revealed that depicted structures were similar in O. sativa, Z mays, S. lycopersicum, and V. vinifera β-LCYs and ϵ-LCYs. Likewise, the predicted binding sites were highly similar between O. sativa, Z mays, S. lycopersicum, and V. vinifera LCYs. Most importantly, analysis elucidated the V/IXGXGXXGXXXA motif for both type of LYC (β-LCY and ϵ-LCY). This motif related to Rossmann fold domain and probably provides a flat platform for binding of FAD in O. sativa, Z mays, S. lycopersicum, and V. vinifera β-LCYs and ϵ-LCYs with conserved structure. In addition to lycopene cyclase domain, the V/IXGXGXXGXXXA motif can be used for exploring LYCs proteins and to annotate the function of unknown proteins containing lycopene cyclase

  17. Adenylyl cyclases in the digestive system

    PubMed Central

    Sabbatini, Maria Eugenia; Gorelick, Fred; Glaser, Shannon

    2015-01-01

    Adenylyl cyclases (ACs) are a group of widely distributed enzymes whose functions are very diverse. There are nine known transmembrane AC isoforms activated by Gαs. Each has its own pattern of expression in the digestive system and differential regulation of function by Ca2+ and other intracellular signals. In addition to the transmembrane isoforms, one AC is soluble and exhibits distinct regulation. In this review, the basic structure, regulation and physiological roles of ACs in the digestive system are discussed. PMID:24521753

  18. Adenylyl cyclases in the digestive system.

    PubMed

    Sabbatini, Maria Eugenia; Gorelick, Fred; Glaser, Shannon

    2014-06-01

    Adenylyl cyclases (ACs) are a group of widely distributed enzymes whose functions are very diverse. There are nine known transmembrane AC isoforms activated by Gαs. Each has its own pattern of expression in the digestive system and differential regulation of function by Ca(2+) and other intracellular signals. In addition to the transmembrane isoforms, one AC is soluble and exhibits distinct regulation. In this review, the basic structure, regulation and physiological roles of ACs in the digestive system are discussed. PMID:24521753

  19. Adenylate kinase complements nucleoside diphosphate kinase deficiency in nucleotide metabolism.

    PubMed Central

    Lu, Q; Inouye, M

    1996-01-01

    Nucleoside diphosphate (NDP) kinase is a ubiquitous nonspecific enzyme that evidently is designed to catalyze in vivo ATP-dependent synthesis of ribo- and deoxyribonucleoside triphosphates from the corresponding diphosphates. Because Escherichia coli contains only one copy of ndk, the structural gene for this enzyme, we were surprised to find that ndk disruption yields bacteria that are still viable. These mutant cells contain a protein with a small amount NDP kinase activity. The protein responsible for this activity was purified and identified as adenylate kinase. This enzyme, also called myokinase, catalyzes the reversible ATP-dependent synthesis of ADP from AMP. We found that this enzyme from E. coli as well as from higher eukaryotes has a broad substrate specificity displaying dual enzymatic functions. Among the nucleoside monophosphate kinases tested, only adenylate kinase was found to have NDP kinase activity. To our knowledge, this is the first report of NDP kinase activity associated with adenylate kinase. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8650159

  20. Inhibition of monoterpene cyclases by inert analogues of geranyl diphosphate and linalyl diphosphate☆

    PubMed Central

    Karp, Frank; Zhao, Yuxin; Santhamma, Bindu; Assink, Bryce; Coates, Robert M.; Croteau, Rodney B.

    2007-01-01

    The tightly coupled nature of the reaction sequence catalyzed by monoterpene synthases has prevented direct observation of the topologically required isomerization step leading from geranyl diphosphate to the enzyme-bound, tertiary allylic intermediate linalyl diphosphate, which then cyclizes to the various monoterpene skeletons. X-ray crystal structures of these enzymes complexed with suitable analogues of the substrate and intermediate could provide a clearer view of this universal, but cryptic, step of monoterpenoid cyclase catalysis. Toward this end, the functionally inert analogues 2-fluorogeranyl diphosphate, (±)-2-fluorolinalyl diphosphate, and (3R)- and (3S)-homolinalyl diphosphates (2,6-dimethyl-2-vinyl-5-heptenyl diphosphates) were prepared, and compared to the previously described substrate analogue 3-azageranyl diphosphate (3-aza-2,3-dihydrogeranyl diphosphate) as inhibitors and potential crystallization aids with two representative monoterpenoid cyclases, (−)-limonene synthase and (+)-bornyl diphosphate synthase. Although these enantioselective synthases readily distinguished between (3R)- and (3S)-homolinalyl diphosphates, both of which were more effective inhibitors than was 3-azageranyl diphosphate, the fluorinated analogues proved to be the most potent competitive inhibitors and have recently yielded informative liganded structures with limonene synthase. PMID:17949678

  1. Cooperative substrate binding by a diguanylate cyclase.

    PubMed

    Oliveira, Maycon C; Teixeira, Raphael D; Andrade, Maxuel O; Pinheiro, Glaucia M S; Ramos, Carlos H I; Farah, Chuck S

    2015-01-30

    XAC0610, from Xanthomonas citri subsp. citri, is a large multi-domain protein containing one GAF (cGMP-specific phosphodiesterases, adenylyl cyclases and FhlA) domain, four PAS (Per-Arnt-Sim) domains and one GGDEF domain. This protein has a demonstrable in vivo and in vitro diguanylate cyclase (DGC) activity that leads to the production of cyclic di-GMP (c-di-GMP), a ubiquitous bacterial signaling molecule. Analysis of a XacΔ0610 knockout strain revealed that XAC0610 plays a role in the regulation of Xac motility and resistance to H2O2. Site-directed mutagenesis of a conserved DGC lysine residue (Lys759 in XAC0610) resulted in a severe reduction in XAC0610 DGC activity. Furthermore, experimental and in silico analyses suggest that XAC0610 is not subject to allosteric product inhibition, a common regulatory mechanism for DGC activity control. Instead, steady-state kinetics of XAC0610 DGC activity revealed a positive cooperative effect of the GTP substrate with a dissociation constant for the binding of the first GTP molecule (K1) approximately 5× greater than the dissociation constant for the binding of the second GTP molecule (K2). We present a general kinetics scheme that should be used when analyzing DGC kinetics data and propose that cooperative GTP binding could be a common, though up to now overlooked, feature of these enzymes that may in some cases offer a physiologically relevant mechanism for regulation of DGC activity in vivo. PMID:25463434

  2. Coordinated changes of adenylate energy charge and ATP/ADP: use in ecotoxicological studies.

    PubMed

    Thébault, M T; Raffin, J P; Picado, A M; Mendonça, E; Skorkowski, E F; Le Gal, Y

    2000-05-01

    The coordinated variations of the adenylate energy charge and ATP/ADP ratio were modeled and a function that depends on the numerical value of the adenylate kinase-catalyzed reaction has been derived. The model allows sensitive detection of the effects of xenobiotics on adenylate kinase and its cellular environment and offers a robust estimation of the direct or indirect effects of pollutants on the adenylate kinase system: data obtained in laboratory studies on shrimp exposed to cadmium and in field studies on oysters either exposed to polychloro-biphenyl compounds or located in a heavily polluted area indicate that xenobiotics affect the adenylate kinase reaction directly or by changing its cellular environment. These results demonstrate that application of the model to the treatment of ecotoxicological data allows detection of energetic changes that would have been missed by simple analysis of the usual energetic parameters, and should overcome problems encountered in using energetic parameters during assessment of pollution monitoring. PMID:10805989

  3. Inhibition of heat-labile cholera and Escherichia coli enterotoxins by brefeldin A.

    PubMed

    Donta, S T; Beristain, S; Tomicic, T K

    1993-08-01

    Cholera enterotoxin and the related heat-labile enterotoxins of Escherichia coli enter their target cells through noncoated vesicles, but how the toxins are processed intracellularly and how they get to their targeted enzyme, adenylate cyclase, remain to be defined. Brefeldin A, an inhibitor of the trans-Golgi network, is shown herein to transiently block the morphologic and enzymatic effects of the toxin at a step distal to the initial binding process but prior to activation of adenylate cyclase by the toxin. It is likely, therefore, that these toxins are processed by the Golgi apparatus before trafficking to the membrane adenylate cyclase. PMID:8392970

  4. Inhibition of heat-labile cholera and Escherichia coli enterotoxins by brefeldin A.

    PubMed Central

    Donta, S T; Beristain, S; Tomicic, T K

    1993-01-01

    Cholera enterotoxin and the related heat-labile enterotoxins of Escherichia coli enter their target cells through noncoated vesicles, but how the toxins are processed intracellularly and how they get to their targeted enzyme, adenylate cyclase, remain to be defined. Brefeldin A, an inhibitor of the trans-Golgi network, is shown herein to transiently block the morphologic and enzymatic effects of the toxin at a step distal to the initial binding process but prior to activation of adenylate cyclase by the toxin. It is likely, therefore, that these toxins are processed by the Golgi apparatus before trafficking to the membrane adenylate cyclase. Images PMID:8392970

  5. Chirally selective, intramolecular interaction observed in an aminoacyl adenylate anhydride

    NASA Astrophysics Data System (ADS)

    Lacey, James C.; Hall, Leo M.; Mullins, Dail W.; Watkins, Charles L.

    1985-06-01

    All earthly creatures use only L-amino acids in template directed protein synthesis. The reason for this exclusive use of the L-isomer is not yet apparent, although recent experiments by Usher and his colleagues have shown some stereoselctivity in the aminoacylation of di- and polynucleotides [1 3]. We have separately reported on intramolecular interactions between hydrophobic amino acid side chains and the adenine ring in aminoacyl adenylates [4]. There was a preferential association of Phe > Leu = Ile > Val with the adenine in these studies, but we made no attempts to address the question of D, L selectivity. Recently, in1H NMR studies of N-acetylphenylalanyl adenylate anhydride, we noticed evidence that both D- and L-isomers of the amino acid were present and, furthermore, that one isomer seemed to be associating with the adenine ring more strongly than the other. Using HPLC, we have separated the two diastereoisomers and have enzymatically determined that the isomer which associates more strongly is the biologically important one, the L-isomer. We present those studies here and discuss the evolutionary significance of this finding.

  6. Adenylate Energy Charge in Escherichia coli During Growth and Starvation

    PubMed Central

    Chapman, Astrid G.; Fall, Lana; Atkinson, Daniel E.

    1971-01-01

    The value of the adenylate energy charge, [(adenosine triphosphate) + ½ (adenosine diphosphate)]/[(adenosine triphosphate) + (adenosine diphosphate) + (adenosine monophosphate)], in Escherichia coli cells during growth is about 0.8. During the stationary phase after cessation of growth, or during starvation in carbon-limited cultures, the energy charge declines slowly to a value of about 0.5, and then falls more rapidly. During the slow decline in energy charge, all the cells are capable of forming colonies, but a rapid fall in viability coincides with the steep drop in energy charge. These results suggest that growth can occur only at energy charge values above about 0.8, that viability is maintained at values between 0.8 and 0.5, and that cells die at values below 0.5. Tabulation of adenylate concentrations previously reported for various organisms and tissues supports the prediction, based on enzyme kinetic observations in vitro, that the energy charge is stabilized near 0.85 in intact metabolizing cells of a wide variety of types. PMID:4333317

  7. The phosphodiesterase 4 inhibitor roflumilast augments the Th17-promoting capability of dendritic cells by enhancing IL-23 production, and impairs their T cell stimulatory activity due to elevated IL-10.

    PubMed

    Bros, Matthias; Montermann, Evelyn; Cholaszczyńska, Anna; Reske-Kunz, Angelika B

    2016-06-01

    Phosphodiesterase 4 (PDE4) inhibitors serve to prevent degradation of the intracellular second messenger cAMP, resulting in broad anti-inflammatory effects on different cell types including immune cells. Agents that elevate cAMP levels via activation of adenylate cyclase have been shown to imprint a Th17-promoting capacity in dendritic cells (DCs). Therefore, we studied the potential of therapeutically relevant PDE inhibitors to induce a pronounced Th17-skewing capacity in DCs. Here we show that mouse bone marrow-derived (BM-) DCs when treated with the PDE4 inhibitor roflumilast (ROF, trade name: Daxas) in the course of stimulation with LPS (ROF-DCs) evoked elevated IL-17 levels in cocultured allogeneic T cells. In addition, as compared with control settings, levels of IFN-γ remained unaltered, while contents of Th2 cytokines (IL-5, IL-10) were diminished. ROF enhanced expression of the Th17-promoting factor IL-23 in BM-DCs. In line, neutralizing antibodies specific for IL-23 or IL-6 when applied to DC/T cell cocultures partially inhibited the IL17-promoting effect of ROF-DCs. Furthermore, ROF-DCs displayed a markedly diminished allogeneic T cell stimulatory capacity due to enhanced production of IL-10, which was restored upon application of IL-10 specific neutralizing antibody to DC/T cell cocultures. Both the IL-17-inducing and impaired T cell stimulatory capacity of BM-DCs were mimicked by a specific activator of protein kinase A, while stimulation of EPACs (exchange proteins of activated cAMP) did not yield such effects. Taken together, our findings suggest that PDE4 inhibitors aside from their broad overall anti-inflammatory effects may enhance the Th17-polarizing capacity in DCs as an unwanted side effect. PMID:27070502

  8. Immunohistochemical Localization of Guanylate Cyclase within Neurons of Rat Brain

    NASA Astrophysics Data System (ADS)

    Ariano, Marjorie A.; Lewicki, John A.; Brandwein, Harvey J.; Murad, Ferid

    1982-02-01

    The immunohistochemical localization of guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] has been examined in rat neocortex, caudate-putamen, and cerebellum by using specific monoclonal antibodies. Immunofluorescence could be seen within somata and proximal dendrites of neurons in these regions. A nuclear immunofluorescence reaction to guanylate cyclase was characteristically absent. The staining pattern for guanylate cyclase was coincident with previously described localizations of cyclic GMP immunofluorescence within medium spiny neurons of the caudate-putamen and pyramidal cells of the neocortex. Cerebellar guanylate cyclase immunoreactivity was primarily confined to Purkinje cells and their primary dendrites, similar to the pattern reported for cyclic GMP-dependent protein kinase localization. Guanylate cyclase immunofluorescence was abolished when the monoclonal antibodies were exposed to purified enzyme prior to incubation of the tissue slices or when control antibody was substituted for the primary antibody. Immunohistochemical localization of cyclic AMP in these same tissues was readily distinguished from that of guanylate cyclase or cyclic GMP, showing uniform fluorescence throughout the cell bodies of neurons and glial elements.

  9. Pituitary Adenlylate Cyclase Activating Peptide Protects Adult Neural Stem Cells from a Hypoglycaemic milieu.

    PubMed

    Mansouri, Shiva; Lietzau, Grazyna; Lundberg, Mathias; Nathanson, David; Nyström, Thomas; Patrone, Cesare

    2016-01-01

    Hypoglycaemia is a common side-effect of glucose-lowering therapies for type-2 diabetic patients, which may cause cognitive/neurological impairment. Although the effects of hypoglycaemia in the brain have been extensively studied in neurons, how hypoglycaemia impacts the viability of adult neural stem cells (NSCs) has been poorly investigated. In addition, the cellular and molecular mechanisms of how hypoglycaemia regulates NSCs survival have not been characterized. Recent work others and us have shown that the pituitary adenylate cyclase-activating polypeptide (PACAP) and the glucagon-like peptide-1 receptor (GLP-1R) agonist Exendin-4 stimulate NSCs survival against glucolipoapoptosis. The aim of this study was to establish an in vitro system where to study the effects of hypoglycaemia on NSC survival. Furthermore, we determine the potential role of PACAP and Exendin-4 in counteracting the effect of hypoglycaemia. A hypoglycaemic in vitro milieu was mimicked by exposing subventricular zone-derived NSC to low levels of glucose. Moreover, we studied the potential involvement of apoptosis and endoplasmic reticulum stress by quantifying protein levels of Bcl-2, cleaved caspase-3 and mRNA levels of CHOP. We show that PACAP via PAC-1 receptor and PKA activation counteracts impaired NSC viability induced by hypoglycaemia. The protective effect induced by PACAP correlated with endoplasmic reticulum stress, Exendin-4 was ineffective. The results show that hypoglycaemia decreases NSC viability and that this effect can be substantially counteracted by PACAP via PAC-1 receptor activation. The data supports a potential therapeutic role of PAC-1 receptor agonists for the treatment of neurological complications, based on neurogenesis impairment by hypoglycaemia. PMID:27305000

  10. Pituitary Adenlylate Cyclase Activating Peptide Protects Adult Neural Stem Cells from a Hypoglycaemic milieu

    PubMed Central

    Mansouri, Shiva; Lietzau, Grazyna; Lundberg, Mathias; Nathanson, David; Nyström, Thomas; Patrone, Cesare

    2016-01-01

    Hypoglycaemia is a common side-effect of glucose-lowering therapies for type-2 diabetic patients, which may cause cognitive/neurological impairment. Although the effects of hypoglycaemia in the brain have been extensively studied in neurons, how hypoglycaemia impacts the viability of adult neural stem cells (NSCs) has been poorly investigated. In addition, the cellular and molecular mechanisms of how hypoglycaemia regulates NSCs survival have not been characterized. Recent work others and us have shown that the pituitary adenylate cyclase-activating polypeptide (PACAP) and the glucagon-like peptide-1 receptor (GLP-1R) agonist Exendin-4 stimulate NSCs survival against glucolipoapoptosis. The aim of this study was to establish an in vitro system where to study the effects of hypoglycaemia on NSC survival. Furthermore, we determine the potential role of PACAP and Exendin-4 in counteracting the effect of hypoglycaemia. A hypoglycaemic in vitro milieu was mimicked by exposing subventricular zone-derived NSC to low levels of glucose. Moreover, we studied the potential involvement of apoptosis and endoplasmic reticulum stress by quantifying protein levels of Bcl-2, cleaved caspase-3 and mRNA levels of CHOP. We show that PACAP via PAC-1 receptor and PKA activation counteracts impaired NSC viability induced by hypoglycaemia. The protective effect induced by PACAP correlated with endoplasmic reticulum stress, Exendin-4 was ineffective. The results show that hypoglycaemia decreases NSC viability and that this effect can be substantially counteracted by PACAP via PAC-1 receptor activation. The data supports a potential therapeutic role of PAC-1 receptor agonists for the treatment of neurological complications, based on neurogenesis impairment by hypoglycaemia. PMID:27305000

  11. A Conserved Glutamate Controls the Commitment to Acyl-adenylate Formation in Asparagine Synthetase†

    PubMed Central

    Meyer, Megan E.; Gutierrez, Jemy A.; Raushel, Frank M.; Richards, Nigel G. J.

    2010-01-01

    Inhibitor docking studies have implicated a conserved glutamate residue (Glu-348) as a general base in the synthetase active site of the enzyme asparagine synthetase B from Escherichia coli (AS-B). We now report steady-state kinetic, isotope transfer and positional isotope exchange experiments for a series of site-directed AS-B mutants in which Glu-348 is substituted by conservative amino acid replacements. We find that formation of the β-aspartyl-AMP intermediate, and therefore the eventual production of asparagine, is dependent on the presence of a carboxylate side chain at this position in the synthetase active site. In addition, Glu-348 may also play a role in mediating the conformational changes needed to (i) coordinate, albeit weakly, the glutaminase and synthetase activities of the enzyme, and (ii) establish the structural integrity of the intramolecular tunnel along which ammonia is translocated. The importance of Glu-348 in mediating acyl-adenylate formation contrasts with the functional role of the cognate residues in β-lactam synthetase (BLS) and carbapenam synthetase (CPS) (Tyr-348 and Tyr-345, respectively), which both likely evolved from asparagine synthetase. Given the similarity of the chemistry catalyzed by AS-B, BLS and CPS, our work highlights the difficulty of predicting the functional outcome of single site mutations on enzymes that catalyze almost identical chemical transformations. PMID:20853825

  12. Glutaminyl Cyclases as Novel Targets for the Treatment of Septic Arthritis

    PubMed Central

    Hellvard, Annelie; Maresz, Katarzyna; Schilling, Stephan; Graubner, Sigrid; Heiser, Ulrich; Jonsson, Roland; Cynis, Holger; Demuth, Hans-Ulrich; Potempa, Jan; Mydel, Piotr

    2013-01-01

    Background. Septic arthritis is a severe and rapidly debilitating disease mainly caused by Staphylococcus aureus. Here, we assess the antiarthritic efficiency of glutaminyl cyclase (QC) inhibitors. Methods. Mice were inoculated with an arthritogenic amount of S. aureus intravenously or by local administration into the knee joint. Animals were treated with QC inhibitors (PBD155 and PQ529) via chow during the experiment. QC and isoQC knockout mice were also analyzed for arthritis symptoms after local administration of bacteria. Results. Both QC inhibitors significantly delayed the onset of clinical signs of arthritis, and inhibitors significantly decreased weight loss in treated animals. Following intraarticular injection of S. aureus, PBD155-treated mice had lower levels of synovitis and bone erosion, as well as less myeloperoxidase in synovial tissue. Fluorescence-activated cell sorter analysis revealed that PBD155 treatment affected the expression pattern of adhesion molecules, preventing the upregulation of cells expressing CD11b/CD18. Conclusion. The compounds investigated here represent a novel class of small molecular antiarthritic inhibitors. In our studies, they exerted strong antiinflammatory actions, and therefore they might be suited for disease-modifying treatment of infectious arthritis. PMID:23204180

  13. Novel hopanoid cyclases from the environment.

    PubMed

    Pearson, Ann; Flood Page, Sarah R; Jorgenson, Tyler L; Fischer, Woodward W; Higgins, Meytal B

    2007-09-01

    Hopanoids are ubiquitous isoprenoid lipids found in modern biota, in recent sediments and in low-maturity sedimentary rocks. Because these lipids primarily are derived from bacteria, they are used as proxies to help decipher geobiological communities. To date, much of the information about sources of hopanoids has come from surveys of culture collections, an approach that does not address the vast fraction of prokaryotic communities that remains uncharacterized. Here we investigated the phylogeny of hopanoid producers using culture-independent methods. We obtained 79 new sequences of squalene-hopene cyclase genes (sqhC) from marine and lacustrine bacterioplankton and analysed them along with all 31 sqhC fragments available from existing metagenomics libraries. The environmental sqhCs average only 60% translated amino acid identity to their closest relatives in public databases. The data imply that the sources of these important geologic biomarkers remain largely unknown. In particular, genes affiliated with known cyanobacterial sequences were not detected in the contemporary environments analysed here, yet the geologic record contains abundant hopanoids apparently of cyanobacterial origin. The data also suggest that hopanoid biosynthesis is uncommon: < 10% of bacterial species may be capable of producing hopanoids. A better understanding of the contemporary distribution of hopanoid biosynthesis may reveal fundamental insight about the function of these compounds, the organisms in which they are found, and the environmental signals preserved in the sedimentary record. PMID:17686016

  14. Purification and Characterization of Allene Oxide Cyclase from Dry Corn Seeds.

    PubMed Central

    Ziegler, J.; Hamberg, M.; Miersch, O.; Parthier, B.

    1997-01-01

    Allene oxide cyclase (AOC; EC 5.3.99.6) catalyzes the cyclization of 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid to 12-oxo- 10,15(Z)-phytodienoic acid, the precursor of jasmonic acid (JA). This soluble enzyme was purified 2000-fold from dry corn (Zea mays L.) kernels to apparent homogeneity. The dimeric protein has a molecular mass of 47 kD. Allene oxide cyclase activity was not affected by divalent ions and was not feedback-regulated by its product, 12-oxo-l0,15(Z)-phytodienoic acid, or by JA. ([plus or minus])-cis- 12,13-Epoxy-9(Z)-octadecenoic acid, a substrate analog, strongly inhibited the enzyme, with 50% inhibition at 20 [mu]M. Modification of the inhibitor, such as methylation of the carboxyl group or a shift in the position of the epoxy group, abolished the inhibitory effect, indicating that both structural elements and their position are essential for binding to AOC. Nonsteroidal anti-inflammatory drugs, which are often used to interfere with JA biosynthesis, did not influence AOC activity. The purified enzyme catalyzed the cyclization of 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid derived from linolenic acid, but not that of 12,13(S)-epoxy-9(Z),11- octadecadienoic acid derived from linoleic acid. PMID:12223729

  15. Neofunctionalization of Chromoplast Specific Lycopene Beta Cyclase Gene (CYC-B) in Tomato Clade.

    PubMed

    Mohan, Vijee; Pandey, Arun; Sreelakshmi, Yellamaraju; Sharma, Rameshwar

    2016-01-01

    The ancestor of tomato underwent whole genome triplication ca. 71 Myr ago followed by widespread gene loss. However, few of the triplicated genes are retained in modern day tomato including lycopene beta cyclase that mediates conversion of lycopene to β-carotene. The fruit specific β-carotene formation is mediated by a chromoplast-specific paralog of lycopene beta cyclase (CYC-B) gene. Presently limited information is available about how the variations in CYC-B gene contributed to its neofunctionalization. CYC-B gene in tomato clade contained several SNPs and In-Dels in the coding sequence (33 haplotypes) and promoter region (44 haplotypes). The CYC-B gene coding sequence in tomato appeared to undergo purifying selection. The transit peptide sequence of CYC-B protein was predicted to have a stronger plastid targeting signal than its chloroplast specific paralog indicating a possible neofunctionalization. In promoter of two Bog (Beta old gold) mutants, a NUPT (nuclear plastid) DNA fragment of 256 bp, likely derived from a S. chilense accession, was present. In transient expression assay, this promoter was more efficient than the "Beta type" promoter. CARGATCONSENSUS box sequences are required for the binding of the MADS-box regulatory protein RIPENING INHIBITOR (RIN). The loss of CARGATCONSENSUS box sequence from CYC-B promoter in tomato may be related to attenuation of its efficiency to promote higher accumulation of β-carotene than lycopene during fruit ripening. PMID:27070417

  16. Neofunctionalization of Chromoplast Specific Lycopene Beta Cyclase Gene (CYC-B) in Tomato Clade

    PubMed Central

    Mohan, Vijee; Pandey, Arun; Sreelakshmi, Yellamaraju; Sharma, Rameshwar

    2016-01-01

    The ancestor of tomato underwent whole genome triplication ca. 71 Myr ago followed by widespread gene loss. However, few of the triplicated genes are retained in modern day tomato including lycopene beta cyclase that mediates conversion of lycopene to β-carotene. The fruit specific β-carotene formation is mediated by a chromoplast-specific paralog of lycopene beta cyclase (CYC-B) gene. Presently limited information is available about how the variations in CYC-B gene contributed to its neofunctionalization. CYC-B gene in tomato clade contained several SNPs and In-Dels in the coding sequence (33 haplotypes) and promoter region (44 haplotypes). The CYC-B gene coding sequence in tomato appeared to undergo purifying selection. The transit peptide sequence of CYC-B protein was predicted to have a stronger plastid targeting signal than its chloroplast specific paralog indicating a possible neofunctionalization. In promoter of two Bog (Beta old gold) mutants, a NUPT (nuclear plastid) DNA fragment of 256 bp, likely derived from a S. chilense accession, was present. In transient expression assay, this promoter was more efficient than the “Beta type” promoter. CARGATCONSENSUS box sequences are required for the binding of the MADS-box regulatory protein RIPENING INHIBITOR (RIN). The loss of CARGATCONSENSUS box sequence from CYC-B promoter in tomato may be related to attenuation of its efficiency to promote higher accumulation of β-carotene than lycopene during fruit ripening. PMID:27070417

  17. Soluble adenylyl cyclase is an acid-base sensor in epithelial base-secreting cells.

    PubMed

    Roa, Jinae N; Tresguerres, Martin

    2016-08-01

    Blood acid-base regulation by specialized epithelia, such as gills and kidney, requires the ability to sense blood acid-base status. Here, we developed primary cultures of ray (Urolophus halleri) gill cells to study mechanisms for acid-base sensing without the interference of whole animal hormonal regulation. Ray gills have abundant base-secreting cells, identified by their noticeable expression of vacuolar-type H(+)-ATPase (VHA), and also express the evolutionarily conserved acid-base sensor soluble adenylyl cyclase (sAC). Exposure of cultured cells to extracellular alkalosis (pH 8.0, 40 mM HCO3 (-)) triggered VHA translocation to the cell membrane, similar to previous reports in live animals experiencing blood alkalosis. VHA translocation was dependent on sAC, as it was blocked by the sAC-specific inhibitor KH7. Ray gill base-secreting cells also express transmembrane adenylyl cyclases (tmACs); however, tmAC inhibition by 2',5'-dideoxyadenosine did not prevent alkalosis-dependent VHA translocation, and tmAC activation by forskolin reduced the abundance of VHA at the cell membrane. This study demonstrates that sAC is a necessary and sufficient sensor of extracellular alkalosis in ray gill base-secreting cells. In addition, this study indicates that different sources of cAMP differentially modulate cell biology. PMID:27335168

  18. Role of adenylyl cyclase in reduced β-adrenoceptor-mediated vasorelaxation during maturation

    PubMed Central

    López-Canales, O.A.; Castillo-Hernandez, M.C.; Vargas-Robles, H.; Rios, A.; López-Canales, J.S.; Escalante, B.

    2016-01-01

    Beta-adrenergic receptor (βAR)-dependent blood vessel relaxation is impaired in older animals and G protein activation has been suggested as the causative mechanism. Here, we investigated the role of βAR subtypes (β1AR, β2AR, and β3AR) and cAMP in maturation-dependent vasorelaxation impairment. Aortic rings from 15 Sprague-Dawley male rats (3 or 9 weeks old) were harvested and left intact or denuded of the endothelium. Vascular relaxation in aortic rings from younger and older groups was compared in the presence of βAR subtype agonists and antagonists along with cAMP and cGMP antagonists. Isolated aortic rings were used to evaluate relaxation responses, protein expression was evaluated by western blot or real time PCR, and metabolites were measured by ELISA. Expression of βAR subtypes and adenylyl cyclase was assessed, and cAMP activity was measured in vascular tissue from both groups. Isoproterenol- and BRL744-dependent relaxation in aortic rings with and without endothelium from 9-week-old rats was impaired compared with younger rats. The β1AR antagonist CGP20712A (10-7 M) did not affect isoproterenol or BRL744-dependent relaxation in arteries from either group. The β2AR antagonist ICI-118,551 (10-7 M) inhibited isoproterenol-dependent aortic relaxation in both groups. The β3AR antagonist SR59230A (10-7 M) inhibited isoproterenol- and BRL744-dependent aortic ring relaxation in younger but not in older rats. All βAR subtypes were expressed in both groups, although β3AR expression was lower in the older group. Adenylyl cyclase (SQ 22536) or protein kinase A (H89) inhibitors prevented isoproterenol-induced relaxation in younger but not in older rats. Production of cAMP was reduced in the older group. Adenylyl cyclase III and RyR3 protein expression was higher in the younger group. In conclusion, altered expression of β3AR and adenylyl cyclase III may be responsible for reduced cAMP production in the older group. PMID:27383122

  19. Role of adenylyl cyclase in reduced β-adrenoceptor-mediated vasorelaxation during maturation.

    PubMed

    López-Canales, O A; Castillo-Hernandez, M C; Vargas-Robles, H; Rios, A; López-Canales, J S; Escalante, B

    2016-07-01

    Beta-adrenergic receptor (βAR)-dependent blood vessel relaxation is impaired in older animals and G protein activation has been suggested as the causative mechanism. Here, we investigated the role of βAR subtypes (β1AR, β2AR, and β3AR) and cAMP in maturation-dependent vasorelaxation impairment. Aortic rings from 15 Sprague-Dawley male rats (3 or 9 weeks old) were harvested and left intact or denuded of the endothelium. Vascular relaxation in aortic rings from younger and older groups was compared in the presence of βAR subtype agonists and antagonists along with cAMP and cGMP antagonists. Isolated aortic rings were used to evaluate relaxation responses, protein expression was evaluated by western blot or real time PCR, and metabolites were measured by ELISA. Expression of βAR subtypes and adenylyl cyclase was assessed, and cAMP activity was measured in vascular tissue from both groups. Isoproterenol- and BRL744-dependent relaxation in aortic rings with and without endothelium from 9-week-old rats was impaired compared with younger rats. The β1AR antagonist CGP20712A (10-7 M) did not affect isoproterenol or BRL744-dependent relaxation in arteries from either group. The β2AR antagonist ICI-118,551 (10-7 M) inhibited isoproterenol-dependent aortic relaxation in both groups. The β3AR antagonist SR59230A (10-7 M) inhibited isoproterenol- and BRL744-dependent aortic ring relaxation in younger but not in older rats. All βAR subtypes were expressed in both groups, although β3AR expression was lower in the older group. Adenylyl cyclase (SQ 22536) or protein kinase A (H89) inhibitors prevented isoproterenol-induced relaxation in younger but not in older rats. Production of cAMP was reduced in the older group. Adenylyl cyclase III and RyR3 protein expression was higher in the younger group. In conclusion, altered expression of β3AR and adenylyl cyclase III may be responsible for reduced cAMP production in the older group. PMID:27383122

  20. Molecular Physiology of Membrane Guanylyl Cyclase Receptors.

    PubMed

    Kuhn, Michaela

    2016-04-01

    cGMP controls many cellular functions ranging from growth, viability, and differentiation to contractility, secretion, and ion transport. The mammalian genome encodes seven transmembrane guanylyl cyclases (GCs), GC-A to GC-G, which mainly modulate submembrane cGMP microdomains. These GCs share a unique topology comprising an extracellular domain, a short transmembrane region, and an intracellular COOH-terminal catalytic (cGMP synthesizing) region. GC-A mediates the endocrine effects of atrial and B-type natriuretic peptides regulating arterial blood pressure/volume and energy balance. GC-B is activated by C-type natriuretic peptide, stimulating endochondral ossification in autocrine way. GC-C mediates the paracrine effects of guanylins on intestinal ion transport and epithelial turnover. GC-E and GC-F are expressed in photoreceptor cells of the retina, and their activation by intracellular Ca(2+)-regulated proteins is essential for vision. Finally, in the rodent system two olfactorial GCs, GC-D and GC-G, are activated by low concentrations of CO2and by peptidergic (guanylins) and nonpeptidergic odorants as well as by coolness, which has implications for social behaviors. In the past years advances in human and mouse genetics as well as the development of sensitive biosensors monitoring the spatiotemporal dynamics of cGMP in living cells have provided novel relevant information about this receptor family. This increased our understanding of the mechanisms of signal transduction, regulation, and (dys)function of the membrane GCs, clarified their relevance for genetic and acquired diseases and, importantly, has revealed novel targets for therapies. The present review aims to illustrate these different features of membrane GCs and the main open questions in this field. PMID:27030537

  1. An intrinsic adenylate kinase activity regulates gating of the ABC transporter CFTR.

    PubMed

    Randak, Christoph; Welsh, Michael J

    2003-12-26

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP binding cassette (ABC) transporter family. Like other ABC transporters, it can hydrolyze ATP. Yet while ATP hydrolysis influences channel gating, it has long seemed puzzling that CFTR would require this reaction because anions flow passively through CFTR. Moreover, no other ion channel is known to require the large energy of ATP hydrolysis to gate. We found that CFTR also has adenylate kinase activity (ATP + AMP <=> ADP + ADP) that regulates gating. When functioning as an adenylate kinase, CFTR showed positive cooperativity for ATP suggesting its two nucleotide binding domains may dimerize. Thus, channel activity could be regulated by two different enzymatic reactions, ATPase and adenylate kinase, that share a common ATP binding site in the second nucleotide binding domain. At physiologic nucleotide concentrations, adenylate kinase activity, rather than ATPase activity may control gating, and therefore involve little energy consumption. PMID:14697202

  2. Characterization of metal and nucleotide liganded forms of adenylate kinase by electrospray ionization mass spectrometry.

    PubMed

    Briand, G; Perrier, V; Kouach, M; Takahashi, M; Gilles, A M; Bârzu, O

    1997-03-15

    Complexes of adenylate kinase from Escherichia coli, Bacillus subtilis, and Bacillus stearothermophilus with the bisubstrate nucleotide analog P1,P5-di(adenosine 5')-pentaphosphate and with metal ions (Zn2+ and/or Mg2+) were analyzed by electrospray ionization mass spectrometry. P1,P5-di(adenosine 5')-pentaphosphate. adenylate kinase complex was detected in the positive mode at pH as low as 3.8. Binding of nucleotide to adenylate kinase stabilizes the overall structure of the protein and preserves the Zn2+ chelated form of the enzyme from the gram-positive organisms. In this way, it is possible in a single mass spectrometry experiment to screen metal-chelating adenylate kinases, without use of radioactively labeled compounds. Binding of Mg2+ to enzyme via P1,P5-di(adenosine 5')-pentaphosphate was also demonstrated by mass spectrometry. Although no amino acid side chain in adenylate kinase is supposed to interact with Mg2+, Asp93 in porcine muscle cytosolic enzyme, equivalent to Asp84 in the E. coli adenylate kinase, was proposed to stabilize the nucleotide.Mg2+ complex via water molecules. PMID:9056261

  3. Aprataxin resolves adenylated RNA–DNA junctions to maintain genome integrity

    SciTech Connect

    Tumbale, Percy; Williams, Jessica S.; Schellenberg, Matthew J.; Kunkel, Thomas A.; Williams, R. Scott

    2013-12-22

    Faithful maintenance and propagation of eukaryotic genomes is ensured by three-step DNA ligation reactions used by ATP-dependent DNA ligases. Paradoxically, when DNA ligases encounter nicked DNA structures with abnormal DNA termini, DNA ligase catalytic activity can generate and/or exacerbate DNA damage through abortive ligation that produces chemically adducted, toxic 5'-adenylated (5'-AMP) DNA lesions. Aprataxin (APTX) reverses DNA adenylation but the context for deadenylation repair is unclear. Here we examine the importance of APTX to RNase-H2-dependent excision repair (RER) of a lesion that is very frequently introduced into DNA, a ribonucleotide. We show that ligases generate adenylated 5' ends containing a ribose characteristic of RNase H2 incision. APTX efficiently repairs adenylated RNA–DNA, and acting in an RNA–DNA damage response (RDDR), promotes cellular survival and prevents S-phase checkpoint activation in budding yeast undergoing RER. Structure–function studies of human APTX–RNA–DNA–AMP–Zn complexes define a mechanism for detecting and reversing adenylation at RNA–DNA junctions. This involves A-form RNA binding, proper protein folding and conformational changes, all of which are affected by heritable APTX mutations in ataxia with oculomotor apraxia 1. Together, these results indicate that accumulation of adenylated RNA–DNA may contribute to neurological disease.

  4. Regulation and organization of adenylyl cyclases and cAMP.

    PubMed Central

    Cooper, Dermot M F

    2003-01-01

    Adenylyl cyclases are a critically important family of multiply regulated signalling molecules. Their susceptibility to many modes of regulation allows them to integrate the activities of a variety of signalling pathways. However, this property brings with it the problem of imparting specificity and discrimination. Recent studies are revealing the range of strategies utilized by the cyclases to solve this problem. Microdomains are a consequence of these solutions, in which cAMP dynamics may differ from the broad cytosol. Currently evolving methodologies are beginning to reveal cAMP fluctuations in these various compartments. PMID:12940771

  5. Regulation of adenylyl cyclase from Blastocladiella emersonii by guanine nucleotides.

    PubMed

    Terenzi, H; Maia, J C

    1993-11-01

    GTP gamma S stimulates adenylyl cyclase in particulate fractions of Blastocladiella emersonii zoospores. Cholera toxin catalyses the ADP-ribosylation of a membrane protein of a molecular weight (46,000) similar to that of the alpha subunit of Gs found in vertebrate cells. A membrane protein of 46 kDa can also be recognized in Western blots by an antipeptide antiserum (RM/1) raised against the C-terminus of G alpha 2-subunits. These results suggest that a G-protein mediates the regulation of Blastocladiella adenylyl cyclase by guanine nucleotides. PMID:8224237

  6. Total synthesis: Towards artificial terpene cyclases

    NASA Astrophysics Data System (ADS)

    Willot, Matthieu; Christmann, Mathias

    2010-07-01

    The plant-derived sesquiterpene englerin A is a potent inhibitor of several renal cancer cell lines. Two recent total syntheses have utilized cationic gold(I)-complexes to coax readily available open-chain precursors into englerin's challenging oxotricyclic core with enzyme-like precision.

  7. Cloning and characterization of the cDNA for lycopene beta-cyclase from tomato reveals decrease in its expression during fruit ripening.

    PubMed

    Pecker, I; Gabbay, R; Cunningham, F X; Hirschberg, J

    1996-02-01

    The cDNA which encodes lycopene cyclase, CrtL, was cloned from tomato (Lycopersicon esculentum cv. VF36) and tobacco (Nicotiana tabacum cv. Samsun NN) and functionally expressed in Escherichia coli. This enzyme converts lycopene to beta-carotene by catalyzing the formation of two beta-rings at each end of the linear carotene. The enzyme interacts with half of the carotenoid molecule and requires a double bond at the C-7,8 (or C-7,8') position. Inhibition in E. coli indicated that lycopene cyclase is the target site for the inhibitor MPTA, 2-(4-methylphenoxy)tri-ethylamine hydrochloride. The primary structure of lycopene cyclase in higher plants is significantly conserved with the enzyme from cyanobacteria but different from that of the non-photosynthetic bacteria Erwinia. mRNA of CrtL and Pds, which encodes phytoene desaturase, was measured in leaves, flowers and ripening fruits of tomato. In contrast to genes which encode enzymes of early steps in the carotenoid biosynthesis pathway, whose transcription increases during the 'breaker' stage of fruit ripening, the level of CrtL mRNA decreases at this stage. Hence, the accumulation of lycopene in tomato fruits is apparently due to a down-regulation of the lycopene cyclase gene that occurs at the breaker stage of fruit development. This conclusion supports the hypothesis that transcriptional regulation of gene expression is a predominant mechanism of regulating carotenogenesis. PMID:8624411

  8. Analysis of the Linker Region Joining the Adenylation and Carrier Protein Domains of the Modular Non-Ribosomal Peptide Synthetases

    PubMed Central

    Miller, Bradley R.; Sundlov, Jesse A.; Drake, Eric J.; Makin, Thomas A.; Gulick, Andrew M.

    2014-01-01

    Non-Ribosomal Peptide Synthetases (NRPSs) are multi-modular proteins capable of producing important peptide natural products. Using an assembly-line process the amino acid substrate and peptide intermediates are passed between the active sites of different catalytic domains of the NRPS while bound covalently to a peptidyl carrier protein (PCP) domain. Examination of the linker sequences that join the NRPS adenylation and PCP domains identified several conserved proline residues that are not found in standalone adenylation domains. We examined the roles of these proline residues and neighboring conserved sequences through mutagenesis and biochemical analysis of the reaction catalyzed by the adenylation domain and the fully reconstituted NRPS pathway. In particular, we identified a conserved LPxP motif at the start of the adenylation-PCP linker. The LPxP motif interacts with a region on the adenylation domain to stabilize a critical catalytic lysine residue belonging to the A10 motif that immediately precedes the linker. Further, this interaction with the C-terminal sub-domain of the adenylation domain may coordinate movement of the PCP with the conformational change of the adenylation domain. Through this work, we extend the conserved A10 motif of the adenylation domain and identify residues that enable proper adenylation domain function. PMID:24975514

  9. A Soluble Adenylyl Cyclase Form Targets to Axonemes and Rescues Beat Regulation in Soluble Adenylyl Cyclase Knockout Mice

    PubMed Central

    Chen, Xi; Baumlin, Nathalie; Buck, Jochen; Levin, Lonny R.; Fregien, Nevis

    2014-01-01

    Ciliary beating is important for effective mucociliary clearance. Soluble adenylyl cyclase (sAC) regulates ciliary beating, and a roughly 50-kD sAC variant is expressed in axonemes. Normal human bronchial epithelial (NHBE) cells express multiple sAC splice variants: full-length sAC; variants with catalytic domain 1 (C1) deletions; and variants with partial C1. One variant, sACex5v2-ex12v2, contains two alternative splices creating new exons 5 (ex5v2) and 12 (ex12v2), encoding a roughly 45-kD protein. It is therefore similar in size to ciliary sAC. The variant increases in expression upon ciliogenesis during differentiation at the air–liquid interface. When expressed in NHBE cells, this variant was targeted to cilia. Exons 5v2–7 were important for ciliary targeting, whereas exons 2–4 prevented it. In vitro, cytoplasmic sACex2-ex12v2 (containing C1 and C2) was the only variant producing cAMP. Ciliary sACex5v2-ex12v2 was not catalytically active. Airway epithelial cells isolated from wild-type mice revealed sAC-dependent ciliary beat frequency (CBF) regulation, analogous to NHBE cells: CBF rescue from HCO3−/CO2–mediated intracellular acidification was sensitive to the sAC inhibitor, KH7. Compared with wild type, sAC C2 knockout (KO) mice revealed lower CBF baseline, and the HCO3−/CO2–mediated CBF decrease was not inhibited by KH7, confirming lack of functional sAC. Human sACex5v2-ex12v2 was targeted to cilia and sACex2-ex12v2 to the cytoplasm in these KO mice. Introduction of the ciliary sACex5v2-ex12v2 variant, but not the cytoplasmic sACex2-ex12v2, restored functional sAC activity in C2 KO mice. Thus, we show, for the first time, a mammalian axonemal targeting sequence that localizes a sAC variant to cilia to regulate CBF. PMID:24874272

  10. Chirally selective, intramolecular interaction observed in an aminoacyl adenylate anhydride

    NASA Technical Reports Server (NTRS)

    Lacey, J. C., Jr.; Hall, L. M.; Mullins, D. W., Jr.; Watkins, C. L.

    1985-01-01

    The interaction between amino acids and nucleotide bases is studied. The proton NMR spectrum of N-acetylphenylalanyl-AMP-anhydride is analyzed H8 and H2 signals, two upfield signals of equal size, and five phenylalanine ring proton signals are observed in the spectrum; the upfield movement of the proton and the racemization of the N-acetyl L-phenylalanine material are examined. The differences in the position of the signals due to the diastereoisomers are investigated. The separation of the D and L amino acyl adenylates using HPLC is described. H-1 NMR spectra of the isomers are examined in order to determine which isomer displays the strongest interaction between the phenyl ring and the adenine ring. The spectra reveal that the L isomer shows the highest upfield change of both H8 and H2 signals. It is noted that the phenyl ring lies over C2 of the adenine ring with the phenyl meta and para protons extended past the adenine ring and the phenyl ortho protons.

  11. Snapshots of tRNA sulphuration via an adenylated intermediate.

    PubMed

    Numata, Tomoyuki; Ikeuchi, Yoshiho; Fukai, Shuya; Suzuki, Tsutomu; Nureki, Osamu

    2006-07-27

    Uridine at the first anticodon position (U34) of glutamate, lysine and glutamine transfer RNAs is universally modified by thiouridylase into 2-thiouridine (s2U34), which is crucial for precise translation by restricting codon-anticodon wobble during protein synthesis on the ribosome. However, it remains unclear how the enzyme incorporates reactive sulphur into the correct position of the uridine base. Here we present the crystal structures of the MnmA thiouridylase-tRNA complex in three discrete forms, which provide snapshots of the sequential chemical reactions during RNA sulphuration. On enzyme activation, an alpha-helix overhanging the active site is restructured into an idiosyncratic beta-hairpin-containing loop, which packs the flipped-out U34 deeply into the catalytic pocket and triggers the activation of the catalytic cysteine residues. The adenylated RNA intermediate is trapped. Thus, the active closed-conformation of the complex ensures accurate sulphur incorporation into the activated uridine carbon by forming a catalytic chamber to prevent solvent from accessing the catalytic site. The structures of the complex with glutamate tRNA further reveal how MnmA specifically recognizes its three different tRNA substrates. These findings provide the structural basis for a general mechanism whereby an enzyme incorporates a reactive atom at a precise position in a biological molecule. PMID:16871210

  12. Adenylate Kinase and AMP Signaling Networks: Metabolic Monitoring, Signal Communication and Body Energy Sensing

    PubMed Central

    Dzeja, Petras; Terzic, Andre

    2009-01-01

    Adenylate kinase and downstream AMP signaling is an integrated metabolic monitoring system which reads the cellular energy state in order to tune and report signals to metabolic sensors. A network of adenylate kinase isoforms (AK1-AK7) are distributed throughout intracellular compartments, interstitial space and body fluids to regulate energetic and metabolic signaling circuits, securing efficient cell energy economy, signal communication and stress response. The dynamics of adenylate kinase-catalyzed phosphotransfer regulates multiple intracellular and extracellular energy-dependent and nucleotide signaling processes, including excitation-contraction coupling, hormone secretion, cell and ciliary motility, nuclear transport, energetics of cell cycle, DNA synthesis and repair, and developmental programming. Metabolomic analyses indicate that cellular, interstitial and blood AMP levels are potential metabolic signals associated with vital functions including body energy sensing, sleep, hibernation and food intake. Either low or excess AMP signaling has been linked to human disease such as diabetes, obesity and hypertrophic cardiomyopathy. Recent studies indicate that derangements in adenylate kinase-mediated energetic signaling due to mutations in AK1, AK2 or AK7 isoforms are associated with hemolytic anemia, reticular dysgenesis and ciliary dyskinesia. Moreover, hormonal, food and antidiabetic drug actions are frequently coupled to alterations of cellular AMP levels and associated signaling. Thus, by monitoring energy state and generating and distributing AMP metabolic signals adenylate kinase represents a unique hub within the cellular homeostatic network. PMID:19468337

  13. ADENYLATE ENERGY CHARGE AND ADENINE NUCLEOTIDE MEASUREMENTS AS INDICATORS OF STRESS IN THE MUSSEL, MYTILUS EDULIS, TREATED WITH DREDGED MATERIAL UNDER LABORATORY CONDITIONS

    EPA Science Inventory

    Adenylate energy charge is an indication of the amount of energy available to an organism from the adenylate pool. t is calculated from measured concentrations of three adenine nucleotides, adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP...

  14. Mechanism of adenylate kinase. Are the essential lysines essential?

    PubMed

    Tian, G C; Yan, H G; Jiang, R T; Kishi, F; Nakazawa, A; Tsai, M D

    1990-05-01

    Using site-specific mutagenesis, we have probed the structural and functional roles of lysine-21 and lysine-27 of adenylate kinase (AK) from chicken muscle expressed in Escherichia coli. The two residues were chosen since according to the nuclear magnetic resonance (NMR) model [Mildvan, A. S., & Fry, D. C. (1987) Adv. Enzymol. 58, 241-313], they are located near the alpha- and the gamma-phosphates, respectively, of adenosine 5'-triphosphate (ATP) in the AK-MgATP complex. In addition, a lysine residue (Lys-21 in the case of AK) along with a glycine-rich loop is considered "essential" in the catalysis of kinases and other nucleotide binding proteins. The Lys-27 to methionine (K27M) mutant showed only slight increases in kcat and Km, but a substantial increase (1.8 kcal/mol) in the free energy of unfolding, relative to the WT AK. For proper interpretation of the steady-state kinetic data, viscosity-dependent kinetics was used to show that the chemical step is partially rate-limiting in the catalysis of AK. Computer modeling suggested that the folded form of K27M could gain stability (relative to the wild type) via hydrophobic interactions of Met-27 with Val-179 and Phe-183 and/or formation of a charge-transfer complex between Met-27 and Phe-183. The latter was supported by an upfield shift of the methyl protons of Met-27 in 1H NMR. Other than this, the 1H NMR spectrum of K27M is very similar to that of WT, suggesting little perturbation in the global or even local conformations.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2161682

  15. Muscarinic cholinergic ligand binding to intact mouse pituitary tumor cells (AtT-20/D16-16) coupling with two biochemical effectors: adenylate cyclase and phosphatidylinositol turnover.

    PubMed

    Akiyama, K; Vickroy, T W; Watson, M; Roeske, W R; Reisine, T D; Smith, T L; Yamamura, H I

    1986-03-01

    (-)-[3H]Quinuclidinyl benzilate (QNB) binding to muscarinic receptors on intact mouse pituitary tumor cells (AtT-20/D16-16) was characterized in an attempt to correlate radioligand binding properties with receptor-coupled biochemical responses. Performing rinse time studies for 2 hr produced a remarkably improved ratio of specific/total (+)-[3H]QNB binding (85%). Kinetic experiments yielded association (k+1) and dissociation (k-1) rate constants of 2.2 X 10(8) M-1 min-1 and 6.8 X 10(-3) min-1, respectively. Receptor occupancy curves demonstrated a uniform population of specific, saturable (-)-[3H]QNB binding sites with a Hill coefficient equal to 1.0 and an apparent dissociation constant (Kd) equal to 34 pM under our conditions. Stereoselectivity was observed with the enantiomers (dexetimide and levetimide) of benzetimide (a factor of 4300). Concentrations of carbachol that produced a half-maximal inhibition of cyclic AMP formation and a concentration of carbachol for producing half-maximal stimulation of phosphatidylinositol turnover in the intact cells were 0.45 and 170 microM, respectively. Schild analysis revealed that pirenzepine, a nonclassical muscarinic antagonist, had a 40-fold greater affinity for reversing carbachol-stimulated phosphatidylinositol turnover (inhibition constant or Ki = 7 nM), compared to its antagonism of the carbachol-mediated inhibition of isoproterenol-stimulated cyclic AMP formation (Ki = 280 nM). Interestingly, pirenzepine inhibited (-)-[3H]QNB binding with a Ki value of 72 nM. In contrast, atropine was nearly equipotent (Ki = 0.3-0.5 nM) in binding studies and in both effector systems. PMID:3005550

  16. Rapid molecular pathotyping of major salmonella enterica serotypes based on single-nucleotide polymorphisms (SNPs) in the adenylate cyclase (cyaA) gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Introduction: Salmonella enterica subsp. enterica serotype Enteriditis (S. Enteriditis) is the leading cause of salmonellosis worldwide, including the USA. Many S. enterica serotypes known to cause foodborne disease are associated with broiler meat contamination. While some serotypes are specific...

  17. Buspirone and gepirone: partial agonists at the 5HT/sub 1/A receptor linked to adenylate cyclase (AC) in rat and guinea pig hippocampal preparations

    SciTech Connect

    Yocca, F.D.; Hyslop, D.K.; Taylor, D.P.; Maayani, S.

    1986-03-01

    The pharmacologic nature of the 5-HT receptor that is negatively linked to AC in membrane preparations from rat and guinea pig (gp) brain in cell culture and in gp hippocampal homogenates positively linked to AC seem to be indistinguishable from the 5HT/sub 1A/ binding site in similar preparations. Affinity values of chemically unrelated but selective drugs for a binding site are useful for taxonomy of functional receptors. The novel anxiolytic drug buspirone (B) and its analog gepirone (G) exhibit selectivity and affinity for spiperone-sensitive (/sup 3/H)-5-HT and (/sup 3/H)-8-OH-DPAT binding sites in gp and rat hippocampus. In the two species tested, B and G were partial agonists (intrinsic activity approx. = 0.5) compared to 5-HT and its potent analog 5-carboxamideotryptamine (5-COAT) at the 5-HT/sub 1A/ receptor linked to AC. The K/sub B/ value of spiperone determined with B and G was indistinguishable from that determined with 5-HT and 5-COAT (20-30 nM). Since B and G exert unique agonist effects at the functional 5HT/sub 1A/ receptor, their structures may be important for identifying chemical groups necessary for recognition and activation of the 5HT/sub 1A/ receptor.

  18. Structure of the DNA Ligase-Adenylate Intermediate: Lysine (ε-amino)-Linked Adenosine Monophosphoramidate*

    PubMed Central

    Gumport, Richard I.; Lehman, I. R.

    1971-01-01

    Proteolytic degradation of the Escherichia coli DNA ligase-adenylate intermediate releases adenosine 5′-monophosphate linked to the ε-amino group of lysine by a phosphoamide bond. Measurements of the rate of hydroxylaminolysis of the ligase-adenylate provide further support for a phosphoamide linkage in the native enzyme. Lysine (ε-amino)-linked adenosine monophosphoramidate has also been isolated from the T4 phage-induced ligase-adenylate intermediate. These results indicate that an initial step of the DNA ligase reaction consists of the nucleophilic attack of the ε-amino group of a lysine residue of the enzyme on the adenylyl phosphorus of DPN or ATP that leads to the formation of enzyme-bound lysine (εamino)-linked adenosine monophosphoramidate. PMID:4944632

  19. Inhibition of monoterpene cyclases by sulfonium analogs of presumptive carbocationic intermediates of the cyclization reaction.

    PubMed

    Croteau, R; Wheeler, C J; Aksela, R; Oehlschlager, A C

    1986-06-01

    The enzymatic cyclization of geranyl pyrophosphate to monoterpenes is thought to proceed through a series of carbocation-pyrophosphate anion paired intermediates. Sulfonium analogs of two putative carbocationic intermediates of the cyclization sequence were shown to be inhibitors of the conversion of the acyclic precursor to the bicyclic monoterpenes (+)-alpha-pinene and (+)-bornyl pyrophosphate by partially purified cyclase preparations from sage (Salvia officinalis). The sulfonium analog of the tertiary allylic, linalyl, intermediate (i.e. methyl-(4-methylpent-3-en-1-yl)vinyl-sulfonium perchlorate) provided respective Ki values of 2.5 microM and 3.0 microM against the cyclization to alpha-pinene and bornyl pyrophosphate at a substrate concentration of 5 microM, whereas the sulfonium analog of the monocyclic, alpha-terpinyl, intermediate (i.e. dimethyl-(4-methylcyclohex-3-en-1-yl) sulfonium iodide) exhibited respective Ki values of 3.4 microM and 3.9 microM against the same two cyclizations. The potency of inhibition in all cases increased with increasing substrate concentration, indicating that the affinity of the enzymes for the sulfonium analogs was increased by the presence of the pyrophosphate ester. Inorganic pyrophosphate at a concentration of 50 microM, which alone had little influence on the cyclizations, increased the effectiveness of inhibition of the sulfonium analogs severalfold, and the apparent Ki for inorganic pyrophosphate was reduced manyfold by the presence of either analog at 5 microM. That the combination of sulfonium analog and pyrophosphate provided synergistic inhibition of the electrophilic cyclizations indicated that the cyclases bind the paired species more tightly than either partner alone. Specificity studies suggested that inhibition by the above sulfonium ion:pyrophosphate pairs was due to both electronic and structural resemblance to intermediates of the reaction. PMID:3011779

  20. Delivery of Large Heterologous Polypeptides across the Cytoplasmic Membrane of Antigen-Presenting Cells by the Bordetella RTX Hemolysin Moiety Lacking the Adenylyl Cyclase Domain

    PubMed Central

    Holubova, Jana; Jelinek, Jiri; Tomala, Jakub; Masin, Jiri; Kosova, Martina; Stanek, Ondrej; Bumba, Ladislav; Michalek, Jaroslav; Kovar, Marek; Sebo, Peter

    2012-01-01

    The Bordetella adenylate cyclase toxin-hemolysin (CyaA; also called ACT or AC-Hly) targets CD11b-expressing phagocytes and translocates into their cytosol an adenylyl cyclase (AC) that hijacks cellular signaling by conversion of ATP to cyclic AMP (cAMP). Intriguingly, insertion of large passenger peptides removes the enzymatic activity but not the cell-invasive capacity of the AC domain. This has repeatedly been exploited for delivery of heterologous antigens into the cytosolic pathway of CD11b-expressing dendritic cells by CyaA/AC− toxoids, thus enabling their processing and presentation on major histocompatibility complex (MHC) class I molecules to cytotoxic CD8+ T lymphocytes (CTLs). We produced a set of toxoids with overlapping deletions within the first 371 residues of CyaA and showed that the structure of the AC enzyme does not contain any sequences indispensable for its translocation across target cell membrane. Moreover, replacement of the AC domain (residues 1 to 371) with heterologous polypeptides of 40, 146, or 203 residues yielded CyaAΔAC constructs that delivered passenger CTL epitopes into antigen-presenting cells (APCs) and induced strong antigen-specific CD8+ CTL responses in vivo in mice and ex vivo in human peripheral blood mononuclear cell cultures. This shows that the RTX (repeats in toxin) hemolysin moiety, consisting of residues 374 to 1706 of CyaA, harbors all structural information involved in translocation of the N-terminal AC domain across target cell membranes. These results decipher the extraordinary capacity of the AC domain of CyaA to transport large heterologous cargo polypeptides into the cytosol of CD11b+ target cells and pave the way for the construction of CyaAΔAC-based polyvalent immunotherapeutic T cell vaccines. PMID:22215742

  1. Clay catalyzed polymerization of amino acid adenylates and its relationship to biochemical reactions

    NASA Technical Reports Server (NTRS)

    Paecht-Horowitz, M.

    1978-01-01

    The adsorption and polymerization of alanine adenylate on montmorillonite at pH 7 when either its interspacial faces or its edger are blocked by an excess of histidine or sodium hexametaphosphate was investigated. Results indicate that alanine adenylate can be adsorbed any place on the interspacial spaces of the clay; however, adsorption of its phosphate part, which is limited to the edges of the clay, is necessary for polymerization to occur. As a result, polymerization takes place only at sites on the interspacial faces bordering the edges.

  2. Activation of the Pacidamycin PacL Adenylation Domain by MbtH-Like Proteins†

    PubMed Central

    Zhang, Wenjun; Heemstra, John R.; Walsh, Christopher T.; Imker, Heidi J.

    2010-01-01

    Nonribosomal peptide synthetase (NRPS) assembly lines are major avenues for the biosynthesis of a vast array of peptidyl natural products. Several hundred bacterial NRPS gene clusters contain a small (~70 residue) protein belonging to the MbtH family for which no function has been defined. Here we show that two strictly conserved Trp residues in MbtH-like proteins contribute to stimulation of amino acid adenylation in some NRPS modules. We also demonstrate that adenylation can be stimulated not only by cognate MbtH-like proteins but also by homologues from disparate natural product pathways. PMID:20964365

  3. Activation of the pacidamycin PacL adenylation domain by MbtH-like proteins.

    PubMed

    Zhang, Wenjun; Heemstra, John R; Walsh, Christopher T; Imker, Heidi J

    2010-11-23

    Nonribosomal peptide synthetase (NRPS) assembly lines are major avenues for the biosynthesis of a vast array of peptidyl natural products. Several hundred bacterial NRPS gene clusters contain a small (∼70-residue) protein belonging to the MbtH family for which no function has been defined. Here we show that two strictly conserved Trp residues in MbtH-like proteins contribute to stimulation of amino acid adenylation in some NRPS modules. We also demonstrate that adenylation can be stimulated not only by cognate MbtH-like proteins but also by homologues from disparate natural product pathways. PMID:20964365

  4. The polymerization of amino acid adenylates on sodium-montmorillonite with preadsorbed polypeptides

    NASA Technical Reports Server (NTRS)

    Paecht-Horowitz, Mella; Eirich, Frederick R.

    1988-01-01

    The spontaneous polymerization of amino acid adenylates on Na-montmorillonite in dilute, neutral suspension, after polypeptides were adsorbed on the clay, is studied. It is found that the degrees of polymerization of the oligopeptides and polypeptides obtained is dependent on the amounts of polypeptides that were preadsorbed. It is concluded that a catalytic activity may derive from c-spacings that offer adsorption sites for the reagent amino acid adenylate within the peripheral recesses of irregularly stacked clay platelets by bringing the anhydride bonds and neutral amino groups into favorable reaction distances.

  5. Requirements for the adenylyl cyclases in the development of Dictyostelium.

    PubMed

    Anjard, C; Söderbom, F; Loomis, W F

    2001-09-01

    It has been suggested that all intracellular signaling by cAMP during development of Dictyostelium is mediated by the cAMP-dependent protein kinase, PKA, since cells carrying null mutations in the acaA gene that encodes adenylyl cyclase can develop so as to form fruiting bodies under some conditions if PKA is made constitutive by overexpressing the catalytic subunit. However, a second adenylyl cyclase encoded by acrA has recently been found that functions in a cell autonomous fashion during late development. We have found that expression of a modified acaA gene rescues acrA- mutant cells indicating that the only role played by ACR is to produce cAMP. To determine whether cells lacking both adenylyl cyclase genes can develop when PKA is constitutive we disrupted acrA in a acaA- PKA-C(over) strain. When developed at high cell densities, acrA- acaA- PKA-C(over) cells form mounds, express cell type-specific genes at reduced levels and secrete cellulose coats but do not form fruiting bodies or significant numbers of viable spores. Thus, it appears that synthesis of cAMP is required for spore differentiation in Dictyostelium even if PKA activity is high. PMID:11566867

  6. Dephosphorylation of sperm guanylate cyclase during sea urchin fertilization

    SciTech Connect

    Ward, G.E.

    1985-01-01

    When intact Arbacia punctulata spermatozoa are exposed to solubilized egg jelly, the electrophoretic mobility of an abundant sperm flagellar membrane protein changes from an apparent molecular mass of 160 kDa to 150 kDa. A. punctulata spermatozoa can be labeled in vivo with /sup 32/P-labeled cells it was demonstrated that the mobility shift of the 160-kDa protein is due to dephosphorylation. The peptide resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH/sub 2/) is the component of egg jelly which is responsible for inducing the dephosphorylation. The 160/150-kdal sperm membrane protein has been purified to homogeneity by affinity chromatography on concanavalin A-agarose, and identified as sperm guanylate cyclase. The enzymatic activity of the guanylate cyclase is tightly coupled to its phosphorylation state. Resact has been shown to act as a potent chemoattractant for A. punctulata spermatozoa. The chemotactic response is concentration-dependent, is abolished by pretreatment of the spermatozoa with resact, and shows an absolute requirement for external calcium. This work represents the first demonstration of animal sperm chemotaxis in response to a precisely-defined molecule of egg origin. The results established a new, biologically meaningful function for resact, and may implicate sperm guanylate cyclase and cGMP in flagellar function and the chemotactic response.

  7. Efficient synthesis of stably adenylated DNA and RNA adapters for microRNA capture using T4 RNA ligase 1.

    PubMed

    Song, Yunke; Liu, Kelvin J; Wang, Tza-Huei

    2015-01-01

    MicroRNA profiling methods have become increasingly important due to the rapid rise of microRNA in both basic and translational sciences. A critical step in many microRNA profiling assays is adapter ligation using pre-adenylated adapters. While pre-adenylated adapters can be chemically or enzymatically prepared, enzymatic adenylation is preferred due to its ease and high yield. However, previously reported enzymatic methods either require tedious purification steps or use thermostable ligases that can generate side products during the subsequent ligation step. We have developed a highly efficient, template- and purification-free, adapter adenylation method using T4 RNA ligase 1. This method is capable of adenylating large amounts of adapter at ~100% efficiency and can efficiently adenylate both DNA and RNA bases. We find that the adenylation reaction speed can differ between DNA and RNA and between terminal nucleotides, leading to bias if reactions are not allowed to run to completion. We further find that the addition of high PEG levels can effectively suppress these differences. PMID:26500066

  8. ADP inhibits function of the ABC transporter cystic fibrosis transmembrane conductance regulator via its adenylate kinase activity.

    PubMed

    Randak, Christoph O; Welsh, Michael J

    2005-02-01

    ADP interacts with the nucleotide-binding domains (NBDs) of the cystic fibrosis transmembrane conductance regulator (CFTR) to inhibit its Cl- channel activity. Because CFTR NBD2 has reversible adenylate kinase activity (ATP + AMP<==> ADP + ADP) that gates the channel, we asked whether ADP might inhibit current through this enzymatic activity. In adenylate kinases, binding of the two ADP molecules is cooperative. Consistent with this hypothesis, CFTR current inhibition showed positive cooperativity for ADP. We also found that ADP inhibition of current was attenuated when we prevented adenylate kinase activity with P1,P5-di(adenosine-5') pentaphosphate. Additional studies suggested that adenylate kinase-dependent inhibition involved phosphotransfer between two nucleotide diphosphates. These data indicate that the adenylate kinase reaction at NBD2 contributed to the inhibitory effect of ADP. Finding that ADP inhibits function via an adenylate kinase activity also helps explain the earlier observation that mutations that disrupt adenylate kinase activity also disrupt ADP inhibition. Thus, the results reveal a previously unrecognized mechanism by which ADP inhibits an ABC transporter. PMID:15684079

  9. ADP inhibits function of the ABC transporter cystic fibrosis transmembrane conductance regulator via its adenylate kinase activity

    PubMed Central

    Randak, Christoph O.; Welsh, Michael J.

    2005-01-01

    ADP interacts with the nucleotide-binding domains (NBDs) of the cystic fibrosis transmembrane conductance regulator (CFTR) to inhibit its Cl- channel activity. Because CFTR NBD2 has reversible adenylate kinase activity (ATP + AMP ⇆ ADP + ADP) that gates the channel, we asked whether ADP might inhibit current through this enzymatic activity. In adenylate kinases, binding of the two ADP molecules is cooperative. Consistent with this hypothesis, CFTR current inhibition showed positive cooperativity for ADP. We also found that ADP inhibition of current was attenuated when we prevented adenylate kinase activity with P1,P5-di(adenosine-5′) pentaphosphate. Additional studies suggested that adenylate kinase-dependent inhibition involved phosphotransfer between two nucleotide diphosphates. These data indicate that the adenylate kinase reaction at NBD2 contributed to the inhibitory effect of ADP. Finding that ADP inhibits function via an adenylate kinase activity also helps explain the earlier observation that mutations that disrupt adenylate kinase activity also disrupt ADP inhibition. Thus, the results reveal a previously unrecognized mechanism by which ADP inhibits an ABC transporter. PMID:15684079

  10. Extracellular Regulation of Sperm Transmembrane Adenylyl Cyclase by a Forward Motility Stimulating Protein

    PubMed Central

    Dey, Souvik; Roy, Debarun; Majumder, Gopal C.; Bhattacharyya, Debdas

    2014-01-01

    Forward motility stimulating factor (FMSF), a glycoprotein isolated from buffalo serum, binds to the surface of the mature sperm cells to promote their progressive motility. This article reports the mode of signal transduction of this extracellular factor in goat sperm. The mechanism was investigated by assaying intracellular second messenger level and forward motility in presence of different pharmacological modulators. Mg++-dependent Forskolin responsive form of transmembrane adenylyl cyclase (tmAC) of goat spermatozoa was probed for its involvement in FMSF action. Dideoxyadenosine, a selective inhibitor of tmACs, was used to identify the role of this enzyme in the scheme of FMSF-signaling. Involvement of the α-subunit of G-protein in this regard has been inspected using GTPγS. Participation of protein kinase A (PKA) and tyrosine kinase was checked using IP20 and genistein, respectively. FMSF promotes tmAC activity in a dose-dependent manner through receptor/G-protein activation to enhance intracellular cAMP and forward motility. Motility boosting effects of this glycoprotein are almost lost in presence of dideoxyadenosine. But, FMSF displayed substantial motility promoting activity when movement of spermatozoa was inhibited with KH7, the specific inhibitor of soluble adenylyl cyclase indicating tmAC to be the primary target of FMSF action. Involvement of cAMP in mediating FMSF action was confirmed by the application of dibutyryl cAMP. Observed motility regulatory effects with IP20 and genistein indicate contribution of PKA and tyrosine kinase in FMSF activity; enhanced phosphorylation of a tyrosine containing ≈50 kDa protein was detected in this regard. FMSF initiates a novel signaling cascade to stimulate tmAC activity that augments intracellular cAMP, which through downstream crosstalk of phosphokinases leads to enhanced forward motility in mature spermatozoa. Thus, this article for the first time describes conventional tmAC-dependent profound activation

  11. Impairment of adenylyl cyclase-mediated glutamatergic synaptic plasticity in the periaqueductal grey in a rat model of neuropathic pain

    PubMed Central

    Ho, Yu-Cheng; Cheng, Jen-Kun; Chiou, Lih-Chu

    2015-01-01

    Key points Long-lasting neuropathic pain has been attributed to elevated neuronal plasticity changes in spinal, peripheral and cortical levels. Here, we found that reduced neuronal plasticity in the ventrolateral periaqueductal grey (vlPAG), a midbrain region important for initiating descending pain inhibition, may also contribute to neuropathic pain. Forskolin- and isoproterenol (isoprenaline)-elicited EPSC potentiation was impaired in the vlPAG of a rat model of neuropathic pain induced by spinal nerve injury. Down-regulation of adenylyl cyclase–cAMP– PKA signalling, due to impaired adenylyl cyclase, but not phosphodiesterase, in glutamatergic terminals may contribute to the hypofunction of excitatory synaptic plasticity in the vlPAG of neuropathic rats and the subsequent descending pain inhibition, ultimately leading to long-lasting neuropathic pain. Our results suggest that drugs that activate adenylyl cyclase in the vlPAG have the potential for relieving neuropathic pain. Abstract Neuropathic pain has been attributed to nerve injury-induced elevation of peripheral neuronal discharges and spinal excitatory synaptic plasticity while little is known about the contribution of neuroplasticity changes in the brainstem. Here, we examined synaptic plasticity changes in the ventrolateral (vl) periaqueductal grey (PAG), a crucial midbrain region for initiating descending pain inhibition, in spinal nerve ligation (SNL)-induced neuropathic rats. In vlPAG slices of sham-operated rats, forskolin, an adenylyl cyclase (AC) activator, produced long-lasting enhancement of EPSCs. This is a presynaptic effect since forskolin decreased the paired-pulse ratio and failure rate of EPSCs, and increased the frequency, but not the amplitude, of miniature EPSCs. Forskolin-induced EPSC potentiation was mimicked by a β-adrenergic agonist (isoproterenol (isoprenaline)), and prevented by an AC inhibitor (SQ 22536) and a cAMP-dependent protein kinase (PKA) inhibitor (H89), but not by a

  12. Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

    SciTech Connect

    Z Zhang; R Zhou; J Sauder; P Tonge; S Burley; S Swaminathan

    2011-12-31

    Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.

  13. Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

    SciTech Connect

    Zhang, Z.; Swaminathan, S.; Zhou, R.; Sauder, J. M.; Tonge, P. J.; Burley, S. K.

    2011-02-18

    Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.

  14. Key Role of the Adenylate Moiety and Integrity of the Adenylate-Binding Site for the NAD(+)/H Binding to Mitochondrial Apoptosis-Inducing Factor.

    PubMed

    Sorrentino, Luca; Calogero, Alessandra Maria; Pandini, Vittorio; Vanoni, Maria Antonietta; Sevrioukova, Irina F; Aliverti, Alessandro

    2015-12-01

    Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with pro-life and pro-death activities, which plays critical roles in mitochondrial energy metabolism and caspase-independent apoptosis. Defects in AIF structure or expression can cause mitochondrial abnormalities leading to mitochondrial defects and neurodegeneration. The mechanism of AIF-induced apoptosis was extensively investigated, whereas the mitochondrial function of AIF is poorly understood. A unique feature of AIF is the ability to form a tight, air-stable charge-transfer (CT) complex upon reaction with NADH and to undergo a conformational switch leading to dimerization, proposed to be important for its vital and lethal functions. Although some aspects of interaction of AIF with NAD(+)/H have been analyzed, its precise mechanism is not fully understood. We investigated how the oxidized and photoreduced wild-type and G307A and -E variants of murine AIF associate with NAD(+)/H and nicotinamide mononucleotide (NMN(+)/H) to determine the role of the adenylate moiety in the binding process. Our results indicate that (i) the adenylate moiety of NAD(+)/H is crucial for the association with AIF and for the subsequent structural reorganization of the complex, but not for protein dimerization, (ii) FAD reduction rather than binding of NAD(+)/H to AIF initiates conformational rearrangement, and (iii) alteration of the adenylate-binding site by the G307E (equivalent to a pathological G308E mutation in human AIF) or G307A replacements decrease the affinity and association rate of NAD(+)/H, which, in turn, perturbs CT complex formation and protein dimerization but has no influence on the conformational switch in the regulatory peptide. PMID:26535916

  15. Soluble Guanylate Cyclase Stimulators: a Novel Treatment Option for Heart Failure Associated with Cardiorenal Syndromes?

    PubMed

    Dubin, Ruth F; Shah, Sanjiv J

    2016-06-01

    Heart failure in the setting of chronic kidney disease (CKD) is an increasingly common scenario and carries a poor prognosis. Clinicians lack tools for primary or secondary heart failure prevention in patients with cardiorenal syndromes. In patients without CKD, angiotensin-converting enzyme inhibitors (ACE-I) or angiotensin receptor blockers (ARB) and statins mitigate cardiovascular risk in large part due to salutary effects on the endothelium. In the setting of CKD, use of these therapies is limited by adverse effects of hyperkalemia in pre-dialysis CKD (ACE-I/ARB), or potential increased risk of stroke in end-stage renal disease (statins). The soluble guanylate cyclase (sGC) stimulators are a novel class of medications that promote endothelial and myocardial function with no known risk of hyperkalemia or stroke. In this review, we discuss the evidence emerging from recent clinical trials of sGC stimulators in pulmonary hypertension and heart failure, the diseased pathways involved in cardiorenal syndromes likely to be restored by sGC stimulators, and several strategies for designing future clinical trials of cardiorenal syndromes that might shorten the timeline for discovery and approval of effective cardiovascular therapies in these high-risk patients. PMID:27118234

  16. Identification of Small Molecules That Antagonize Diguanylate Cyclase Enzymes To Inhibit Biofilm Formation

    PubMed Central

    Sambanthamoorthy, Karthik; Sloup, Rudolph E.; Parashar, Vijay; Smith, Joshua M.; Kim, Eric E.; Semmelhack, Martin F.; Neiditch, Matthew B.

    2012-01-01

    Bacterial biofilm formation is responsible for numerous chronic infections, causing a severe health burden. Many of these infections cannot be resolved, as bacteria in biofilms are resistant to the host's immune defenses and antibiotic therapy. New strategies to treat biofilm-based infections are critically needed. Cyclic di-GMP (c-di-GMP) is a widely conserved second-messenger signal essential for biofilm formation. As this signaling system is found only in bacteria, it is an attractive target for the development of new antibiofilm interventions. Here, we describe the results of a high-throughput screen to identify small-molecule inhibitors of diguanylate cyclase (DGC) enzymes that synthesize c-di-GMP. We report seven small molecules that antagonize these enzymes and inhibit biofilm formation by Vibrio cholerae. Moreover, two of these compounds significantly reduce the total concentration of c-di-GMP in V. cholerae, one of which also inhibits biofilm formation by Pseudomonas aeruginosa in a continuous-flow system. These molecules represent the first compounds described that are able to inhibit DGC activity to prevent biofilm formation. PMID:22850508

  17. Crystallization of the class IV adenylyl cyclase from Yersinia pestis

    SciTech Connect

    Smith, Natasha; Kim, Sook-Kyung; Reddy, Prasad T.; Gallagher, D. Travis

    2006-03-01

    The class IV adenylyl cyclase from Y. pestis has been crystallized in an orthorhombic form suitable for structure determination. The class IV adenylyl cyclase from Yersinia pestis has been cloned and crystallized in both a triclinic and an orthorhombic form. An amino-terminal His-tagged construct, from which the tag was removed by thrombin, crystallized in a triclinic form diffracting to 1.9 Å, with one dimer per asymmetric unit and unit-cell parameters a = 33.5, b = 35.5, c = 71.8 Å, α = 88.7, β = 82.5, γ = 65.5°. Several mutants of this construct crystallized but diffracted poorly. A non-His-tagged native construct (179 amino acids, MW = 20.5 kDa) was purified by conventional chromatography and crystallized in space group P2{sub 1}2{sub 1}2{sub 1}. These crystals have unit-cell parameters a = 56.8, b = 118.6, c = 144.5 Å, diffract to 3 Å and probably have two dimers per asymmetric unit and V{sub M} = 3.0 Å{sup 3} Da{sup −1}. Both crystal forms appear to require pH below 5, complicating attempts to incorporate nucleotide ligands into the structure. The native construct has been produced as a selenomethionine derivative and crystallized for phasing and structure determination.

  18. Receptor guanylyl cyclases in Inka cells targeted by eclosion hormone.

    PubMed

    Chang, Jer-Cherng; Yang, Ruey-Bing; Adams, Michael E; Lu, Kuang-Hui

    2009-08-11

    A signature of eclosion hormone (EH) action in insect ecdysis is elevation of cGMP in Inka cells, leading to massive release of ecdysis triggering hormone (ETH) and ecdysis initiation. Although this aspect of EH-induced signal transduction is well known, the receptor mediating this process has not been identified. Here, we describe a receptor guanylyl cyclase BdmGC-1 and its isoform BdmGC-1B in the Oriental fruit fly Bactrocera dorsalis that are activated by EH. The B form exhibits the conserved domains and putative N-glycosylation sites found in BdmGC-1, but possesses an additional 46-amino acid insertion in the extracellular domain and lacks the C-terminal tail of BdmGC-1. Combined immunolabeling and in situ hybridization reveal that BdmGC-1 is expressed in Inka cells. Heterologous expression of BdmGC-1 in HEK cells leads to robust increases in cGMP following exposure to low picomolar concentrations of EH. The B-isoform responds only to higher EH concentrations, suggesting different physiological roles of these cyclases. We propose that BdmGC-1 and BdmGC-1B are high- and low-affinity EH receptors, respectively. PMID:19666575

  19. Role of soluble adenylyl cyclase in the heart

    PubMed Central

    Chen, Jonathan; Levin, Lonny R.

    2012-01-01

    This review discusses the potential place of soluble adenylyl cyclase (sAC) in the framework of signaling in the cardiovascular system. cAMP has been studied as a critical and pleiotropic second messenger in cardiomyocytes, endothelial cells, and smooth muscle vascular cells for many years. It is involved in the transduction of signaling by catecholamines, prostaglandins, adenosine, and glucagon, just to name a few. These hormones can act via cAMP by binding to a G protein-coupled receptor on the plasma membrane with subsequent activation of a heterotrimeric G protein and its downstream effector, transmembrane adenylyl cyclase. This has long been the canonical standard for cAMP production in a cell. However, the relatively recent discovery of a unique source of cAMP, sAC, creates the potential for a shift in this signaling paradigm. In fact, sAC has been shown to play a role in apoptosis in coronary endothelial cells and cardiomyocytes. Additionally, it links nutrient utilization with ATP production in the liver and brain, which suggests one of many potential roles for sAC in cardiac function. The possibility of producing cAMP from a source distal to the plasma membrane provides a critical new building block for reconstructing the cellular signaling infrastructure. PMID:22058150

  20. The Crystal Structure of the Adenylation Enzyme VinN Reveals a Unique β-Amino Acid Recognition Mechanism*

    PubMed Central

    Miyanaga, Akimasa; Cieślak, Jolanta; Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2014-01-01

    Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp230 residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes. PMID:25246523

  1. The crystal structure of the adenylation enzyme VinN reveals a unique β-amino acid recognition mechanism.

    PubMed

    Miyanaga, Akimasa; Cieślak, Jolanta; Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2014-11-01

    Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp(230) residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes. PMID:25246523

  2. Role of CFTR's intrinsic adenylate kinase activity in gating of the Cl(-) channel.

    PubMed

    Randak, Christoph O; Welsh, Michael J

    2007-12-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-)channel in the ATP-binding cassette (ABC) transporter protein family. CFTR features the modular design characteristic of ABC transporters, which includes two membrane-spanning domains forming the channel pore, and two ABC nucleotide-binding domains that interact with ATP and contain the enzymatic activity coupled to normal gating. Like other ABC transporters CFTR is an ATPase (ATP + H(2)O --> ADP + Pi). Recent work has shown that CFTR also possesses intrinsic adenylate kinase activity (ATP + AMP left arrow over right arrow ADP + ADP). This finding raises important questions: How does AMP influence CFTR gating? Why does ADP inhibit CFTR current? Which enzymatic activity gates CFTR in vivo? Are there implications for other ABC transporters? This minireview attempts to shed light on these questions by summarizing recent advances in our understanding of the role of the CFTR adenylate kinase activity for channel gating. PMID:17965924

  3. Adenylyl cyclase regulation in heart failure due to myocardial infarction in rats.

    PubMed

    Bräunig, Jörg H; Albrecht-Küpper, Barbara; Seifert, Roland

    2014-04-01

    Cardiac adenylyl cyclase (AC) activity was described to be differentially regulated in left and right ventricles (LVs and RVs) of rats with heart failure (HF) due to LV myocardial infarction (MI) (Sethi et al. Am J Physiol 272:H884-H893, 1997). AC activities in LVs and RVs were increased and decreased respectively in rats 8 and 16 weeks post MI under basal and stimulatory conditions including AC activation via β-adrenergic receptors (β-ARs), stimulatory G protein (Gs), and direct AC activation with forskolin (FS). The current study aimed to detect alterations in rat heart AC activities in a comparable model of HF 9 weeks post LV MI. Therefore, cardiac AC activities were measured under basal and β-AR-, Gs-, or FS-stimulated conditions as well as under inhibition with various MANT [2'(3')-O-(N-methylanthraniloyl)]-nucleotide AC inhibitors and the P-site AC inhibitors NKY80 [2-amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazolinone] and vidarabine (9-β-D-arabinosyladenine, AraAde). Basal and stimulated AC activities along with AC inhibition experiments did not reveal evidence for changes in AC activity in LVs and RVs from MI group animals despite the presence of congestive HF. However, our study is indeterminate. Further studies are required to identify the factors responsible for previously described changes in cardiac AC activity in MI induced HF and to elucidate the role of altered AC regulation in the pathophysiology of HF. In order to detect small changes in AC regulation, larger group sizes than the ones used in our present study are required. PMID:24276219

  4. The Function of Guanylate Cyclase 1 and Guanylate Cyclase 2 in Rod and Cone Photoreceptors*S

    PubMed Central

    Baehr, Wolfgang; Karan, Sukanya; Maeda, Tadao; Luo, Dong-Gen; Li, Sha; Darin Bronson, J.; Watt, Carl B.; Yau, King-Wai; Frederick, Jeanne M.; Palczewski, Krzysztof

    2007-01-01

    Retinal guanylate cyclases 1 and 2 (GC1 and GC2) are responsible for synthesis of cyclic GMP in rods and cones, but their individual contributions to phototransduction are unknown. We report here that the deletion of both GC1 and GC2 rendered rod and cone photoreceptors nonfunctional and unstable. In the rod outer segments of GC double knock-out mice, guanylate cyclase-activating proteins 1 and 2, and cyclic GMP phosphodiesterase were undetectable, although rhodopsin and transducin α-subunit were mostly unaffected. Outer segment membranes of GC1−/− and GC double knock-out cones were destabilized and devoid of cone transducin (α- and γ-subunits), cone phosphodiesterase, and G protein-coupled receptor kinase 1, whereas cone pigments were present at reduced levels. Real time reverse transcription-PCR analyses demonstrated normal RNA transcript levels for the down-regulated proteins, indicating that down-regulation is posttranslational. We interpret these results to demonstrate an intrinsic requirement of GCs for stability and/or transport of a set of membrane-associated phototransduction proteins. PMID:17255100

  5. XLF-Cernunnos promotes DNA ligase IV-XRCC4 re-adenylation following ligation.

    PubMed

    Riballo, Enriqueta; Woodbine, Lisa; Stiff, Thomas; Walker, Sarah A; Goodarzi, Aaron A; Jeggo, Penny A

    2009-02-01

    XLF-Cernunnos (XLF) is a component of the DNA ligase IV-XRCC4 (LX) complex, which functions during DNA non-homologous end joining (NHEJ). Here, we use biochemical and cellular approaches to probe the impact of XLF on LX activities. We show that XLF stimulates adenylation of LX complexes de-adenylated by pyrophosphate or following LX decharging during ligation. XLF enhances LX ligation activity in an ATP-independent and dependent manner. ATP-independent stimulation can be attributed to enhanced end-bridging. Whilst ATP alone fails to stimulate LX ligation activity, addition of XLF and ATP promotes ligation in a manner consistent with XLF-stimulated readenylation linked to ligation. We show that XLF is a weakly bound partner of the tightly associated LX complex and, unlike XRCC4, is dispensable for LX stability. 2BN cells, which have little, if any, residual XLF activity, show a 3-fold decreased ability to repair DNA double strand breaks covering a range of complexity. These findings strongly suggest that XLF is not essential for NHEJ but promotes LX adenylation and hence ligation. We propose a model in which XLF, by in situ recharging DNA ligase IV after the first ligation event, promotes double stranded ligation by a single LX complex. PMID:19056826

  6. Role of soluble guanylyl cyclase-cyclic GMP signaling in tumor cell proliferation

    PubMed Central

    Mujoo, Kalpana; Sharin, Vladislav G.; Martin, Emil; Choi, Byung-Kwon; Sloan, Courtney; Nikonoff, Lubov E.; Kots, Alexander Y; Murad, Ferid

    2010-01-01

    Our previous studies demonstrate a differential expression of nitric oxide (NO) signaling components in ES cells and our recent study demonstrated an enhanced differentiation of ES cells into myocardial cells with NO donors and soluble guanylyl cyclase (sGC) activators. Since NO-cGMP pathway exhibits a diverse role in cancer, we were interested in evaluating the role of the NO receptor sGC and other components of the pathway in regulation of the tumor cell proliferation. Our results demonstrate a differential expression of the sGC subunits, NOS-1 and PKG mRNA and protein levels in various human cancer models. In contrast to sGCα1, robust levels of sGC β1 were observed in OVCAR-3 (ovarian) and MDA-MB-468 (breast) cancer cells which correlated well with the sGC activity and a marked increase in cGMP levels upon exposure to the combination of a NO donor and a sGC activator. NOC-18 (DETA NONOate; NO donor), BAY41-2272 (3-(4-Amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine); sGC activator), NOC-18+BAY41-2272, IBMX (3-Isobutyl-1-methylxanthine; phosphodiesterase inhibitor) and 8-bromo-cGMP (cGMP analog) caused growth inhibition and apoptosis in various cancer cell lines. To elucidate the molecular mechanisms involved in growth inhibition, we evaluated the effect of activators/inhibitors on ERK phosphorylation. Our studies indicate that BAY41-2272 or the combination NOC18+BAY41-2272 caused inhibition of the basal ERK1/2 phosphorylation in OVCAR-3 (high sGC activity), SK-OV-3 and SK-Br-3 (low sGC activity) cell lines and in some cases the inhibition was rescued by the sGC inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). These studies suggest that the effects of activators/inhibitors of NO-sGC-cGMP in tumor cell proliferation is mediated by both cGMP-dependent and independent mechanisms. PMID:19948239

  7. Targeting soluble guanylate cyclase for the treatment of pulmonary hypertension

    PubMed Central

    Lasker, George F; Maley, Jason H; Pankey, Edward A; Kadowitz, Philip J

    2011-01-01

    Pulmonary arterial hypertension is a disease characterized by a sustained increase in pulmonary arterial pressure leading to right heart failure. Current treatments focus on endothelial dysfunction and an aberrant regulatory pathway for vascular tone. Unfortunately, a large proportion of patients are unresponsive to conventional vasodilator therapy. Investigations are ongoing into the effects of experimental therapies targeting the signal transduction pathway that mediates vasodilation. Here, we briefly discuss the pathophysiology of pulmonary hypertension and endothelial dysfunction, along with current treatments. We then present a focused review of recent animal studies and human trials examining the use of activators and stimulators of soluble guanylate cyclase for the treatment of pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension. PMID:21510726

  8. Tetrahydrobiopterin protects soluble guanylate cyclase against oxidative inactivation.

    PubMed

    Schmidt, Kurt; Neubauer, Andrea; Kolesnik, Bernd; Stasch, Johannes-Peter; Werner, Ernst R; Gorren, Antonius C F; Mayer, Bernd

    2012-09-01

    Tetrahydrobiopterin (BH4) is a major endogenous vasoprotective agent that improves endothelial function by increasing nitric oxide (NO) synthesis and scavenging of superoxide and peroxynitrite. Therefore, administration of BH4 is considered a promising therapy for cardiovascular diseases associated with endothelial dysfunction and oxidative stress. Here we report on a novel function of BH4 that might contribute to the beneficial vascular effects of the pteridine. Treatment of cultured porcine aortic endothelial cells with nitroglycerin (GTN) or 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) resulted in heme oxidation of soluble guanylate cyclase (sGC), as evident from diminished NO-induced cGMP accumulation that was paralleled by increased cGMP response to a heme- and NO-independent activator of soluble guanylate cyclase [4-([(4-carboxybutyl)[2-(5-fluoro-2-([4'-(trifluoromethyl)biphenyl-4-yl]methoxy)phenyl)ethyl]amino]methyl)benzoic acid (BAY 60-2770)]. Whereas scavenging of superoxide and/or peroxynitrite with superoxide dismutase, tiron, Mn(III)tetrakis(4-benzoic acid)porphyrin, and urate had no protective effects, supplementation of the cells with BH4, either by application of BH4 directly or of its precursors dihydrobiopterin or sepiapterin, completely prevented the inhibition of NO-induced cGMP accumulation by GTN and ODQ. Tetrahydroneopterin had the same effect, and virtually identical results were obtained with RFL-6 fibroblasts, suggesting that our observation reflects a general feature of tetrahydropteridines that is unrelated to NO synthase function and not limited to endothelial cells. Protection of sGC against oxidative inactivation may contribute to the known beneficial effects of BH4 in cardiovascular disorders associated with oxidative stress. PMID:22648973

  9. Effect of 3' terminal adenylic acid residue on the uridylation of human small RNAs in vitro and in frog oocytes.

    PubMed Central

    Chen, Y; Sinha, K; Perumal, K; Reddy, R

    2000-01-01

    It is known that several small RNAs including human and Xenopus signal recognition particle (SRP) RNA, U2 small nuclear RNA (snRNA) and 7SK RNAs are posttranscriptionally adenylated, whereas U6 snRNA and ribosomal 5S RNA are posttranscriptionally uridylated on their 3' ends. In this study, we provide evidence that a small fraction of U6 snRNA and 5S ribosomal RNA molecules from human as well as Xenopus oocytes contain a single posttranscriptionally added adenylic acid residue on their 3' ends. These data show that U6 snRNA and 5S rRNAs are posttranscriptionally modified on their 3' ends by both uridylation and adenylation. Although the SRP RNA, 7SK RNA, 5S RNA, and U6 snRNA with the uridylic acid residue on their 3' ends were readily uridylated, all these RNAs with posttranscriptionally added adenylic acid residue on their 3' ends were not uridylated in vitro, or when U6 snRNA with 3' A(OH) was injected into Xenopus oocytes. These results show that the presence of a single posttranscriptionally added adenylic acid residue on the 3' end of SRP RNA, U6 snRNA, 5S rRNA, or 7SK RNA prevents 3' uridylation. These data also show that adenylation and uridylation are two competing processes that add nucleotides on the 3' end of some small RNAs and suggest that one of the functions of the 3' adenylation may be to negatively affect the 3' uridylation of small RNAs. PMID:10999605

  10. Effects of catecholamines on rat myocardial metabolism. II. Influence of catecholamines on 32p-incorporation into rat myocardial adenylic nucleotides and their turn-over.

    PubMed

    Merouze, P; Gaudemer, Y; Gautheron, D

    1975-01-01

    1. The influence of catecholamines (adrenaline and noradrenaline) on 32Pi incorporation into intracellular phosphate and adenylic nucleotides has been studied on rat myocardium slices; consequently, the turn-over of nucleotides could be determined and compared under the influence of these two hormones. 2. In order to specify the site of action of these catecholamines, several inhibitors and activators of energetic metabolism were included in the incubation medium: 3'5'-AMP, caffein, ouabain, oligomycin, rotenone + antimycin. 3. Both catecholamines favour Pi exchanges between intra and extracellular spaces; ATP turn-over is greatly increased, while ADP turn-over is slightly decreased, and 32P-incorporation into ADP is increased. 4. 3'5'-AMP and caffein are without effect on Pi penetration; however, caffein increases catecholamine effects on this penetration. ATP turn-over is slightly increased by 3'5'-AMP or caffein. 5. Ouabain decreases ATP turn-over but does not prevent the adrenaline induced acceleration. Inhibitors of oxidative phosphorylation and electron transport decrease ATP-turn-over severely; this inhibition is not released by catecholamines. 6. It is concluded that the catecholamine effects observed are dependent on the oxidative phosphorylations process. The increase of Pi exchange by catecholamines may be related to the increase of extracellular space and cation translocations we observed with the hormones. PMID:173417

  11. Structural and biochemical analysis of the essential diadenylate cyclase CdaA from Listeria monocytogenes.

    PubMed

    Rosenberg, Jonathan; Dickmanns, Achim; Neumann, Piotr; Gunka, Katrin; Arens, Johannes; Kaever, Volkhard; Stülke, Jörg; Ficner, Ralf; Commichau, Fabian M

    2015-03-01

    The recently identified second messenger cyclic di-AMP (c-di-AMP) is involved in several important cellular processes, such as cell wall metabolism, maintenance of DNA integrity, ion transport, transcription regulation, and allosteric regulation of enzyme function. Interestingly, c-di-AMP is essential for growth of the Gram-positive model bacterium Bacillus subtilis. Although the genome of B. subtilis encodes three c-di-AMP-producing diadenlyate cyclases that can functionally replace each other, the phylogenetically related human pathogens like Listeria monocytogenes and Staphylococcus aureus possess only one enzyme, the diadenlyate cyclase CdaA. Because CdaA is also essential for growth of these bacteria, the enzyme is a promising target for the development of novel antibiotics. Here we present the first crystal structure of the L. monocytogenes CdaA diadenylate cyclase domain that is conserved in many human pathogens. Moreover, biochemical characterization of the cyclase revealed an unusual metal cofactor requirement. PMID:25605729

  12. Structural and Biochemical Analysis of the Essential Diadenylate Cyclase CdaA from Listeria monocytogenes*

    PubMed Central

    Rosenberg, Jonathan; Dickmanns, Achim; Neumann, Piotr; Gunka, Katrin; Arens, Johannes; Kaever, Volkhard; Stülke, Jörg; Ficner, Ralf; Commichau, Fabian M.

    2015-01-01

    The recently identified second messenger cyclic di-AMP (c-di-AMP) is involved in several important cellular processes, such as cell wall metabolism, maintenance of DNA integrity, ion transport, transcription regulation, and allosteric regulation of enzyme function. Interestingly, c-di-AMP is essential for growth of the Gram-positive model bacterium Bacillus subtilis. Although the genome of B. subtilis encodes three c-di-AMP-producing diadenlyate cyclases that can functionally replace each other, the phylogenetically related human pathogens like Listeria monocytogenes and Staphylococcus aureus possess only one enzyme, the diadenlyate cyclase CdaA. Because CdaA is also essential for growth of these bacteria, the enzyme is a promising target for the development of novel antibiotics. Here we present the first crystal structure of the L. monocytogenes CdaA diadenylate cyclase domain that is conserved in many human pathogens. Moreover, biochemical characterization of the cyclase revealed an unusual metal cofactor requirement. PMID:25605729

  13. A Constitutively “Phosphorylated” Guanylyl Cyclase-linked Atrial Natriuretic Peptide Receptor Mutant Is Resistant to Desensitization

    PubMed Central

    Potter, Lincoln R.; Hunter, Tony

    1999-01-01

    Dephosphorylation of the natriuretic peptide receptor-A (NPR-A) is hypothesized to mediate its desensitization in response to atrial natriuretic peptide (ANP) binding. Recently, we identified six phosphorylation sites within the kinase homology domain of NPR-A and determined that the conversion of these residues to alanine abolished the ability of the receptor to be phosphorylated or to be activated by ANP and ATP. In an attempt to generate a form of NPR-A that mimics a fully phosphorylated receptor but that is resistant to dephosphorylation, we engineered a receptor variant (NPR-A-6E) containing glutamate substitutions at all six phosphorylation sites. Consistent with the known ability of negatively charged glutamate residues to substitute functionally, in some cases, for phosphorylated residues, we found that NPR-A-6E was activated 10-fold by ANP and ATP. As determined by guanylyl cyclase assays, the hormone-stimulated activity of the wild-type receptor declined over time in membrane preparations in vitro, and this loss was blocked by the serine/threonine protein phosphatase inhibitor microcystin. In contrast, the activity of NPR-A-6E was more linear with time and was unaffected by microcystin. The nonhydrolyzable ATP analogue adenosine 5′-(β,γ-imino)-triphosphate was half as effective as ATP in stimulating the wild-type receptor but was equally as potent in stimulating NPR-A-6E, suggesting that ATP is required to keep the wild-type but not 6E variant phosphorylated. Finally, the desensitization of NPR-A-6E in whole cells was markedly blunted compared with that of the wild-type receptor, consistent with its inability to shed the negative charge from its kinase homology domain via dephosphorylation. These data provide the first direct test of the requirement for dephosphorylation in guanylyl cyclase desensitization and they indicate that it is an essential component of this process. PMID:10359598

  14. Nicorandil stimulates a Na(+)/Ca(2+) exchanger by activating guanylate cyclase in guinea pig cardiac myocytes.

    PubMed

    Wei, Jiazhang; Watanabe, Yasuhide; Takeuchi, Kazuhiko; Yamashita, Kanna; Tashiro, Miyuki; Kita, Satomi; Iwamoto, Takahiro; Watanabe, Hiroshi; Kimura, Junko

    2016-04-01

    Nicorandil, a hybrid of an ATP-sensitive K(+) (KATP) channel opener and a nitrate generator, is used clinically for the treatment of angina pectoris. This agent has been reported to exert antiarrhythmic actions by abolishing both triggered activity and spontaneous automaticity in an in vitro study. It is well known that delayed afterdepolarizations (DADs) are caused by the Na(+)/Ca(2+) exchange current (I NCX). In this study, we investigated the effect of nicorandil on the cardiac Na(+)/Ca(2+) exchanger (NCX1). We used the whole-cell patch clamp technique and the Fura-2/AM (Ca(2+) indicator) method to investigate the effect of nicorandil on I NCX in isolated guinea pig ventricular myocytes and CCL39 fibroblast cells transfected with dog heart NCX1. Nicorandil enhanced I NCX in a concentration-dependent manner. The EC50 (half-maximum concentration for enhancement of the drug) values were 15.0 and 8.7 μM for the outward and inward components of I NCX, respectively. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), a membrane-permeable analog of guanosine 3',5'-cyclic monophosphate (cGMP), enhanced I NCX. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylate cyclase inhibitor (10 μM), completely abolished the nicorandil-induced I NCX increase. Nicorandil increased I NCX in CCL39 cells expressing wild-type NCX1 but did not affect mutant NCX1 without a long intracellular loop between transmembrane segments (TMSs) 5 and 6. Nicorandil at 100 μM abolished DADs induced by electrical stimulation with ouabain. Nicorandil enhanced the function of NCX1 via guanylate cyclase and thus may accelerate Ca(2+) exit via NCX1. This may partially contribute to the cardioprotection by nicorandil in addition to shortening action potential duration (APD) by activating KATP channels. PMID:26631169

  15. Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase

    SciTech Connect

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W.

    2011-09-20

    The structure of ent-copalyl diphosphate synthase reveals three {alpha}-helical domains ({alpha}, {beta} and {gamma}), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the {beta}{gamma} domains in ent-copalyl diphosphate synthase but exclusively in the {alpha} domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.

  16. [Soluble guanylate cyclase in the molecular mechanism underlying the therapeutic action of drugs].

    PubMed

    Piatakova, N V; Severina, I S

    2012-01-01

    The influence of ambroxol--a mucolytic drug--on the activity of human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase and activation of both enzymes by NO-donors (sodium nitroprusside and Sin-1) were investigated. Ambroxol in the concentration range from 0.1 to 10 microM had no effect on the basal activity of both enzymes. Ambroxol inhibited in a concentration-dependent manner the sodium nitroprusside-induced human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase with the IC50 values 3.9 and 2.1 microM, respectively. Ambroxol did not influence the stimulation of both enzymes by protoporphyrin IX. The influence of artemisinin--an antimalarial drug--on human platelet soluble guanylate cyclase activity and the enzyme activation by NO-donors were investigated. Artemisinin (0.1-100 microM) had no effect on the basal activity of the enzyme. Artemisinin inhibited in a concentration-dependent manner the sodium nitroprusside-induced activation of human platelet guanylate cyclase with an IC50 value 5.6 microM. Artemisinin (10 microM) also inhibited (by 71 +/- 4.0%) the activation of the enzyme by thiol-dependent NO-donor the derivative of furoxan, 3,4-dicyano-1,2,5-oxadiazolo-2-oxide (10 microM), but did not influence the stimulation of soluble guanylate cyclase by protoporphyrin IX. It was concluded that the sygnalling system NO-soluble guanylate cyclase-cGMP is involved in the molecular mechanism of the therapeutic action of ambroxol and artemisinin. PMID:22642150

  17. Mechanisms of relaxant activity of the nitric oxide-independent soluble guanylyl cyclase stimulator BAY 41-2272 in rat tracheal smooth muscle.

    PubMed

    Toque, Haroldo A; Mónica, Fabíola Z T; Morganti, Rafael P; De Nucci, Gilberto; Antunes, Edson

    2010-10-25

    The soluble guanylyl cyclase is expressed in airway smooth muscle, and agents that stimulate this enzyme activity cause airway smooth muscle relaxation and bronchodilation. The compound 5-Cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272) is a potent nitric oxide (NO)-independent soluble guanylyl cyclase stimulator, but little is known about its effects in airway smooth muscle. Therefore, this study aimed to investigate the mechanisms underlying the relaxations of rat tracheal smooth muscle induced by BAY 41-2272. Tracheal rings were mounted in 10-ml organ baths for isometric force recording. BAY 41-2272 concentration-dependently relaxed carbachol-precontracted tracheal rings (pEC(50)=6.68+/-0.14). Prior incubation with the NO synthesis inhibitor l-NAME (100 microM) or the soluble guanylyl cyclase inhibitor ODQ (10 microM) caused significant rightward shifts in the concentration-response curves to BAY 41-2272. Sodium nitroprusside caused concentration-dependent relaxations, which were greatly potentiated by BAY 41-2272 and completely inhibited by ODQ. In addition, BAY 41-2272 shifted to the right the tracheal contractile responses to either carbachol (0.01-1 microM) or electrical field stimulation (EFS, 1-32 Hz). BAY 41-2272 (1 microM) also caused a marked rightward shift and decreased the maximal contractile responses to extracellular CaCl2, and such effect was not modified by pretreatment with ODQ. In addition, BAY 41-2272 (up to 1 microM) significantly increased the cGMP levels, and that was abolished by ODQ. Our results indicate that BAY 41-2272 causes cGMP-dependent rat tracheal smooth muscle relaxations in a synergistic fashion with exogenous NO. BAY 41-2272 has also an additional mechanism independently of soluble guanylyl cyclase activation possibly involving Ca(2+) entry blockade. PMID:20670622

  18. 2'-Phosphate cyclase activity of RtcA: a potential rationale for the operon organization of RtcA with an RNA repair ligase RtcB in Escherichia coli and other bacterial taxa.

    PubMed

    Das, Ushati; Shuman, Stewart

    2013-10-01

    RNA terminal phosphate cyclase catalyzes the ATP-dependent conversion of a 3'-phosphate RNA end to a 2',3'-cyclic phosphate via covalent enzyme-(histidinyl-Nε)-AMP and RNA(3')pp(5')A intermediates. Here, we report that Escherichia coli RtcA (and its human homolog Rtc1) are capable of cyclizing a 2'-phosphate RNA end in high yield. The rate of 2'-phosphate cyclization by RtcA is five orders of magnitude slower than 3'-phosphate cyclization, notwithstanding that RtcA binds with similar affinity to RNA3'p and RNA2'p substrates. These findings expand the functional repertoire of RNA cyclase and suggest that phosphate geometry during adenylate transfer to RNA is a major factor in the kinetics of cyclization. RtcA is coregulated in an operon with an RNA ligase, RtcB, that splices RNA 5'-OH ends to either 3'-phosphate or 2',3'-cyclic phosphate ends. Our results suggest that RtcA might serve an end healing function in an RNA repair pathway, by converting RNA 2'-phosphates, which cannot be spliced by RtcB, to 2',3'-cyclic phosphates that can be sealed. The rtcBA operon is controlled by the σ(54) coactivator RtcR encoded by an adjacent gene. This operon arrangement is conserved in diverse bacterial taxa, many of which have also incorporated the RNA-binding protein Ro (which is implicated in RNA quality control under stress conditions) as a coregulated component of the operon. PMID:23945037

  19. Prenatal exposure to cocaine decreases adenylyl cyclase activity in embryonic mouse striatum.

    PubMed

    Unterwald, Ellen M; Ivkovic, Sanja; Cuntapay, Marie; Stroppolo, Antonella; Guinea, Barbara; Ehrlich, Michelle E

    2003-12-30

    Adenylyl cyclase activity was measured in the striatum of naive mice as a function of age and in mice exposed in utero to cocaine. In naive Swiss-Webster mice, basal and forskolin-stimulated adenylyl cyclase activity increased gradually from embryonic day 13 (E13) until 2-3 weeks of age when activity peaked before decreasing slightly to adult levels. The ability of the dopamine D1 receptor agonist, SKF 82958, to stimulate adenylyl cyclase activity also increased in magnitude until P15. In a separate study, pregnant Swiss-Webster mice were injected twice daily with cocaine (15 mg/kg, s.c.) or an equal volume of saline from E10 to E17. Adenylyl cyclase activity was measured in the striatum of E18 embryos. Basal adenylyl cyclase activity was significantly reduced following prenatal exposure to cocaine. Likewise, the ability of forskolin or SKF 82958 to stimulate adenylyl cyclase was attenuated following cocaine exposure. DeltaFosB was not induced, contrary to what is seen in adult mice. These results demonstrate a functional change in a critical signal transduction pathway following chronic in utero exposure to cocaine that might have profound effects of the development of the brain. Alterations in the cAMP system may underlie some of the deficits seen in humans exposed in utero to cocaine. PMID:14741752

  20. Cloning, chromosomal mapping, and expression of human fetal brain type I adenylyl cyclase

    SciTech Connect

    Villacres, E.C.; Xia, Z.; Bookbinder, L.H.; Edelhoff, S.; Disteche, C.M.; Storm, D.R.

    1993-05-01

    The neural-specific calmodulin-sensitive adenylyl cyclase (type I), which was first cloned from bovine brain, has been implicated in learning and memory. The objective of this study was to clone and determine the chromosomal localization of human fetal brain type I adenylyl cyclase. A 3.8-kb cDNA clone was isolated that contained sequence coinciding with the 3{prime} end 2553 nucleotides of the bovine open reading frame. This clone shows 87% nucleotide and 92% translated amino acid sequence identity to the bovine clone. The most significant sequence differences were in the carboxy-terminal 100 amino acid residues. This region contains one of several possible calmodulin binding domains and the only putative cAMP-dependent protein kinase A phosphorylation site. A chimera was constructed that contained the 5{prime} half of the bovine type I adenylyl cyclase and the 3{prime} half of the human type I adenylyl cyclase. The activity of the chimeric gene product and its sensitivity to calmodulin and calcium were indistinguishable from those of the bovine type I adenylyl cyclase. In situ hybridization was used to localize the human type I adenylyl cyclase gene to the proximal portion of the short arm of chromosome 7. 36 refs., 4 figs.

  1. Bithionol Potently Inhibits Human Soluble Adenylyl Cyclase through Binding to the Allosteric Activator Site.

    PubMed

    Kleinboelting, Silke; Ramos-Espiritu, Lavoisier; Buck, Hannes; Colis, Laureen; van den Heuvel, Joop; Glickman, J Fraser; Levin, Lonny R; Buck, Jochen; Steegborn, Clemens

    2016-04-29

    The signaling molecule cAMP regulates functions ranging from bacterial transcription to mammalian memory. In mammals, cAMP is synthesized by nine transmembrane adenylyl cyclases (ACs) and one soluble AC (sAC). Despite similarities in their catalytic domains, these ACs differ in regulation. Transmembrane ACs respond to G proteins, whereas sAC is uniquely activated by bicarbonate. Via bicarbonate regulation, sAC acts as a physiological sensor for pH/bicarbonate/CO2, and it has been implicated as a therapeutic target, e.g. for diabetes, glaucoma, and a male contraceptive. Here we identify the bisphenols bithionol and hexachlorophene as potent, sAC-specific inhibitors. Inhibition appears mostly non-competitive with the substrate ATP, indicating that they act via an allosteric site. To analyze the interaction details, we solved a crystal structure of an sAC·bithionol complex. The structure reveals that the compounds are selective for sAC because they bind to the sAC-specific, allosteric binding site for the physiological activator bicarbonate. Structural comparison of the bithionol complex with apo-sAC and other sAC·ligand complexes along with mutagenesis experiments reveals an allosteric mechanism of inhibition; the compound induces rearrangements of substrate binding residues and of Arg(176), a trigger between the active site and allosteric site. Our results thus provide 1) novel insights into the communication between allosteric regulatory and active sites, 2) a novel mechanism for sAC inhibition, and 3) pharmacological compounds targeting this allosteric site and utilizing this mode of inhibition. These studies provide support for the future development of sAC-modulating drugs. PMID:26961873

  2. Characterization and Inhibition of a Class II Diterpene Cyclase from Mycobacterium tuberculosis

    PubMed Central

    Mann, Francis M.; Prisic, Sladjana; Hu, Huayou; Xu, Meimei; Coates, Robert M.; Peters, Reuben J.

    2009-01-01

    Mycobacterium tuberculosis remains a widespread and devastating human pathogen, whose ability to infiltrate macrophage host cells from the human immune system is an active area of investigation. We have recently reported the discovery of a novel diterpene from M. tuberculosis, edaxadiene, whose ability to arrest phagosomal maturation in isolation presumably contributes to this critical process in M. tuberculosis infections. (Mann, F. M., Xu, M., Chen, X., Fulton, D. B., Russell, D. G., and Peters, R. J. (2009) J. Am. Chem. Soc., in press). Here, we present characterization of the class II diterpene cyclase that catalyzes the committed step in edaxadiene biosynthesis, i.e. the previously identified halimadienyl-diphosphate synthase (HPS; EC 5.5.1.16). Intriguingly, our kinetic analysis suggests a potential biochemical regulatory mechanism that triggers edaxadiene production upon phagosomal engulfment. Furthermore, we report characterization of potential HPS inhibitors: specifically, two related transition state analogs (15-aza-14,15-dihydrogeranylgeranyl diphosphate (7a) and 15-aza-14,15-dihydrogeranylgeranyl thiolodiphosphate (7b)) that exhibit very tight binding. Although arguably not suitable for clinical use, these nevertheless provide a basis for pharmaceutical design against this intriguing biosynthetic pathway. Finally, we provide evidence indicating that this pathway exists only in M. tuberculosis and is not functional in the closely related Mycobacterium bovis because of an inactivating frameshift in the HPS-encoding gene. Thus, we hypothesize that the inability to produce edaxadiene may be a contributing factor in the decreased infectivity and/or virulence of M. bovis relative to M. tuberculosis in humans. PMID:19574210

  3. Functional properties of a naturally occurring isoform of soluble guanylyl cyclase.

    PubMed Central

    Russwurm, M; Behrends, S; Harteneck, C; Koesling, D

    1998-01-01

    Soluble guanylyl cyclase (sGC), the target enzyme of the signalling molecule NO, contains one prosthetic haem group and consists of an alpha and a beta subunit. So far, only the alpha1beta1 heterodimer has been shown to exist in different cells and tissues, and most biochemical studies of sGC have been performed with the alpha1 beta1 heterodimer. Here we demonstrate for the first time the natural occurrence of the alpha2 subunit on the protein level. The alpha2 subunit co-precipitated with the beta1 subunit from human placenta, showing the existence of the alpha2 beta1 isoform in vivo. The new enzyme was expressed in and purified from cells from the Spodoptera frugiperda ovary cell line Sf 9. Spectral analysis showed that the alpha2 beta1 heterodimer contains a prosthetic haem group revealing the same characteristics as the haem in the alpha1 beta1 form. The kinetic properties of both isoforms and sensitivity towards NO were indistinguishable. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of sGC, abolished NO-stimulated activity of both heterodimers. The new NO-independent activator, 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), increased the maximal NO-stimulated activity of the new isoform, caused a leftward-shift in the NO concentration-response curve and turned CO into an effective activator, as it did for the alpha1 beta1 heterodimer (200-fold activation). In summary, the differences in primary structure of both alpha subunits are contrasted by their functional similarity. Further studies will be needed to elucidate the physiological purpose of the new isoform. PMID:9742221

  4. On the Dynamics of the Adenylate Energy System: Homeorhesis vs Homeostasis

    PubMed Central

    De la Fuente, Ildefonso M.; Cortés, Jesús M.; Valero, Edelmira; Desroches, Mathieu; Rodrigues, Serafim; Malaina, Iker; Martínez, Luis

    2014-01-01

    Biochemical energy is the fundamental element that maintains both the adequate turnover of the biomolecular structures and the functional metabolic viability of unicellular organisms. The levels of ATP, ADP and AMP reflect roughly the energetic status of the cell, and a precise ratio relating them was proposed by Atkinson as the adenylate energy charge (AEC). Under growth-phase conditions, cells maintain the AEC within narrow physiological values, despite extremely large fluctuations in the adenine nucleotides concentration. Intensive experimental studies have shown that these AEC values are preserved in a wide variety of organisms, both eukaryotes and prokaryotes. Here, to understand some of the functional elements involved in the cellular energy status, we present a computational model conformed by some key essential parts of the adenylate energy system. Specifically, we have considered (I) the main synthesis process of ATP from ADP, (II) the main catalyzed phosphotransfer reaction for interconversion of ATP, ADP and AMP, (III) the enzymatic hydrolysis of ATP yielding ADP, and (IV) the enzymatic hydrolysis of ATP providing AMP. This leads to a dynamic metabolic model (with the form of a delayed differential system) in which the enzymatic rate equations and all the physiological kinetic parameters have been explicitly considered and experimentally tested in vitro. Our central hypothesis is that cells are characterized by changing energy dynamics (homeorhesis). The results show that the AEC presents stable transitions between steady states and periodic oscillations and, in agreement with experimental data these oscillations range within the narrow AEC window. Furthermore, the model shows sustained oscillations in the Gibbs free energy and in the total nucleotide pool. The present study provides a step forward towards the understanding of the fundamental principles and quantitative laws governing the adenylate energy system, which is a fundamental element for

  5. Adenylate Energy Pool and Energy Charge in Maturing Rape Seeds 1

    PubMed Central

    Ching, Te May; Crane, Jim M.; Stamp, David L.

    1974-01-01

    A study of energy state and chemical composition of pod walls and seeds of maturing rape (Brassica napus L.) was conducted on two varieties, Victor and Gorczanski. Total adenosine phosphates, ATP, and adenylate energy charge increased with increasing cell number and cellular synthesis during the early stages, remained high at maximum dry weight accumulation and maximum substrate influx time, and decreased with ripening. A temporal control of energy supply and ATP concentration is evident in developing tissues with determined functions; whereas the association of a high energy charge and active cellular biosynthesis occurs only in tissues with a stabilized cell number. PMID:16658964

  6. Clinico-pathological correlation in adenylate kinase 5 autoimmune limbic encephalitis.

    PubMed

    Ng, Adeline S L; Kramer, Joel; Centurion, Alejandro; Dalmau, Josep; Huang, Eric; Cotter, Jennifer A; Geschwind, Michael D

    2015-10-15

    Autoantibodies associated with autoimmune limbic encephalitis (ALE) have been well-characterized, with intracellular neuronal antibodies being less responsive to immunotherapy than antibodies to cell surface antigens. Adenylate kinase 5 (AK5) is a nucleoside monophosphate kinase vital for neuronal-specific metabolism and is located intracellularly in the cytosol and expressed exclusively in the brain. Antibodies to AK5 had been previously identified but were not known to be associated with human disease prior to the report of two patients with AK5-related ALE (Tuzun et al., 2007). We present the complete clinical picture for one of these patients and the first reported neuropathology for AK5 ALE. PMID:26439959

  7. Pyridopyrimidine derivatives as inhibitors of cyclic nucleotide synthesis: Application for treatment of diarrhea

    PubMed Central

    Kots, Alexander Y.; Choi, Byung-Kwon; Estrella-Jimenez, Maria E.; Warren, Cirle A.; Gilbertson, Scott R.; Guerrant, Richard L.; Murad, Ferid

    2008-01-01

    Acute secretory diarrhea induced by infection with enterotoxigenic strains of Escherichia coli involves binding of stable toxin (STa) to its receptor on the intestinal brush border, guanylyl cyclase type C (GC-C). Intracellular cGMP is elevated, inducing increase in chloride efflux and subsequent accumulation of fluid in the intestinal lumen. We have screened a library of compounds and identified a pyridopyrimidine derivatives {5-(3-bromophenyl)-1,3-dimethyl-5,11-dihydro-1H-indeno[2′,1′:5,6]pyrido[2,3-d]pyrimidine-2,4,6-trione; BPIPP} as an inhibitor of GC-C that can suppress STa-stimulated cGMP accumulation by decreasing GC-C activation in intact T84 human colorectal carcinoma cells. BPIPP inhibited stimulation of guanylyl cyclases, including types A and B and soluble isoform in various cells. BPIPP suppressed stimulation of adenylyl cyclase and significantly decreased the activities of adenylyl cyclase toxin of Bordetella pertussis and edema toxin of Bacillus anthracis. The effects of BPIPP on cyclic nucleotide synthesis were observed only in intact cells. The mechanism of BPIPP-dependent inhibition appears to be complex and indirect, possibly associated with phospholipase C and tyrosine-specific phosphorylation. BPIPP inhibited chloride-ion transport stimulated by activation of guanylyl or adenylyl cyclases and suppressed STa-induced fluid accumulation in an in vivo rabbit intestinal loop model. Thus, BPIPP may be a promising lead compound for treatment of diarrhea and other diseases. PMID:18559851

  8. Conventional and Unconventional Mechanisms for Soluble Guanylyl Cyclase Signaling.

    PubMed

    Gao, Yuansheng

    2016-05-01

    Soluble guanylyl cyclase (sGC) is the principal enzyme in mediating the biological actions of nitric oxide. On activation, sGC converts guanosine triphosphate to guanosine 3',5'-cyclic monophosphate (cGMP), which mediates diverse physiological processes including vasodilation, platelet aggregation, and myocardial functions predominantly by acting on cGMP-dependent protein kinases. Cyclic GMP has long been considered as the sole second messenger for sGC action. However, emerging evidence suggests that, in addition to cGMP, other nucleoside 3',5'-cyclic monophosphates (cNMPs) are synthesized by sGC in response to nitric oxide stimulation, and some of these nucleoside 3',5'-cyclic monophosphates are involved in various physiological activities. For example, inosine 3',5'-cyclic monophosphate synthesized by sGC may play a critical role in hypoxic augmentation of vasoconstriction. The involvement of cytidine 3',5'-cyclic monophosphate and uridine 3',5'-cyclic monophosphate in certain cardiovascular activities is also implicated. PMID:26452163

  9. Human recombinant soluble guanylyl cyclase: Expression, purification, and regulation

    PubMed Central

    Lee, Yu-Chen; Martin, Emil; Murad, Ferid

    2000-01-01

    The α1- and β1-subunits of human soluble guanylate cyclase (sGC) were coexpressed in the Sf9 cells/baculovirus system. In addition to the native enzyme, constructs with hexahistidine tag at the amino and carboxyl termini of each subunit were coexpressed. This permitted the rapid and efficient purification of active recombinant enzyme on a nickel-affinity column. The enzyme has one heme per heterodimer and was readily activated with the NO donor sodium nitroprusside or 3-(5′-hydroxymethyl-2′furyl)-1-benzyl-indazole (YC-1). Sodium nitroprusside and YC-1 treatment potentiated each other in combination and demonstrated a remarkable 2,200-fold stimulation of the human recombinant sGC. The effects were inhibited with 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1one (ODQ). The kinetics of the recombinant enzyme with respect to GTP was examined. The products of the reaction, cGMP and pyrophosphate, inhibited the enzyme. The extent of inhibition by cGMP depended on the activation state of the enzyme, whereas inhibition by pyrophosphate was not affected by the enzyme state. Both reaction products displayed independent binding and cooperativity with respect to enzyme inhibition. The expression of large quantities of active enzyme will facilitate structural characterization of the protein. PMID:10995472

  10. Catalytic Mechanism of Mammalian Adenylyl Cyclase: A Computational Investigation.

    PubMed

    Hahn, David K; Tusell, Jose R; Sprang, Stephen R; Chu, Xi

    2015-10-13

    Adenylyl cyclase (AC) catalyzes the synthesis of cyclic AMP, an important intracellular regulatory molecule, from ATP. We propose a catalytic mechanism for class III mammalian AC based on density functional theory calculations. We employ a model of the AC active site derived from a crystal structure of mammalian AC activated by Gα·GTP and forskolin at separate allosteric sites. We compared the calculated activation free energies for 13 possible reaction sequences involving proton transfer, nucleophilic attack, and elimination of pyrophosphate. The proposed most probable mechanism is initiated by deprotonation of 3'OH and water-mediated transfer of the 3'H to the γ-phosphate. Proton transfer is followed by changes in coordination of the two magnesium ion cofactors and changes in the conformation of ATP to enhance the role of 3'O as a nucleophile and to bring 3'O close to Pα. The subsequent phosphoryl transfer step is concerted and rate-limiting. Comparison of the enzyme-catalyzed and nonenzymatic reactions reveals that the active site residues lower the free energy barrier for both phosphoryl transfer and proton transfer and significantly shift the proton transfer equilibrium. Calculations for mutants K1065A and R1029A demonstrate that K1065 plays a significant role in shifting the proton transfer equilibrium, whereas R1029 is important for making the transition state of concerted phosphoryl transfer tight rather than loose. PMID:26393535

  11. Structure of RNA 3'-phosphate cyclase bound to substrate RNA.

    PubMed

    Desai, Kevin K; Bingman, Craig A; Cheng, Chin L; Phillips, George N; Raines, Ronald T

    2014-10-01

    RNA 3'-phosphate cyclase (RtcA) catalyzes the ATP-dependent cyclization of a 3'-phosphate to form a 2',3'-cyclic phosphate at RNA termini. Cyclization proceeds through RtcA-AMP and RNA(3')pp(5')A covalent intermediates, which are analogous to intermediates formed during catalysis by the tRNA ligase RtcB. Here we present a crystal structure of Pyrococcus horikoshii RtcA in complex with a 3'-phosphate terminated RNA and adenosine in the AMP-binding pocket. Our data reveal that RtcA recognizes substrate RNA by ensuring that the terminal 3'-phosphate makes a large contribution to RNA binding. Furthermore, the RNA 3'-phosphate is poised for in-line attack on the P-N bond that links the phosphorous atom of AMP to N(ε) of His307. Thus, we provide the first insights into RNA 3'-phosphate termini recognition and the mechanism of 3'-phosphate activation by an Rtc enzyme. PMID:25161314

  12. Expression of soluble adenylyl cyclase in acral melanomas.

    PubMed

    Li, H; Kim, S M; Savkovic, V; Jin, S A; Choi, Y D; Yun, S J

    2016-06-01

    Soluble adenylyl cyclase (sAC) regulates melanocytic cells, and is a diagnostic marker for pigmented skin lesions. Because only a few studies on sAC expression in acral melanomas have been performed, we investigated the histopathological significance of sAC expression in 33 cases of acral melanoma, and assessed its diagnostic value in distinguishing melanoma in situ (MIS, n = 17) from acral invasive melanomas (n = 16) and melanocytic naevi (n = 11). Acral melanomas exhibited more marked nuclear immunopositivity compared with acral melanocytic naevi. sAC expression significantly correlated with the nuclear morphology of melanocytes and melanoma cells, namely, hyperchromatic nuclei and prominent nucleoli within vesicular nuclei. sAC expression was predominantly observed in the hyperchromatic nuclei of MIS and the prominent nucleoli invasive melanomas, respectively. In vitro culture models of melanocytes and melanoma cell lines exhibited sAC staining patterns similar to those of acral melanomas. Differentiation induction showed that nuclear and nucleolar expression varied depending on cell morphology. sAC immunostaining may be useful for the differential diagnosis of acral melanocytic lesions, and sAC expressed in the nucleus and nucleolus might be related to cytological and nuclear changes associated with invasion and progression of acral melanomas. PMID:26290224

  13. Human recombinant soluble guanylyl cyclase: expression, purification, and regulation

    NASA Technical Reports Server (NTRS)

    Lee, Y. C.; Martin, E.; Murad, F.

    2000-01-01

    The alpha1- and beta1-subunits of human soluble guanylate cyclase (sGC) were coexpressed in the Sf9 cells/baculovirus system. In addition to the native enzyme, constructs with hexahistidine tag at the amino and carboxyl termini of each subunit were coexpressed. This permitted the rapid and efficient purification of active recombinant enzyme on a nickel-affinity column. The enzyme has one heme per heterodimer and was readily activated with the NO donor sodium nitroprusside or 3-(5'-hydroxymethyl-2'furyl)-1-benzyl-indazole (YC-1). Sodium nitroprusside and YC-1 treatment potentiated each other in combination and demonstrated a remarkable 2,200-fold stimulation of the human recombinant sGC. The effects were inhibited with 1H-(1,2, 4)oxadiazole(4,3-a)quinoxalin-1one (ODQ). The kinetics of the recombinant enzyme with respect to GTP was examined. The products of the reaction, cGMP and pyrophosphate, inhibited the enzyme. The extent of inhibition by cGMP depended on the activation state of the enzyme, whereas inhibition by pyrophosphate was not affected by the enzyme state. Both reaction products displayed independent binding and cooperativity with respect to enzyme inhibition. The expression of large quantities of active enzyme will facilitate structural characterization of the protein.

  14. Pharmacokinetic interaction profile of riociguat, a new soluble guanylate cyclase stimulator, in vitro.

    PubMed

    Rickert, Verena; Haefeli, Walter Emil; Weiss, Johanna

    2014-08-01

    Riociguat is a new soluble guanylate cyclase stimulator under development for pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension. So far, the interaction potential of riociguat with other drugs is nearly unknown. Therefore, we assessed in vitro the potency of riociguat to inhibit important drug metabolising enzymes (cytochrome P450 (CYP) 3A4, CYP2C19, and CYP2D6) and drug transporters (P-glycoprotein (P-gp/ABCB1), breast cancer resistance protein (BCRP/ABCG2), and organic anion transporting polypeptides (OATP) 1B1 and 1B3). In addition we evaluated its substrate characteristics for P-gp, BCRP, and the multidrug resistance-associated protein 1 (MRP1/ABCC1). We also assessed riociguat's inducing properties on important drug metabolising enzymes and transporters and investigated its ability to activate the pregnane-X-receptor (PXR). Riociguat was identified as a weak to moderate inhibitor of P-gp (f2-value: 11.7 ± 4.8 μM), BCRP (IC50 = 46.2 ± 20.3 μM), OATP1B1 (IC50 = 34.1 ± 3.15 μM), OATP1B3 (IC50 = 50.3 ± 7.5 μM), CYP2D6 (IC50 = 12.4 ± 0.74 μM), and CYP2C19 (IC50 = 46.1 ± 7.14 μM). Furthermore, it induced mRNA expression of BCRP/ABCG2 (3-fold at 20 μM) and to a lesser extent of CYP3A4 (2.3-fold at 20 μM), UGT1A4, and ABCB11. The only weak inducing properties were confirmed by weak activation of PXR. Considering its systemic concentrations its interaction potential as a perpetrator drug seems to be low. In contrast, our data suggest that riociguat is a P-gp substrate and might therefore act as a victim drug when co-administered with strong P-gp inductors or inhibitors. PMID:24657506

  15. Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120.

    PubMed

    Frébortová, Jitka; Greplová, Marta; Seidl, Michael F; Heyl, Alexander; Frébort, Ivo

    2015-01-01

    Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants. PMID:26376297

  16. Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120

    PubMed Central

    Frébortová, Jitka; Greplová, Marta; Seidl, Michael F.; Heyl, Alexander; Frébort, Ivo

    2015-01-01

    Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants. PMID:26376297

  17. Adenylate kinase isozyme 2 is essential for growth and development of Drosophila melanogaster.

    PubMed

    Fujisawa, Koichi; Murakami, Ryutaro; Horiguchi, Taigo; Noma, Takafumi

    2009-05-01

    Adenylate kinases are phylogenetically widespread, highly conserved, and involved in energy metabolism and energy transfer. Of these, adenylate kinase (AK) isozyme 2 is uniquely localized in the mitochondrial intermembrane space and its physiological role remains largely unknown. In this study, we selected Drosophila melanogaster to analyze its role in vivo. AK isozyme cDNAs were cloned and their gene expressions were characterized in D. melanogaster. The deduced amino acid sequences contain highly conserved motifs for P-loop, NMP binding, and LID domains of AKs. In addition, the effects of AK2 gene knockout on phenotype of AK2 mutants were examined using P-element technology. Although homozygous AK2 mutated embryos developed without any visible defects, their growth ceased and they died before reaching the third instar larval stage. Maternally provided AK2 mRNA was detected in fertilized eggs, and weak AK2 activity was observed in first and second instar larvae of the homozygous AK2 mutants, suggesting that maternally provided AK2 is sufficient for embryonic development. Disappearance of AK2 activity during larval stages resulted in growth arrest and eventual death. These results demonstrate that AK2 plays a critical role in adenine nucleotide metabolism in the mitochondrial intermembrane space and is essential for growth in D. melanogaster. PMID:19416704

  18. Crystal structure of histidyl-tRNA synthetase from Escherichia coli complexed with histidyl-adenylate.

    PubMed Central

    Arnez, J G; Harris, D C; Mitschler, A; Rees, B; Francklyn, C S; Moras, D

    1995-01-01

    The crystal structure at 2.6 A of the histidyl-tRNA synthetase from Escherichia coli complexed with histidyl-adenylate has been determined. The enzyme is a homodimer with a molecular weight of 94 kDa and belongs to the class II of aminoacyl-tRNA synthetases (aaRS). The asymmetric unit is composed of two homodimers. Each monomer consists of two domains. The N-terminal catalytic core domain contains a six-stranded antiparallel beta-sheet sitting on two alpha-helices, which can be superposed with the catalytic domains of yeast AspRS, and GlyRS and SerRS from Thermus thermophilus with a root-mean-square difference on the C alpha atoms of 1.7-1.9 A. The active sites of all four monomers are occupied by histidyl-adenylate, which apparently forms during crystallization. The 100 residue C-terminal alpha/beta domain resembles half of a beta-barrel, and provides an independent domain oriented to contact the anticodon stem and part of the anticodon loop of tRNA(His). The modular domain organization of histidyl-tRNA synthetase reiterates a repeated theme in aaRS, and its structure should provide insight into the ability of certain aaRS to aminoacylate minihelices and other non-tRNA molecules. Images PMID:7556055

  19. Leveraging the Mechanism of Oxidative Decay for Adenylate Kinase to Design Structural and Functional Resistances

    PubMed Central

    Howell, Stanley C.; Richards, David H.; Mitch, William A.; Wilson, Corey J.

    2016-01-01

    Characterization of the mechanisms underlying hypohalous acid (i.e., hypochlorous acid or hypobromous acid) degradation of proteins is important for understanding how the immune system deactivates pathogens during infections, and damages human tissues during inflammatory diseases. Proteins are particularly important hypohalous acid reaction targets in pathogens and in host tissues, as evidenced by the detection of chlorinated and brominated oxidizable residues. While a significant amount of work has been conducted for reactions of hypohalous acids with a range of individual amino acids and small peptides, the assessment of oxidative decay in full-length proteins has lagged in comparison. The most rigorous test of our understanding of oxidative decay of proteins is the rational redesign of proteins with conferred resistances to the decay of structure and function. Toward this end, in this study we experimentally determined a putative mechanism of oxidative decay using adenylate kinase as the model system. In turn, we leveraged this mechanism to rationally design new proteins and experimentally test each system for oxidative resistance to loss of structure and function. From our extensive assessment of secondary-structure, protein hydrodynamics and enzyme activity upon hypochlorous acid or hypobromous acid challenge, we have identified two key strategies for conferring structural and functional resistance. Namely, the design of proteins (adenylate kinase enzymes) that are resistant to oxidation requires complementary consideration of protein stability and the modification (elimination) of certain oxidizable residues proximal to catalytic sites. PMID:26266833

  20. Leveraging the Mechanism of Oxidative Decay for Adenylate Kinase to Design Structural and Functional Resistances.

    PubMed

    Howell, Stanley C; Richards, David H; Mitch, William A; Wilson, Corey J

    2015-10-16

    Characterization of the mechanisms underlying hypohalous acid (i.e., hypochlorous acid or hypobromous acid) degradation of proteins is important for understanding how the immune system deactivates pathogens during infections and damages human tissues during inflammatory diseases. Proteins are particularly important hypohalous acid reaction targets in pathogens and in host tissues, as evidenced by the detection of chlorinated and brominated oxidizable residues. While a significant amount of work has been conducted for reactions of hypohalous acids with a range of individual amino acids and small peptides, the assessment of oxidative decay in full-length proteins has lagged in comparison. The most rigorous test of our understanding of oxidative decay of proteins is the rational redesign of proteins with conferred resistances to the decay of structure and function. Toward this end, in this study, we experimentally determined a putative mechanism of oxidative decay using adenylate kinase as the model system. In turn, we leveraged this mechanism to rationally design new proteins and experimentally test each system for oxidative resistance to loss of structure and function. From our extensive assessment of secondary structure, protein hydrodynamics, and enzyme activity upon hypochlorous acid or hypobromous acid challenge, we have identified two key strategies for conferring structural and functional resistance, namely, the design of proteins (adenylate kinase enzymes) that are resistant to oxidation requires complementary consideration of protein stability and the modification (elimination) of certain oxidizable residues proximal to catalytic sites. PMID:26266833

  1. RNA Mimicry by the Fap7 Adenylate Kinase in Ribosome Biogenesis

    PubMed Central

    Réty, Stéphane; Lebaron, Simon; Deschamps, Patrick; Bareille, Joseph; Jombart, Julie; Robert-Paganin, Julien; Delbos, Lila; Chardon, Florian; Zhang, Elodie; Charenton, Clément; Tollervey, David; Leulliot, Nicolas

    2014-01-01

    During biogenesis of the 40S and 60S ribosomal subunits, the pre-40S particles are exported to the cytoplasm prior to final cleavage of the 20S pre-rRNA to mature 18S rRNA. Amongst the factors involved in this maturation step, Fap7 is unusual, as it both interacts with ribosomal protein Rps14 and harbors adenylate kinase activity, a function not usually associated with ribonucleoprotein assembly. Human hFap7 also regulates Cajal body assembly and cell cycle progression via the p53–MDM2 pathway. This work presents the functional and structural characterization of the Fap7–Rps14 complex. We report that Fap7 association blocks the RNA binding surface of Rps14 and, conversely, Rps14 binding inhibits adenylate kinase activity of Fap7. In addition, the affinity of Fap7 for Rps14 is higher with bound ADP, whereas ATP hydrolysis dissociates the complex. These results suggest that Fap7 chaperones Rps14 assembly into pre-40S particles via RNA mimicry in an ATP-dependent manner. Incorporation of Rps14 by Fap7 leads to a structural rearrangement of the platform domain necessary for the pre-rRNA to acquire a cleavage competent conformation. PMID:24823650

  2. Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains.

    PubMed

    Ishikawa, Fumihiro; Miyamoto, Kengo; Konno, Sho; Kasai, Shota; Kakeya, Hideaki

    2015-12-18

    A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the Escherichia coli expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe-protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the kcat/Km value with the noncognate substrate Sal and a corresponding 48-fold decrease in the kcat/Km value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique

  3. Molecular identification and functional characterization of an adenylyl cyclase from the honeybee.

    PubMed

    Wachten, Sebastian; Schlenstedt, Jana; Gauss, Renate; Baumann, Arnd

    2006-03-01

    Cyclic AMP (cAMP) serves as an important messenger in virtually all organisms. In the honeybee (Apis mellifera), cAMP-dependent signal transduction has been implicated in behavioural processes as well as in learning and memory. Key components of cAMP-signalling cascades are adenylyl cyclases. However, the molecular identities and biochemical properties of adenylyl cyclases are completely unknown in the honeybee. We have cloned a cDNA (Amac3) from honeybee brain that encodes a membrane-bound adenylyl cyclase. The Amac3 gene is an orthologue of the Drosophila ac39E gene. The corresponding proteins share an overall amino acid similarity of approximately 62%. Phylogenetically, AmAC3 belongs to group 1 adenylyl cyclases. Heterologously expressed AmAC3 displays basal enzymatic activity and efficient coupling to endogenous G protein signalling pathways. Stimulation of beta-adrenergic receptors induces AmAC3 activity with an EC(50) of about 3.1 microm. Enzymatic activity is also increased by forskolin (EC(50) approximately 15 microm), a specific agonist of membrane-bound adenylyl cyclases. Similar to certain biogenic amine receptor genes of the honeybee, Amac3 transcripts are expressed in many somata of the brain, especially in mushroom body neurones. These results suggest that the enzyme serves in biogenic amine signal transduction cascades and in higher brain functions that contribute to learning and memory of the bee. PMID:16464235

  4. Role of soluble guanylate cyclase in the molecular mechanism underlying the physiological effects of nitric oxide.

    PubMed

    Severina, I S

    1998-07-01

    In this review the molecular mechanisms underlying the antihypertensive and antiaggregatory actions of nitric oxide (NO) are discussed. It has been shown that these effects are directly connected with the activation of soluble guanylate cyclase and the accumulation of cyclic 3;,5;-guanosine monophosphate (cGMP). The mechanism of guanylate cyclase activation by NO is analyzed, especially the role and biological significance of the nitrosyl--heme complex formed as a result of interaction of guanylate cyclase heme with NO and the role of sulfhydryl groups of the enzyme in this process. Using new approaches for studying the antihypertensive and antiaggregatory actions of nitric oxide in combination with the newly obtained data on the regulatory role of guanylate cyclase in the platelet aggregation process, the most important results were obtained regarding the molecular bases providing for a directed search for and creation of new effective antihypertensive and antiaggregatory preparations. In studying the molecular mechanism for directed activation of soluble guanylate cyclase by new NO donors, a series of hitherto unknown enzyme activators generating NO and involved in the regulation of hemostasis and vascular tone were revealed. PMID:9721331

  5. Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides

    PubMed Central

    Gagne, Steve J.; Stout, Jake M.; Liu, Enwu; Boubakir, Zakia; Clark, Shawn M.; Page, Jonathan E.

    2012-01-01

    Δ9-Tetrahydrocannabinol (THC) and other cannabinoids are responsible for the psychoactive and medicinal properties of Cannabis sativa L. (marijuana). The first intermediate in the cannabinoid biosynthetic pathway is proposed to be olivetolic acid (OA), an alkylresorcinolic acid that forms the polyketide nucleus of the cannabinoids. OA has been postulated to be synthesized by a type III polyketide synthase (PKS) enzyme, but so far type III PKSs from cannabis have been shown to produce catalytic byproducts instead of OA. We analyzed the transcriptome of glandular trichomes from female cannabis flowers, which are the primary site of cannabinoid biosynthesis, and searched for polyketide cyclase-like enzymes that could assist in OA cyclization. Here, we show that a type III PKS (tetraketide synthase) from cannabis trichomes requires the presence of a polyketide cyclase enzyme, olivetolic acid cyclase (OAC), which catalyzes a C2–C7 intramolecular aldol condensation with carboxylate retention to form OA. OAC is a dimeric α+β barrel (DABB) protein that is structurally similar to polyketide cyclases from Streptomyces species. OAC transcript is present at high levels in glandular trichomes, an expression profile that parallels other cannabinoid pathway enzymes. Our identification of OAC both clarifies the cannabinoid pathway and demonstrates unexpected evolutionary parallels between polyketide biosynthesis in plants and bacteria. In addition, the widespread occurrence of DABB proteins in plants suggests that polyketide cyclases may play an overlooked role in generating plant chemical diversity. PMID:22802619

  6. Phylogenetic analysis of the triterpene cyclase protein family in prokaryotes and eukaryotes suggests bidirectional lateral gene transfer.

    PubMed

    Frickey, Tancred; Kannenberg, Elmar

    2009-05-01

    Functional constraints to modifications in triterpene cyclase amino acid sequences make them good candidates for evolutionary studies on the phylogenetic relatedness of these enzymes in prokaryotes as well as in eukaryotes. In this study, we used a set of identified triterpene cyclases, a group of mainly bacterial squalene cyclases and a group of predominantly eukaryotic oxidosqualene cyclases, as seed sequences to identify 5288 putative triterpene cyclase homologues in publicly available databases. The Cluster Analysis of Sequences software was used to detect groups of sequences with increased pairwise sequence similarity. The sequences fall into two main clusters, a bacterial and a eukaryotic. The conserved, informative regions of a multiple sequence alignment of the family were used to construct a neighbour-joining phylogenetic tree using the AsaturA and maximum likelihood phylogenetic tree using the PhyML software. Both analyses showed that most of the triterpene cyclase sequences were similarly grouped to the accepted taxonomic relationships of the organism the sequences originated from, supporting the idea of vertical transfer of cyclase genes from parent to offspring as the main evolutionary driving force in this protein family. However, a small group of sequences from three bacterial species (Stigmatella, Gemmata and Methylococcus) grouped with an otherwise purely eukaryotic cluster of oxidosqualene cyclases, while a small group of sequences from seven fungal species and a sequence from the fern Adiantum grouped consistently with a cluster of otherwise purely bacterial squalene cyclases. This suggests that lateral gene transfer may have taken place, entailing a transfer of oxidosqualene cyclases from eukaryotes to bacteria and a transfer of squalene cyclase from bacteria to an ancestor of the group of Pezizomycotina fungi. PMID:19207562

  7. Functional role of the Ti plasmid-encoded catabolic mannopine cyclase in mannityl opine catabolism by Agrobacterium spp.

    PubMed Central

    Hong, S B; Farrand, S K

    1994-01-01

    Catabolic mannopine (MOP) cyclase encoded by Ti or Ri plasmids lactonizes MOP to agropine (AGR). The gene of the octopine-type Ti plasmid pTi15955 encoding the catabolic MOP cyclase enzyme previously was localized to a 1.6-kb segment within a cosmid clone, pYDH208. A subclone containing only this region complemented the AGR catabolism-negative phenotype conferred by a derivative of the octopine-type plasmid pTiB6S3 containing a Tn7 insertion in the region encoding the MOP cyclase enzyme. Uptake assays of strains harboring pRiA4 or pArA4a, along with complementation analyses, indicate that MOP cyclase is not sufficient for catabolism of AGR but that the strains must also express an AGR transport system. To determine the requirement for MOP cyclase in opine catabolism unequivocally, a site-specific, nonpolar deletion mutation abolishing only MOP cyclase activity was introduced into pYDH208, a cosmid clone that confers utilization of MOP, AGR, and mannopinic acid (MOA). Strains harboring this MOP cyclase-negative mutant clone, pYDPH208, did not utilize AGR but continued to utilize MOP. Growth on AGR was restored in this strain upon introduction of clones encoding the pTi15955-derived catabolic or anabolic MOP cyclase genes. The induction pattern of MOA catabolism shown by strain NT1 harboring the MOP cyclase-deficient pYDPH208 suggests that AGR is converted into MOP by MOP cyclase and that MOP, but not AGR, induces catabolism of MOA. Genetic and biochemical analyses of MOP and AGR metabolism suggest that only the conversion of AGR to MOP is directly involved in catabolism of AGR, even though the reaction catalyzed by MOP cyclase predominantly lies in the lactonization of MOP to AGR. Images PMID:8206835

  8. Quaternary Structure Controls Ligand Dynamics in Soluble Guanylate Cyclase*

    PubMed Central

    Yoo, Byung-Kuk; Lamarre, Isabelle; Martin, Jean-Louis; Negrerie, Michel

    2012-01-01

    Soluble guanylate cyclase (sGC) is the mammalian endogenous nitric oxide (NO) receptor. The mechanisms of activation and deactivation of this heterodimeric enzyme are unknown. For deciphering them, functional domains can be overexpressed. We have probed the dynamics of the diatomic ligands NO and CO within the isolated heme domain β1(190) of human sGC by piconanosecond absorption spectroscopy. After photo-excitation of nitrosylated sGC, only NO geminate rebinding occurs in 7.5 ps. In β1(190), both photo-dissociation of 5c-NO and photo-oxidation occur, contrary to sGC, followed by NO rebinding (7 ps) and back-reduction (230 ps and 2 ns). In full-length sGC, CO geminate rebinding to the heme does not occur. In contrast, CO geminately rebinds to β1(190) with fast multiphasic process (35, 171, and 18 ns). We measured the bimolecular association rates kon = 0.075 ± 0.01 × 106 m−1·s−1 for sGC and 0.83 ± 0.1 × 106 m−1·s−1 for β1(190). These different dynamics reflect conformational changes and less proximal constraints in the isolated heme domain with respect to the dimeric native sGC. We concluded that the α-subunit and the β1(191–619) domain exert structural strains on the heme domain. These strains are likely involved in the transmission of the energy and relaxation toward the activated state after Fe2+-His bond breaking. This also reveals the heme domain plasticity modulated by the associated domains and subunit. PMID:22223482

  9. Human soluble guanylate cyclase: functional expression and revised isoenzyme family.

    PubMed Central

    Zabel, U; Weeger, M; La, M; Schmidt, H H

    1998-01-01

    Soluble guanylate cyclase (sGC), a heterodimeric (alpha/beta) haem protein that converts GTP to the second messenger cGMP, functions as the receptor for nitric oxide (NO) and nitrovasodilator drugs. Three distinct cDNA species of each subunit (alpha1-alpha3, beta1-beta3) have been reported from various species. From human sources, none of these have been expressed as functionally active enzyme. Here we describe the expression of human alpha/beta heterodimeric sGC in Sf9 cells yielding active recombinant enzyme that was stimulated by the nitrovasodilator sodium nitroprusside or the NO-independent activator 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1). At the protein level, both alpha and beta subunits were detected in human tissues, suggesting co-expression also in vivo. Moreover, resequencing of the human cDNA clones [originally termed alpha3 and beta3; Giuili, Scholl, Bulle and Guellaen (1992) FEBS Lett. 304, 83-88] revealed several sequencing errors in human alpha3; correction of these eliminated major regions of divergence from rat and bovine alpha1. As human beta3 also displays more than 98% similarity to rat and bovine beta1 at the amino acid level, alpha3 and beta3 represent the human homologues of rat and bovine alpha1 and beta1, and the isoenzyme family is decreased to two isoforms for each subunit (alpha1, alpha2; beta1, beta2). Having access to the human key enzyme of NO signalling will now permit the study of novel sGC-modulating compounds with therapeutic potential. PMID:9742212

  10. Allostery in Recombinant Soluble Guanylyl Cyclase from Manduca sexta*

    PubMed Central

    Hu, Xiaohui; Murata, Lauren B.; Weichsel, Andrzej; Brailey, Jacqueline L.; Roberts, Sue A.; Nighorn, Alan; Montfort, William R.

    2008-01-01

    Soluble guanylyl/guanylate cyclase (sGC), the primary biological receptor for nitric oxide, is required for proper development and health in all animals. We have expressed heterodimeric full-length and N-terminal fragments of Manduca sexta sGC in Escherichia coli, the first time this has been accomplished for any sGC, and have performed the first functional analyses of an insect sGC. Manduca sGC behaves much like its mammalian counterparts, displaying a 170-fold stimulation by NO and sensitivity to compound YC-1. YC-1 reduces the NO and CO off-rates for the ∼100-kDa N-terminal heterodimeric fragment and increases the CO affinity by ∼50-fold to 1.7 μm. Binding of NO leads to a transient six-coordinate intermediate, followed by release of the proximal histidine to yield a five-coordinate nitrosyl complex (k6-5 = 12.8 s-1). The conversion rate is insensitive to nucleotides, YC-1, and changes in NO concentration up to ∼30 μm. NO release is biphasic in the absence of YC-1 (koff1 = 0.10 s-1 and koff2 = 0.0015 s-1); binding of YC-1 eliminates the fast phase but has little effect on the slower phase. Our data are consistent with a model for allosteric activation in which sGC undergoes a simple switch between two conformations, with an open or a closed heme pocket, integrating the influence of numerous effectors to give the final catalytic rate. Importantly, YC-1 binding occurs in the N-terminal two-thirds of the protein. Homology modeling and mutagenesis experiments suggest the presence of an H-NOX domain in the α subunit with importance for heme binding. PMID:18515359

  11. Functional characterization of transmembrane adenylyl cyclases from the honeybee brain.

    PubMed

    Balfanz, Sabine; Ehling, Petra; Wachten, Sebastian; Jordan, Nadine; Erber, Joachim; Mujagic, Samir; Baumann, Arnd

    2012-06-01

    The second messenger cAMP has a pivotal role in animals' physiology and behavior. Intracellular concentrations of cAMP are balanced by cAMP-synthesizing adenylyl cyclases (ACs) and cAMP-cleaving phosphodiesterases. Knowledge about ACs in the honeybee (Apis mellifera) is rather limited and only an ortholog of the vertebrate AC3 isoform has been functionally characterized, so far. Employing bioinformatics and functional expression we characterized two additional honeybee genes encoding membrane-bound (tm)ACs. The proteins were designated AmAC2t and AmAC8. Unlike the common structure of tmACs, AmAC2t lacks the first transmembrane domain. Despite this unusual topography, AmAC2t-activity could be stimulated by norepinephrine and NKH477 with EC(50s) of 0.07 μM and 3 μM. Both ligands stimulated AmAC8 with EC(50s) of 0.24 μM and 3.1 μM. In brain cryosections, intensive staining of mushroom bodies was observed with specific antibodies against AmAC8, an expression pattern highly reminiscent of the Drosophila rutabaga AC. In a current release of the honeybee genome database we identified three additional tmAC- and one soluble AC-encoding gene. These results suggest that (1) the AC-gene family in honeybees is comparably large as in other species, and (2) based on the restricted expression of AmAC8 in mushroom bodies, this enzyme might serve important functions in honeybee behavior. PMID:22426196

  12. Estradiol rapidly inhibits soluble guanylyl cyclase expression in rat uterus

    PubMed Central

    Krumenacker, Joshua S.; Hyder, Salman M.; Murad, Ferid

    2001-01-01

    Previous reports that investigated the regulation of the NO/soluble guanylyl cyclase (sGC)/cGMP pathway by estrogenic compounds have focused primarily on the levels of NO, NO-producing enzymes, and cGMP in various tissues. In this study, we demonstrate that 17β-estradiol (E2) regulates the α1 and β1 subunits of the NO receptor, sGC, at the mRNA and protein levels in rat uterus. Using real-time quantitative PCR, we found that within 1 h of in vivo E2 administration to rats, sGC mRNA levels begin to diminish. After 3 h, there is a maximal diminution of sGC mRNA expression (sGC α1 10% and sGC β1 33% of untreated). This effect was blocked by the estrogen receptor antagonist, ICI 182,780, indicating that estrogen receptor is required. The effect of E2 also was observed in vitro with incubations of uterine tissue, indicating that the response does not depend on the secondary release of other hormones or factors from other tissues. Puromycin did not block the effect, suggesting the effects occur because of preexisting factors in uterine tissues and do not require new protein synthesis. Using immunoblot analysis, we found that sGC protein levels also were reduced by E2 over a similar time course as the sGC mRNA. We conclude that sGC plays a vital role in the NO/sGC/cGMP regulatory pathway during conditions of elevated estrogen levels in the rat uterus as a result of the reduction of sGC expression. PMID:11209068

  13. Heme deficiency of soluble guanylate cyclase induces gastroparesis

    PubMed Central

    COSYNS, S. M. R.; DHAESE, I.; THOONEN, R.; BUYS, E. S.; VRAL, A.; BROUCKAERT, P.; LEFEBVRE, R. A.

    2016-01-01

    Background Soluble guanylate cyclase (sGC) is the principal target of nitric oxide (NO) to control gastrointestinal motility. The consequence on nitrergic signaling and gut motility of inducing a heme-free status of sGC, as induced by oxidative stress, was investigated. Methods sGCβ1H105F knock-in (apo-sGC) mice, which express heme-free sGC that has basal activity, but cannot be stimulated by NO, were generated. Key Results Diethylenetriamine NONOate did not increase sGC activity in gastrointestinal tissue of apo-sGC mice. Exogenous NO did not induce relaxation in fundic, jejunal and colonic strips, and pyloric rings of apo-sGC mice. The stomach was enlarged in apo-sGC mice with hypertrophy of the muscularis externa of the fundus and pylorus. In addition, gastric emptying and intestinal transit were delayed and whole-gut transit time was increased in the apo-sGC mice, while distal colonic transit time was maintained. The nitrergic relaxant responses to electrical field stimulation at 1–4 Hz were abolished in fundic and jejunal strips from apo-sGC mice, but in pyloric rings and colonic strips, only the response at 1 Hz was abolished, indicating the contribution of other transmitters than NO. Conclusions & Inferences The results indicate that the gastrointestinal consequences of switching from a native sGC to a heme-free sGC, which cannot be stimulated by NO, are most pronounced at the level of the stomach establishing a pivotal role of the activation of sGC by NO in normal gastric functioning. In addition, delayed intestinal transit was observed, indicating that nitrergic activation of sGC also plays a role in the lower gastrointestinal tract. PMID:23551931

  14. Cloning and Characterization of Oxidosqualene Cyclases from Kalanchoe daigremontiana

    PubMed Central

    Wang, Zhonghua; Yeats, Trevor; Han, Hong; Jetter, Reinhard

    2010-01-01

    The first committed step in triterpenoid biosynthesis is the cyclization of oxidosqualene to polycyclic alcohols or ketones C30H50O. It is catalyzed by single oxidosqualene cyclase (OSC) enzymes that can carry out varying numbers of carbocation rearrangements and, thus, generate triterpenoids with diverse carbon skeletons. OSCs from diverse plant species have been cloned and characterized, the large majority of them catalyzing relatively few rearrangement steps. It was recently predicted that special OSCs must exist that can form friedelin, the pentacyclic triterpenoid whose formation involves the maximum possible number of rearrangement steps. The goal of the present study, therefore, was to clone a friedelin synthase from Kalanchoe daigremontiana, a plant species known to accumulate this triterpenoid in its leaf surface waxes. Five OSC cDNAs were isolated, encoding proteins with 761–779 amino acids and sharing between 57.4 and 94.3% nucleotide sequence identity. Heterologous expression in yeast and GC-MS analyses showed that one of the OSCs generated the steroid cycloartenol together with minor side products, whereas the other four enzymes produced mixtures of pentacyclic triterpenoids dominated by lupeol (93%), taraxerol (60%), glutinol (66%), and friedelin (71%), respectively. The cycloartenol synthase was found expressed in all leaf tissues, whereas the lupeol, taraxerol, glutinol, and friedelin synthases were expressed only in the epidermis layers lining the upper and lower surfaces of the leaf blade. It is concluded that the function of these enzymes is to form respective triterpenoid aglycones destined to coat the leaf exterior, probably as defense compounds against pathogens or herbivores. PMID:20610397

  15. Molecular Characterization of Tick Salivary Gland Glutaminyl Cyclase

    PubMed Central

    Adamson, Steven W.; Browning, Rebecca E.; Chao, Chien-Chung; Bateman, Robert C.; Ching, Wei-Mei; Karim, Shahid

    2013-01-01

    Glutaminyl cyclase (QC) catalyzes the cyclization of N-terminal glutamine residues into pyroglutamate. This post-translational modification extends the half-life of peptides and, in some cases, is essential in binding to their cognate receptor. Due to its potential role in the post-translational modification of tick neuropeptides, we report the molecular, biochemical and physiological characterization of salivary gland QC during the prolonged blood-feeding of the black-legged tick (Ixodes scapularis) and the gulf-coast tick (Amblyomma maculatum). QC sequences from I. scapularis and A. maculatum showed a high degree of amino acid identity to each other and other arthropods and residues critical for zinc-binding/catalysis (D159, E202, and H330) or intermediate stabilization (E201, W207, D248, D305, F325, and W329) are conserved. Analysis of QC transcriptional gene expression kinetics depicts an upregulation during the blood-meal of adult female ticks prior to fast feeding phases in both I. scapularis and A. maculatum suggesting a functional link with blood meal uptake. QC enzymatic activity was detected in saliva and extracts of tick salivary glands and midguts. Recombinant QC was shown to be catalytically active. Furthermore, knockdown of QC-transcript by RNA interference resulted in lower enzymatic activity, and small, unviable egg masses in both studied tick species as well as lower engorged tick weights for I. scapularis. These results suggest that the post-translational modification of neurotransmitters and other bioactive peptides by QC is critical to oviposition and potentially other physiological processes. Moreover, these data suggest that tick-specific QC-modified neurotransmitters/hormones or other relevant parts of this system could potentially be used as novel physiological targets for tick control. PMID:23770496

  16. Estradiol rapidly inhibits soluble guanylyl cyclase expression in rat uterus

    NASA Technical Reports Server (NTRS)

    Krumenacker, J. S.; Hyder, S. M.; Murad, F.

    2001-01-01

    Previous reports that investigated the regulation of the NO/soluble guanylyl cyclase (sGC)/cGMP pathway by estrogenic compounds have focused primarily on the levels of NO, NO-producing enzymes, and cGMP in various tissues. In this study, we demonstrate that 17beta-estradiol (E2) regulates the alpha(1) and beta(1) subunits of the NO receptor, sGC, at the mRNA and protein levels in rat uterus. Using real-time quantitative PCR, we found that within 1 h of in vivo E2 administration to rats, sGC mRNA levels begin to diminish. After 3 h, there is a maximal diminution of sGC mRNA expression (sGC alpha(1) 10% and sGC beta(1) 33% of untreated). This effect was blocked by the estrogen receptor antagonist, ICI 182,780, indicating that estrogen receptor is required. The effect of E2 also was observed in vitro with incubations of uterine tissue, indicating that the response does not depend on the secondary release of other hormones or factors from other tissues. Puromycin did not block the effect, suggesting the effects occur because of preexisting factors in uterine tissues and do not require new protein synthesis. Using immunoblot analysis, we found that sGC protein levels also were reduced by E2 over a similar time course as the sGC mRNA. We conclude that sGC plays a vital role in the NO/sGC/cGMP regulatory pathway during conditions of elevated estrogen levels in the rat uterus as a result of the reduction of sGC expression.

  17. Learning our ABCs: Rad50 directs MRN repair functions via adenylate kinase activity from the conserved ATP binding cassette.

    PubMed

    Williams, R Scott; Tainer, John A

    2007-03-23

    In groundbreaking work, Bhaskara et al. (2007) demonstrate in a recent issue of Molecular Cell that the Mre11/Rad50/Nbs1 (MRN) complex harbors distinct, yet chemically related, ATPase and adenylate kinase catalytic activities that together orchestrate multiple requisite MRN functional and conformational states in dsDNA break repair sensing and signaling with general implications for ABC ATPases. PMID:17386254

  18. MELATONIN-INDUCED SUPPRESSION OF PC12 CELL GROWTH IS MEDIATED BY ITS GI COUPLED TRANSMEMBRANE RECEPTORS. (R826248)

    EPA Science Inventory

    The effects of pertussis toxin, an uncoupler of Gi protein from adenylate cyclase, and luzindole, a competitive inhibitor of melatonin receptor binding, were examined for their ability to inhibit melatonin-induced suppression of PC12 cell growth. Both agents inhibited the mela...

  19. ATP and AMP Mutually Influence Their Interaction with the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) at Separate Binding Sites*

    PubMed Central

    Randak, Christoph O.; Dong, Qian; Ver Heul, Amanda R.; Elcock, Adrian H.; Welsh, Michael J.

    2013-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP ⇆ 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5′-triphosphate (8-N3-ATP) and 8-azidoadenosine 5′-monophosphate (8-N3-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N3-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N3-ATP at ATP-binding site 2. The adenylate kinase active center probe P1,P5-di(adenosine-5′) pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2. PMID:23921386

  20. ATP and AMP mutually influence their interaction with the ATP-binding cassette (ABC) adenylate kinase cystic fibrosis transmembrane conductance regulator (CFTR) at separate binding sites.

    PubMed

    Randak, Christoph O; Dong, Qian; Ver Heul, Amanda R; Elcock, Adrian H; Welsh, Michael J

    2013-09-20

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP &lrarr2; 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5'-triphosphate (8-N3-ATP) and 8-azidoadenosine 5'-monophosphate (8-N3-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N3-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N3-ATP at ATP-binding site 2. The adenylate kinase active center probe P(1),P(5)-di(adenosine-5') pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2. PMID:23921386

  1. Purification and physiological evaluation of a guanylate cyclase activating protein from retinal rods.

    PubMed Central

    Gorczyca, W A; Gray-Keller, M P; Detwiler, P B; Palczewski, K

    1994-01-01

    In retinal rods light triggers a cascade of enzymatic reactions that increases cGMP hydrolysis and generates an electrical signal by causing closure of cGMP-gated ion channels in the photoreceptor outer segment. This leads to a decrease in internal Ca, which activates guanylate cyclase and promotes photoresponse recovery by stimulating the resynthesis of cGMP. We report here that the activation of guanylate cyclase by low Ca is mediated by an approximately 20-kDa protein purified from bovine rod outer segments by using DEAE-Sepharose, hydroxylapatite, and reverse-phase chromatographies. In a reconstituted system, this protein restores the Ca-sensitive regulation of guanylate cyclase and when dialyzed into functionally intact lizard rod outer segment decreases the sensitivity, time to peak, and recovery time of the flash response. Images PMID:7909609

  2. Molecular study of a squalene cyclase homolog gene in Bacillus subtilis

    NASA Astrophysics Data System (ADS)

    Bosak, T.; Pearson, A.; Losick, R.

    2005-12-01

    Polycyclic triterpenoids such as hopanes and steranes are formed by enzymatic cyclization of linear isoprenoid precursors by squalene cyclases and oxidosqualene cyclases. Due to their amazing preservation potential, polycyclic triterpenoids have been used to indicate the source of organic matter in oils and sediments for decades, although many cannot be attributed to known organisms and genes. To bridge the gap between the genomic database and the geochemical record, we are using molecular tools to study the expression, intracellular localization, and products of a squalene cyclase homolog found in Bacillus subtilis, a Gram-positive soil bacterium. We find that the gene is expressed during sporulation and is localized to the spore coat. Our results may help to understand the source of some previously unassigned natural products, and they may also provide clues to the physiological role of triterpenoids in the Bacillales.

  3. P2 purinergic receptor activation of neuronal nitric oxide synthase and guanylyl cyclase in the dorsal facial area of the medulla increases blood flow in the common carotid arteries of cats.

    PubMed

    Hung, Y-W; Leung, Y-M; Lin, N-N; Lee, T J-F; Kuo, J-S; Tung, K-C; Gong, C-L

    2015-02-12

    In the dorsal facial area (DFA) of the medulla, an activation of either P2 purinergic receptor or nitric oxide synthase (NOS) results in the release of glutamate, leading to an increase in blood flow of the common carotid artery (CCA). It is not known whether activation of the P2 receptor by ATP may mediate activation of NOS/guanylyl cyclase to cause glutamate release and/or whether L-Arg (nitric oxide (NO) precursor) may also cause ATP release from any other neuron, to cause an increase in CCA flow. We demonstrated that microinjections of P2 receptor agonists (ATP, α,β-methylene ATP) or NO precursor (L-arginine) into the DFA increased CCA blood flow. The P2-induced CCA blood flow increase was dose-dependently reduced by pretreatment with NG-nitro-arginine methyl ester (L-NAME, a non-specific NOS inhibitor), 7-nitroindazole (7-NI, a relatively selective neuronal NOS inhibitor) or methylene blue (MB, a guanylyl cyclase inhibitor) but not by that with D-NAME (an isomer of L-NAME) or N5-(1-iminoethyl)-L-ornithine (L-NIO, a potent endothelial NOS inhibitor). Involvement of glutamate release in these responses were substantiated by microdialysis studies, in which perfusions of ATP into the DFA increased the glutamate concentration in dialysates, but co-perfusion of ATP with L-NAME or 7-NI did not. Nevertheless, the arginine-induced CCA blood flow increase was abolished by combined pretreatment of L-NAME and MB, but not affected by pretreatment with a selective P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). In conclusion, ATP activation of the P2 receptor in the DFA induced activation of neuronal NOS/guanylyl cyclase, which causes glutamate release leading to an increase in CCA blood flow. However, arginine activation of neuronal NOS/guanylyl cyclase, which also caused glutamate release and CCA blood flow increase, did not induce activation of P2 receptors. These findings provide important information for drug design and

  4. Synechocystis Strain PCC 6803 cya2, a Prokaryotic Gene That Encodes a Guanylyl Cyclase

    PubMed Central

    Ochoa de Alda, Jesús A. G.; Ajlani, Ghada; Houmard, Jean

    2000-01-01

    Synechocystis strain PCC 6803 exhibits similar levels of cyclic AMP (cAMP) and cyclic GMP (cGMP). A thorough analysis of its genome showed that Cya2 (Sll0646) has all the sequence determinants required in terms of activity and purine specificity for being a guanylyl cyclase. Insertional mutagenesis of cya2 caused a marked reduction in cGMP content without altering the cAMP content. Thus, Cya2 represents the first example of a prokaryotic guanylyl cyclase. PMID:10851002

  5. The influence of various cations on the catalytic properties of clays. [polymerization of alanine adenylate

    NASA Technical Reports Server (NTRS)

    Paecht-Horowitz, M.

    1978-01-01

    The polymerization of alanine adenylate in the presence of the sodium form of various clays was studied, and hectorite was found to cause more polymerization than nontronite and montmorillonite (in that order) although the differences were not great. The effect on polymerization of presaturating montmorillonite with different cations was determined. Hectorite, with increased basicity of the interspatial planes, allows polymerization of lysine, which montmorillonite does not. The general trend is that, for the same amino acid, higher degrees of polymerization are obtained when the cation in the octahedral lattice of the clay is divalent rather than trivalent. With the exchangeable cations the order is reversed, for a reason that is explained. The main role of clays in the polymerization mechanism of amino acids is concentration and neutralization of charges.

  6. Alterations in adenylate ratios in plant cells after accelerated ion irradiation.

    PubMed

    Vasilenko, A; Sidorenko, P G

    1996-01-01

    Levels of adenylate metabolism have been studied in cells of Nicotiana tabacum growing in vitro, and in root apex extracts of Pisum sativum irradiated at the 95-in. isochronous cyclotron U-240, Institute for Nuclear Research, Ukrainian National Academy of Sciences, Kyiv. Particle beams of accelerated helium ions with energy 9.34 keV/micrometer were used. Replacement and rapid freezing of the irradiated plants samples in liquid nitrogen were carried out with a manipulator and a remote control system. After doses of 5, 20, 50, and 100 Gy of gamma-irradiation, as well as 50 and 100 Gy 4He irradiation, the cellular ATP/ADP ratio increased during early stages of the response. This effect was absent at higher doses and after exposure to sparesly-ionizing radiation, when a rapid decline in the cellular ATP concentration and the ATP/ADP ratio occurred. PMID:11538989

  7. Minimum Free Energy Path of Ligand-Induced Transition in Adenylate Kinase

    PubMed Central

    Matsunaga, Yasuhiro; Fujisaki, Hiroshi; Terada, Tohru; Furuta, Tadaomi; Moritsugu, Kei; Kidera, Akinori

    2012-01-01

    Large-scale conformational changes in proteins involve barrier-crossing transitions on the complex free energy surfaces of high-dimensional space. Such rare events cannot be efficiently captured by conventional molecular dynamics simulations. Here we show that, by combining the on-the-fly string method and the multi-state Bennett acceptance ratio (MBAR) method, the free energy profile of a conformational transition pathway in Escherichia coli adenylate kinase can be characterized in a high-dimensional space. The minimum free energy paths of the conformational transitions in adenylate kinase were explored by the on-the-fly string method in 20-dimensional space spanned by the 20 largest-amplitude principal modes, and the free energy and various kinds of average physical quantities along the pathways were successfully evaluated by the MBAR method. The influence of ligand binding on the pathways was characterized in terms of rigid-body motions of the lid-shaped ATP-binding domain (LID) and the AMP-binding (AMPbd) domains. It was found that the LID domain was able to partially close without the ligand, while the closure of the AMPbd domain required the ligand binding. The transition state ensemble of the ligand bound form was identified as those structures characterized by highly specific binding of the ligand to the AMPbd domain, and was validated by unrestrained MD simulations. It was also found that complete closure of the LID domain required the dehydration of solvents around the P-loop. These findings suggest that the interplay of the two different types of domain motion is an essential feature in the conformational transition of the enzyme. PMID:22685395

  8. Retigeric Acid B Attenuates the Virulence of Candida albicans via Inhibiting Adenylyl Cyclase Activity Targeted by Enhanced Farnesol Production

    PubMed Central

    Chang, Wenqiang; Li, Ying; Zhang, Li; Cheng, Aixia; Lou, Hongxiang

    2012-01-01

    Candida albicans, the most prevalent fungal pathogen, undergoes yeast-to-hyphal switch which has long been identified as a key fungal virulence factor. We showed here that the lichen-derived small molecule retigeric acid B (RAB) acted as an inhibitor that significantly inhibited the filamentation of C. albicans, leading to the prolonged survival of nematodes infected by C. albicans. Quantitative real-time PCR analysis and intracellular cAMP measurement revealed RAB regulated the Ras1-cAMP-Efg1 pathway by reducing cAMP level to inhibit the hyphae formation. Confocal microscopic observation showed RAB induced the expression of Dpp3, synthesizing more farnesol, which was confirmed by gas chromatography-mass spectroscopy detection. An adenylyl cyclase activity assay demonstrated RAB could repress the activity of Cdc35 through stimulating farnesol synthesis, thus causing a decrease in cAMP synthesis, leading to retarded yeast-to-hyphal transition. Moreover, reduced levels of intracellular cAMP resulted in the inhibition of downstream adhesins. Together, these findings indicate that RAB stimulates farnesol production that directly inhibits the Cdc35 activity, reducing the synthesis of cAMP and thereby causing the disruption of the morphologic transition and attenuating the virulence of C. albicans. Our work illustrates the underlying mechanism of RAB-dependent inhibition of the yeast-to-hyphal switch and provides a potential application in treating the infection of C. albicans. PMID:22848547

  9. Crystal structures of human soluble adenylyl cyclase reveal mechanisms of catalysis and of its activation through bicarbonate

    PubMed Central

    Kleinboelting, Silke; Diaz, Ana; Moniot, Sebastien; van den Heuvel, Joop; Weyand, Michael; Levin, Lonny R.; Buck, Jochen; Steegborn, Clemens

    2014-01-01

    cAMP is an evolutionary conserved, prototypic second messenger regulating numerous cellular functions. In mammals, cAMP is synthesized by one of 10 homologous adenylyl cyclases (ACs): nine transmembrane enzymes and one soluble AC (sAC). Among these, only sAC is directly activated by bicarbonate (HCO3−); it thereby serves as a cellular sensor for HCO3−, carbon dioxide (CO2), and pH in physiological functions, such as sperm activation, aqueous humor formation, and metabolic regulation. Here, we describe crystal structures of human sAC catalytic domains in the apo state and in complex with substrate analog, products, and regulators. The activator HCO3− binds adjacent to Arg176, which acts as a switch that enables formation of the catalytic cation sites. An anionic inhibitor, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, inhibits sAC through binding to the active site entrance, which blocks HCO3− activation through steric hindrance and trapping of the Arg176 side chain. Finally, product complexes reveal small, local rearrangements that facilitate catalysis. Our results provide a molecular mechanism for sAC catalysis and cellular HCO3− sensing and a basis for targeting this system with drugs. PMID:24567411

  10. Purification and assay of cell-invasive form of calmodulin-sensitive adenylyl cyclase from Bordetella pertussis

    SciTech Connect

    Masure, H.R.; Donovan, M.G.; Storm, D.R.

    1991-01-01

    An invasive form of the CaM-sensitive adenylyl cyclase from Bordetella pertussis can be isolated from bacterial culture supernatants. This isolation is achieved through the use of QAE-Sephadex anion-exchange chromatography. It has been demonstrated that the addition of exogenous Ca{sup 2}{sup +} to the anion-exchange gradient buffers will affect elution from the column and will thereby affect the isolation of invasive adenylyl cyclase. This is probably due to a Ca2(+)-dependent interaction of the catalytic subunit with another component in the culture supernatant. Two peaks of adenylyl cyclase activity are obtained. The Pk1 adenylyl cyclase preparation is able to cause significant increases in intracellular cAMP levels in animal cells. This increase occurs rapidly and in a dose-dependent manner in both N1E-115 mouse neuroblastoma cells and human erythrocytes. The Pk2 adenylyl cyclase has catalytic activity but is not cell invasive. This material can serve, therefore, as a control to ensure that the cAMP which is measured is, indeed, intracellular. A second control is to add exogenous CaM to the Pk1 adenylyl cyclase preparation. The 45-kDa catalytic subunit-CaM complex is not cell invasive. Although the mechanism for membrane translocation of the adenylyl cyclase is unknown, there is evidence that the adenylyl cyclase enters animal cells by a mechanism distinct from receptor-mediated endocytosis. Calmodulin-sensitive adenylyl cyclase activity can be removed from preparations of the adenylyl cyclase that have been subjected to SDS-polyacrylamide gel electrophoresis. This property of the enzyme has enabled purification of the catalytic subunit to apparent homogeneity. The purified catalytic subunit from culture supernatants has a predicted molecular weight of 45,000. This polypeptide interacts directly with Ca{sup 2}{sup +} and this interaction may be important for its invasion into animal cells.

  11. Multiple lineage specific expansions within the guanylyl cyclase gene family

    PubMed Central

    Fitzpatrick, David A; O'Halloran, Damien M; Burnell, Ann M

    2006-01-01

    Background Guanylyl cyclases (GCs) are responsible for the production of the secondary messenger cyclic guanosine monophosphate, which plays important roles in a variety of physiological responses such as vision, olfaction, muscle contraction, homeostatic regulation, cardiovascular and nervous function. There are two types of GCs in animals, soluble (sGCs) which are found ubiquitously in cell cytoplasm, and receptor (rGC) forms which span cell membranes. The complete genomes of several vertebrate and invertebrate species are now available. These data provide a platform to investigate the evolution of GCs across a diverse range of animal phyla. Results In this analysis we located GC genes from a broad spectrum of vertebrate and invertebrate animals and reconstructed molecular phylogenies for both sGC and rGC proteins. The most notable features of the resulting phylogenies are the number of lineage specific rGC and sGC expansions that have occurred during metazoan evolution. Among these expansions is a large nematode specific rGC clade comprising 21 genes in C. elegans alone; a vertebrate specific expansion in the natriuretic receptors GC-A and GC-B; a vertebrate specific expansion in the guanylyl GC-C receptors, an echinoderm specific expansion in the sperm rGC genes and a nematode specific sGC clade. Our phylogenetic reconstruction also shows the existence of a basal group of nitric oxide (NO) insensitive insect and nematode sGCs which are regulated by O2. This suggests that the primordial eukaryotes probably utilized sGC as an O2 sensor, with the ligand specificity of sGC later switching to NO which provides a very effective local cell-to-cell signalling system. Phylogenetic analysis of the sGC and bacterial heme nitric oxide/oxygen binding protein domain supports the hypothesis that this domain originated from a cyanobacterial source. Conclusion The most salient feature of our phylogenies is the number of lineage specific expansions, which have occurred within

  12. Elevation of Soluble Guanylate Cyclase Suppresses Proliferation and Survival of Human Breast Cancer Cells

    PubMed Central

    Chen, Chen-Yu; Shiah, Shine-Gwo; Kung, Hsing-Jien; King, Kuang-Liang; Su, Liang-Chen; Chang, Shi-Chuan; Chang, Chung-Ho

    2015-01-01

    Nitric oxide (NO) is an essential signaling molecule in biological systems. Soluble guanylate cyclase (sGC), composing of α1 and β1 subunit, is the receptor for NO. Using radioimmunoassay, we discovered that activation of sGC by treatment with bradykinin or sodium nitroprusside (SNP) is impaired in MCF-7 and MDA-MB-231 breast cancer cells as compared to normal breast epithelial 184A1 cells. The 184A1 cells expressed both sGC α1 and sGCβ1 mRNAs. However, levels of sGCβ1 mRNAs were relatively lower in MCF-7 cells while both mRNA of sGC subunits were absent in MDA-MB-231 cells. Treatment with DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-aza-dC) increased mRNA levels of both sGCα1 and sGCβ1 in MDA-MB-231 cells but only sGCβ1 mRNAs in MCF-7 cells. The 5-aza-dC treatment increased the SNP-induced cGMP production in MCF-7 and MDA-MB-231, but not in 184A1 cells. Bisulfite sequencing revealed that the promoter of sGCα1 in MDA-MB-231 cells and promoter of sGCβ1 in MCF-7 cells were methylated. Promoter hypermethylation of sGCα1 and sGCβ1 was found in 1 out of 10 breast cancer patients. Over-expression of both sGC subunits in MDA-MB-231 cells induced apoptosis and growth inhibition in vitro as well as reduced tumor incidence and tumor growth rate of MDA-MB-231 xenografts in nude mice. Elevation of sGC reduced protein abundance of Bcl-2, Bcl-xL, Cdc2, Cdc25A, Cyclin B1, Cyclin D1, Cdk6, c-Myc, and Skp2 while increased protein expression of p53. Our study demonstrated that down-regulation of sGC, partially due to promoter methylation, provides growth and survival advantage in human breast cancer cells. PMID:25928539

  13. A critical electrostatic interaction mediates inhibitor recognition by human asparagine synthetase.

    PubMed

    Ikeuchi, Hideyuki; Meyer, Megan E; Ding, Yun; Hiratake, Jun; Richards, Nigel G J

    2009-09-15

    The first sulfoximine-based inhibitor of human asparagine synthetase (ASNS) with nanomolar potency has been shown to suppress proliferation of asparaginase-resistant MOLT-4 cells in the presence of L-asparaginase. This validates literature hypotheses concerning the viability of human ASNS as a target for new drugs against acute lymphoblastic leukemia and ovarian cancer. Developing structure-function relationships for this class of human ASNS inhibitors has proven difficult, however, primarily because of the absence of rapid synthetic procedures for constructing highly functionalized sulfoximines. We now report conditions for the efficient preparation of these compounds by coupling sulfoxides and sulfamides in the presence of a rhodium catalyst. Access to this methodology has permitted the construction of two new adenylated sulfoximines, which were expected to exhibit similar binding affinity and better bioavailability than the original human ASNS inhibitor. Steady-state kinetic characterization of these compounds, however, has revealed the importance of a localized negative charge on the inhibitor that mimics that of the phosphate group in a key acyl-adenylate reaction intermediate. These experiments place an important constraint on the design of sulfoximine libraries for screening experiments to obtain ASNS inhibitors with increased potency and bioavailability. PMID:19683931

  14. Proteasome inhibitors.

    PubMed

    Teicher, Beverly A; Tomaszewski, Joseph E

    2015-07-01

    Proteasome inhibitors have a 20 year history in cancer therapy. The first proteasome inhibitor, bortezomib (Velcade, PS-341), a break-through multiple myeloma treatment, moved rapidly through development from bench in 1994 to first approval in 2003. Bortezomib is a reversible boronic acid inhibitor of the chymotrypsin-like activity of the proteasome. Next generation proteasome inhibitors include carfilzomib and oprozomib which are irreversible epoxyketone proteasome inhibitors; and ixazomib and delanzomib which are reversible boronic acid proteasome inhibitors. Two proteasome inhibitors, bortezomib and carfilzomib are FDA approved drugs and ixazomib and oprozomib are in late stage clinical trials. All of the agents are potent cytotoxics. The disease focus for all the proteasome inhibitors is multiple myeloma. This focus arose from clinical observations made in bortezomib early clinical trials. Later preclinical studies confirmed that multiple myeloma cells were indeed more sensitive to proteasome inhibitors than other tumor cell types. The discovery and development of the proteasome inhibitor class of anticancer agents has progressed through a classic route of serendipity and scientific investigation. These agents are continuing to have a major impact in their treatment of hematologic malignancies and are beginning to be explored as potential treatment agent for non-cancer indications. PMID:25935605

  15. A Soluble Guanylate Cyclase Mediates Negative Signaling by Ammonium on Expression of Nitrate Reductase in Chlamydomonas[W

    PubMed Central

    de Montaigu, Amaury; Sanz-Luque, Emanuel; Galván, Aurora; Fernández, Emilio

    2010-01-01

    Nitrate assimilation in plants and related organisms is a highly regulated and conserved pathway in which the enzyme nitrate reductase (NR) occupies a central position. Although some progress has been made in understanding the regulation of the protein, transcriptional regulation of the NR gene (NIA1) is poorly understood. This work describes a mechanism for the ammonium-mediated repression of NIA1. We report the characterization of a mutant defective in the repression of NIA1 and NR in response to ammonium and show that a gene (CYG56) coding for a nitric oxide (NO)-dependent guanylate cyclase (GC) was interrupted in this mutant. NO donors, cGMP analogs, a phosphodiesterase inhibitor isobutylmethylxanthine (IBMX), and a calcium ionophore (A23187) repress the expression of NIA1 in Chlamydomonas reinhardtii wild-type cells and also repress the expression of other ammonium-sensitive genes. In addition, the GC inhibitors LY83,583 (6-anilino-5,8-quinolinedione) and ODQ (1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one) release cells from ammonium repression. Intracellular NO and cGMP levels were increased in the presence of ammonium in wild-type cells. In the cyg56 mutant, NIA1 transcription was less sensitive to NO donors and A23187, but responded like the wild type to IBMX. Results presented here suggest that CYG56 participates in ammonium-mediated NIA1 repression through a pathway that involves NO, cGMP, and calcium and that similar mechanisms might be occurring in plants. PMID:20442374

  16. Cinaciguat, a novel activator of soluble guanylate cyclase, protects against ischemia/reperfusion injury: role of hydrogen sulfide

    PubMed Central

    Salloum, Fadi N.; Das, Anindita; Samidurai, Arun; Hoke, Nicholas N.; Chau, Vinh Q.; Ockaili, Ramzi A.; Stasch, Johannes-Peter

    2012-01-01

    Cinaciguat (BAY 58–2667) is a novel nitric oxide (NO)-independent activator of soluble guanylate cyclase (sGC), which induces cGMP-generation and vasodilation in diseased vessels. We tested the hypothesis that cinaciguat might trigger protection against ischemia/reperfusion (I/R) in the heart and adult cardiomyocytes through cGMP/protein kinase G (PKG)-dependent generation of hydrogen sulfide (H2S). Adult New Zealand White rabbits were pretreated with 1 or 10 μg/kg cinaciguat (iv) or 10% DMSO (vehicle) 15 min before I/R or with 10 μg/kg cinaciguat (iv) at reperfusion. Additionally, adult male ICR mice were treated with either cinaciguat (10 μg/kg ip) or vehicle 30 min before I/R or at the onset of reperfusion (10 μg/kg iv). The PKG inhibitor KT5283 (KT; 1 mg/kg ip) or dl-propargylglycine (PAG; 50 mg/kg ip) the inhibitor of the H2S-producing enzyme cystathionine-γ-lyase (CSE) were given 10 and 30 min before cinaciguat. Cardiac function and infarct size were assessed by echocardiography and tetrazolium staining, respectively. Primary adult mouse cardiomyocytes were isolated and treated with cinaciguat before simulated ischemia/reoxygenation. Cinaciguat caused 63 and 41% reduction of infarct size when given before I/R and at reperfusion in rabbits, respectively. In mice, cinaciguat pretreatment caused a more robust 80% reduction in infarct size vs. 63% reduction when given at reperfusion and preserved cardiac function following I/R, which were blocked by KT and PAG. Cinaciguat also caused an increase in myocardial PKG activity and CSE expression. In cardiomyocytes, cinaciguat (50 nM) reduced necrosis and apoptosis and increased H2S levels, which was abrogated by KT. Cinaciguat is a novel molecule to induce H2S generation and a powerful protection against I/R injury in heart. PMID:22268103

  17. Soluble Guanylate Cyclase Stimulation Prevents Fibrotic Tissue Remodeling and Improves Survival in Salt-Sensitive Dahl Rats

    PubMed Central

    Geschka, Sandra; Kretschmer, Axel; Sharkovska, Yuliya; Evgenov, Oleg V.; Lawrenz, Bettina; Hucke, Andreas; Hocher, Berthold; Stasch, Johannes-Peter

    2011-01-01

    Background A direct pharmacological stimulation of soluble guanylate cyclase (sGC) is an emerging therapeutic approach to the management of various cardiovascular disorders associated with endothelial dysfunction. Novel sGC stimulators, including riociguat (BAY 63-2521), have a dual mode of action: They sensitize sGC to endogenously produced nitric oxide (NO) and also directly stimulate sGC independently of NO. Little is known about their effects on tissue remodeling and degeneration and survival in experimental malignant hypertension. Methods and Results Mortality, hemodynamics and biomarkers of tissue remodeling and degeneration were assessed in Dahl salt-sensitive rats maintained on a high salt diet and treated with riociguat (3 or 10 mg/kg/d) for 14 weeks. Riociguat markedly attenuated systemic hypertension, improved systolic heart function and increased survival from 33% to 85%. Histological examination of the heart and kidneys revealed that riociguat significantly ameliorated fibrotic tissue remodeling and degeneration. Correspondingly, mRNA expression of the pro-fibrotic biomarkers osteopontin (OPN), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and plasminogen activator inhibitor-1 (PAI-1) in the myocardium and the renal cortex was attenuated by riociguat. In addition, riociguat reduced plasma and urinary levels of OPN, TIMP-1, and PAI-1. Conclusions Stimulation of sGC by riociguat markedly improves survival and attenuates systemic hypertension and systolic dysfunction, as well as fibrotic tissue remodeling in the myocardium and the renal cortex in a rodent model of pressure and volume overload. These findings suggest a therapeutic potential of sGC stimulators in diseases associated with impaired cardiovascular and renal functions. PMID:21789188

  18. A Short History of cGMP, Guanylyl Cyclases, and cGMP-Dependent Protein Kinases

    PubMed Central

    Kots, Alexander Y.; Martin, Emil; Sharina, Iraida G.

    2014-01-01

    Here, we review the early studies on cGMP, guanylyl cyclases, and cGMP-dependent protein kinases to facilitate understanding of development of this exciting but complex field of research encompassing pharmacology, biochemistry, physiology, and molecular biology of these important regulatory molecules. PMID:19089322

  19. Determinants for the activation and autoinhibition of the diguanylate cyclase response regulator WspR

    PubMed Central

    De, Nabanita; Navarro, Marcos V.A.S.; Raghavan, Rahul V.; Sondermann, Holger

    2009-01-01

    The bacterial second messenger c-di-GMP controls secretion, cell adhesion and motility leading to biofilm formation and increased cytotoxicity. Diguanylate cyclases containing GGDEF and phosphodiesterases containing EAL or HD-GYP domains have been identified as the enzymes controlling cellular c-di-GMP levels, yet less is known regarding the molecular mechanisms governing regulation and signaling specificity. We recently determined a product-inhibition pathway for the diguanylate cyclase response regulator WspR from Pseudomonas, a potent molecular switch that controls biofilm formation. In WspR, catalytic activity is modulated by a helical stalk motif that connects its phospho-receiver (REC) and GGDEF domains. The stalks facilitate the formation of distinct oligomeric states that contribute to both activation and autoinhibition. Here, we provide novel insights into the regulation of diguanylate cyclase activity in WspR based on the crystal structures of full-length WspR, the isolated GGDEF domain, and an artificially dimerized catalytic domain. The structures highlight that inhibition is achieved by restricting the mobility of rigid GGDEF domains, mediated by c-di-GMP binding to an inhibitory site at the GGDEF domain. Kinetic measurements and biochemical characterization corroborate a model in which the activation of WspR requires the formation of a tetrameric species. Tetramerization occurs spontaneously at high protein concentration or upon addition of the phosphomimetic compound beryllium fluoride. Our analyses elucidate common and WspR-specific mechanisms for the fine-tuning of diguanylate cyclase activity. PMID:19695263

  20. Soluble guanylyl cyclase is involved in PDT-induced injury of crayfish glial cells

    NASA Astrophysics Data System (ADS)

    Kovaleva, V. D.; Uzdensky, A. B.

    2016-04-01

    Photodynamic therapy (PDT) is a potential tool for selective destruction of malignant brain tumors. However, not only malignant but also healthy neurons and glial cells may be damaged during PDT. Nitric oxide is an important modulator of cell viability and intercellular neuroglial communications. NO have been already shown to participate in PDT-induced injury of neurons and glial cells. As soluble guanylyl cyclase is the only known receptor for NO, we have studied the possible role of soluble guanylyl cyclase in the regulation of survival and death of neurons and surrounding glial cells under photo-oxidative stress induced by photodynamic treatment (PDT). The crayfish stretch receptor consisting of a single identified sensory neuron enveloped by glial cells is a simple but informative model object. It was photosensitized with alumophthalocyanine photosens (10 nM) and irradiated with a laser diode (670 nm, 0.4 W/cm2). Using inhibitory analysis we have shown that during PDT soluble guanylyl cyclase, probably, has proapoptotic and antinecrotic effect on the glial cells of the isolated crayfish stretch receptor. Proapoptotic effect of soluble guanylyl cyclase could be mediated by protein kinase G (PKG). Thus, the involvement of NO/sGC/cGMP/PKG signaling pathway in PDT-induced apoptosis of glial cells was indirectly demonstrated.

  1. Squalene-hopene cyclase from Methylococcus capsulatus (Bath): a bacterium producing hopanoids and steroids.

    PubMed

    Tippelt, A; Jahnke, L; Poralla, K

    1998-03-30

    We report the cloning and characterisation of the Methylococcus capsulatus shc gene, which encodes the squalene-hopene cyclase (SHC). This enzyme catalyses the complex cyclization of squalene to the pentacyclic triterpene skeleton of hopanoids and represents the key reaction in this biosynthesis. Using a combination of PCR amplification and DNA hybridization, two overlapping 2.6 kb PstI and 3.3 kb SalI DNA fragments were cloned bearing a 1962 bp open reading frame encoding a 74 kDa protein with 654 amino acids and a predicted isoelectric point at about pH 6.3. The deduced amino acid sequence of the M. capsulatus shc gene showed significant similarity to known prokaryotic SHCs and to a lesser degree to the related eukaryotic oxidosqualene cyclases (OSCs). Like other triterpene cyclases, the M. capsulatus SHC contains seven so-called QW-motifs as well as an aspartate-rich domain. The recombinant M. capsulatus SHC was expressed in Escherichia coli and in vitro activity of the recombinant cyclase was demonstrated using crude cell-free lysate or solubilized membrane preparation. The cyclization products hop-22-ene and hopan-22-ol (diplopterol) were identified by GC and GC-MS. PMID:9555026

  2. Demonstration of phosphoryl group transfer indicates that the ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) exhibits adenylate kinase activity.

    PubMed

    Randak, Christoph O; Ver Heul, Amanda R; Welsh, Michael J

    2012-10-19

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane-spanning adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter. ABC transporters and other nuclear and cytoplasmic ABC proteins have ATPase activity that is coupled to their biological function. Recent studies with CFTR and two nonmembrane-bound ABC proteins, the DNA repair enzyme Rad50 and a structural maintenance of chromosome (SMC) protein, challenge the model that the function of all ABC proteins depends solely on their associated ATPase activity. Patch clamp studies indicated that in the presence of physiologically relevant concentrations of adenosine 5'-monophosphate (AMP), CFTR Cl(-) channel function is coupled to adenylate kinase activity (ATP+AMP <==> 2 ADP). Work with Rad50 and SMC showed that these enzymes catalyze both ATPase and adenylate kinase reactions. However, despite the supportive electrophysiological results with CFTR, there are no biochemical data demonstrating intrinsic adenylate kinase activity of a membrane-bound ABC transporter. We developed a biochemical assay for adenylate kinase activity, in which the radioactive γ-phosphate of a nucleotide triphosphate could transfer to a photoactivatable AMP analog. UV irradiation could then trap the (32)P on the adenylate kinase. With this assay, we discovered phosphoryl group transfer that labeled CFTR, thereby demonstrating its adenylate kinase activity. Our results also suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for adenylate kinase activity. These biochemical data complement earlier biophysical studies of CFTR and indicate that the ABC transporter CFTR can function as an adenylate kinase. PMID:22948143

  3. Demonstration of Phosphoryl Group Transfer Indicates That the ATP-binding Cassette (ABC) Transporter Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Exhibits Adenylate Kinase Activity*

    PubMed Central

    Randak, Christoph O.; Ver Heul, Amanda R.; Welsh, Michael J.

    2012-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane-spanning adenosine 5′-triphosphate (ATP)-binding cassette (ABC) transporter. ABC transporters and other nuclear and cytoplasmic ABC proteins have ATPase activity that is coupled to their biological function. Recent studies with CFTR and two nonmembrane-bound ABC proteins, the DNA repair enzyme Rad50 and a structural maintenance of chromosome (SMC) protein, challenge the model that the function of all ABC proteins depends solely on their associated ATPase activity. Patch clamp studies indicated that in the presence of physiologically relevant concentrations of adenosine 5′-monophosphate (AMP), CFTR Cl− channel function is coupled to adenylate kinase activity (ATP+AMP ⇆ 2 ADP). Work with Rad50 and SMC showed that these enzymes catalyze both ATPase and adenylate kinase reactions. However, despite the supportive electrophysiological results with CFTR, there are no biochemical data demonstrating intrinsic adenylate kinase activity of a membrane-bound ABC transporter. We developed a biochemical assay for adenylate kinase activity, in which the radioactive γ-phosphate of a nucleotide triphosphate could transfer to a photoactivatable AMP analog. UV irradiation could then trap the 32P on the adenylate kinase. With this assay, we discovered phosphoryl group transfer that labeled CFTR, thereby demonstrating its adenylate kinase activity. Our results also suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for adenylate kinase activity. These biochemical data complement earlier biophysical studies of CFTR and indicate that the ABC transporter CFTR can function as an adenylate kinase. PMID:22948143

  4. NO-Mediated [Ca2+]cyt Increases Depend on ADP-Ribosyl Cyclase Activity in Arabidopsis1[OPEN

    PubMed Central

    Hotta, Carlos T.; Davey, Matthew P.; Dodd, Antony N.

    2016-01-01

    Cyclic ADP ribose (cADPR) is a Ca2+-mobilizing intracellular second messenger synthesized from NAD by ADP-ribosyl cyclases (ADPR cyclases). In animals, cADPR targets the ryanodine receptor present in the sarcoplasmic/endoplasmic reticulum to promote Ca2+ release from intracellular stores to increase the concentration of cytosolic free Ca2+ in Arabidopsis (Arabidopsis thaliana), and cADPR has been proposed to play a central role in signal transduction pathways evoked by the drought and stress hormone, abscisic acid, and the circadian clock. Despite evidence for the action of cADPR in Arabidopsis, no predicted proteins with significant similarity to the known ADPR cyclases have been reported in any plant genome database, suggesting either that there is a unique route for cADPR synthesis or that a homolog of ADPR cyclase with low similarity might exist in plants. We sought to determine whether the low levels of ADPR cyclase activity reported in Arabidopsis are indicative of a bona fide activity that can be associated with the regulation of Ca2+ signaling. We adapted two different fluorescence-based assays to measure ADPR cyclase activity in Arabidopsis and found that this activity has the characteristics of a nucleotide cyclase that is activated by nitric oxide to increase cADPR and mobilize Ca2+. PMID:26932235

  5. Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942.

    PubMed

    Cunningham, F X; Sun, Z; Chamovitz, D; Hirschberg, J; Gantt, E

    1994-08-01

    A gene encoding the enzyme lycopene cyclase in the cyanobacterium Synechococcus sp strain PCC7942 was mapped by genetic complementation, cloned, and sequenced. This gene, which we have named crtL, was expressed in strains of Escherichia coli that were genetically engineered to accumulate the carotenoid precursors lycopene, neurosporene, and zeta-carotene. The crtL gene product converts the acyclic hydrocarbon lycopene into the bicyclic beta-carotene, an essential component of the photosynthetic apparatus in oxygen-evolving organisms and a source of vitamin A in human and animal nutrition. The enzyme also converts neurosporene to the monocyclic beta-zeacarotene but does not cyclize zeta-carotene, indicating that desaturation of the 7-8 or 7'-8' carbon-carbon bond is required for cyclization. The bleaching herbicide 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA) effectively inhibits both cyclization reactions. A mutation that confers resistance to MPTA in Synechococcus sp PCC7942 was identified as a point mutation in the promoter region of crtL. The deduced amino acid sequence of lycopene cyclase specifies a polypeptide of 411 amino acids with a molecular weight of 46,125 and a pI of 6.0. An amino acid sequence motif indicative of FAD utilization is located at the N terminus of the polypeptide. DNA gel blot hybridization analysis indicated a single copy of crtL in Synechococcus sp PCC7942. Other than the FAD binding motif, the predicted amino acid sequence of the cyanobacterial lycopene cyclase bears little resemblance to the two known lycopene cyclase enzymes from nonphotosynthetic bacteria. Preliminary results from DNA gel blot hybridization experiments suggest that, like two earlier genes in the pathway, the Synechococcus gene encoding lycopene cyclase is homologous to plant and algal genes encoding this enzyme. PMID:7919981

  6. Cloning and characterization of the gene encoding Halobacterium halobium adenylate kinase.

    PubMed

    Song, S; Inouye, S; Kawai, M; Fukami-Kobayashi, K; Gõ, M; Nakazawa, A

    1996-10-10

    The gene (AK) encoding adenylate kinase (AK) of Halobacterium halobium was cloned. AK consisted of 648 bp and coded for 216 amino acids (aa). S1 mapping and primer extension experiments indicated that the transcription start point (tsp) was located immediately upstream from the start codon. The TAT-like promoter sequence was found at a position 20-24 bp upstream from tsp. The most striking property of the enzyme was a putative Zn finger-like structure with four cysteines. It might contribute to the structural stability of the molecule in high-salt conditions. Phylogenetic analysis indicated two lineages of the AK family, the short and long types which diverged a long time ago, possibly before the separation of prokaryotes and eukaryotes. Although the H. halobium AK belongs to the long-type AK lineage, it is located in an intermediary position between the two lineages of the phylogenetic tree, indicating early divergence of the gene along the long-type lineage. PMID:8917077

  7. Identification of antifungal niphimycin from Streptomyces sp. KP6107 by screening based on adenylate kinase assay.

    PubMed

    Kim, Hye Yoon; Kim, Jeong Do; Hong, Jin Sung; Ham, Jong Hyun; Kim, Beom Seok

    2013-07-01

    Microbial culture extracts are used for natural product screening to find antifungal lead compounds. A microbial culture extract library was constructed using 343 actinomycete isolates to examine the value of the adenylate kinase (AK) assay for screening to identify antifungal metabolites that disrupt cell integrity in plant pathogenic fungi. A culture extract of Streptomyces sp. strain KP6107 lysed cells of Fusarium oxysporum f.sp. lycopersici which resulted in high AK activity. The active ingredient N-1 was purified from the culture extract using various chromatographic procedures and identified to be the guanidyl-polyol macrolide antibiotic, niphimycin, which is a potent fungal cell membrane disruptor. Niphimycin showed broad-spectrum antifungal activity against Alternaria mali, Aspergillus oryzae, Colletotrichum coccodes, Colletotrichum gloeosporioides, Cercospora canescens, Cylindrocarpon destructans, F. oxysporum f.sp. cucumerinum, F. oxysporum f.sp. lycopersici, and Rhizoctonia solani at concentrations of 8-64 µg ml(-1). Anthracnose development in pepper plants was completely inhibited by treatment with 50 µg ml(-1) niphimycin, which was as effective as chlorothalonil. These results show that the AK assay is an efficient and selective tool in screening for cell membrane/wall disruptors of plant pathogenic fungi. PMID:22915202

  8. Energy landscape and multiroute folding of topologically complex proteins adenylate kinase and 2ouf-knot.

    PubMed

    Li, Wenfei; Terakawa, Tsuyoshi; Wang, Wei; Takada, Shoji

    2012-10-30

    While fast folding of small proteins has been relatively well characterized by experiments and theories, much less is known for slow folding of larger proteins, for which recent experiments suggested quite complex and rich folding behaviors. Here, we address how the energy landscape theory can be applied to these slow folding reactions. Combining the perfect-funnel approximation with a multiscale method, we first extended our previous atomic-interaction based coarse grained (AICG) model to take into account local flexibility of protein molecules. Using this model, we then investigated the energy landscapes and folding routes of two proteins with complex topologies: a multidomain protein adenylate kinase (AKE) and a knotted protein 2ouf-knot. In the AKE folding, consistent with experimental results, the kinetic free energy surface showed several substates between the fully unfolded and native states. We characterized the structural features of these substates and transitions among them, finding temperature-dependent multiroute folding. For protein 2ouf-knot, we found that the improved atomic-interaction based coarse-grained model can spontaneously tie a knot and fold the protein with a probability up to 96%. The computed folding rate of the knotted protein was much slower than that of its unknotted counterpart, in agreement with experimental findings. Similar to the AKE case, the 2ouf-knot folding exhibited several substates and transitions among them. Interestingly, we found a dead-end substate that lacks the knot, thus suggesting backtracking mechanisms. PMID:22753508

  9. The adenylate energy charge as a new and useful indicator of capture stress in chondrichthyans.

    PubMed

    Guida, Leonardo; Walker, Terence I; Reina, Richard D

    2016-02-01

    Quantifying the physiological stress response of chondrichthyans to capture has assisted the development of fishing practices conducive to their survival. However, currently used indicators of stress show significant interspecific and intraspecific variation in species' physiological responses and tolerances to capture. To improve our understanding of chondrichthyan stress physiology and potentially reduce variation when quantifying the stress response, we investigated the use of the adenylate energy charge (AEC); a measure of available metabolic energy. To determine tissues sensitive to metabolic stress, we extracted samples of the brain, heart, liver, white muscle and blood from gummy sharks (Mustelus antarcticus) immediately following gillnet capture and after 3 h recovery under laboratory conditions. Capture caused significant declines in liver, white muscle and blood AEC, whereas no decline was detected in the heart and brain AEC. Following 3 h of recovery from capture, the AEC of the liver and blood returned to "unstressed" levels (control values) whereas white muscle AEC was not significantly different to that immediately after capture. Our results show that the liver is most sensitive to metabolic stress and white muscle offers a practical method to sample animals non-lethally for determination of the AEC. The AEC is a highly informative indicator of stress and unlike current indicators, it can directly measure the change in available energy and thus the metabolic stress experienced by a given tissue. Cellular metabolism is highly conserved across organisms and, therefore, we think the AEC can also provide a standardised form of measuring capture stress in many chondrichthyan species. PMID:26660290

  10. The DUSP26 phosphatase activator adenylate kinase 2 regulates FADD phosphorylation and cell growth

    NASA Astrophysics Data System (ADS)

    Kim, Hyunjoo; Lee, Ho-June; Oh, Yumin; Choi, Seon-Guk; Hong, Se-Hoon; Kim, Hyo-Jin; Lee, Song-Yi; Choi, Ji-Woo; Su Hwang, Deog; Kim, Key-Sun; Kim, Hyo-Joon; Zhang, Jianke; Youn, Hyun-Jo; Noh, Dong-Young; Jung, Yong-Keun

    2014-02-01

    Adenylate kinase 2 (AK2), which balances adenine nucleotide pool, is a multi-functional protein. Here we show that AK2 negatively regulates tumour cell growth. AK2 forms a complex with dual-specificity phosphatase 26 (DUSP26) phosphatase and stimulates DUSP26 activity independently of its AK activity. AK2/DUSP26 phosphatase protein complex dephosphorylates fas-associated protein with death domain (FADD) and regulates cell growth. AK2 deficiency enhances cell proliferation and induces tumour formation in a xenograft assay. This anti-growth function of AK2 is associated with its DUSP26-stimulating activity. Downregulation of AK2 is frequently found in tumour cells and human cancer tissues showing high levels of phospho-FADDSer194. Moreover, reconstitution of AK2 in AK2-deficient tumour cells retards both cell proliferation and tumourigenesis. Consistent with this, AK2+/- mouse embryo fibroblasts exhibit enhanced cell proliferation with a significant alteration in phospho-FADDSer191. These results suggest that AK2 is an associated activator of DUSP26 and suppresses cell proliferation by FADD dephosphorylation, postulating AK2 as a negative regulator of tumour growth.

  11. Effects of Temperature on Infected Cell O2 Concentration and Adenylate Levels in Attached Soybean Nodules.

    PubMed Central

    Kuzma, M. M.; Topunov, A. F.; Layzell, D. B.

    1995-01-01

    To assess the role of O2 in the regulation of nodule metabolism following a decrease or an increase in temperature, the fractional oxygenation of leghemoglobin (FOL) was measured in soybean (Glycine max L. Merr.) nodules during rapid and gradual changes in temperature from 20[deg]C to either 15 or 25[deg]C. The affinity of leghemoglobin for O2 was also measured at each temperature and the values were used to calculate the infected cell O2 concentration (Oi). After nodules were transferred to 15[deg]C, FOL and Oi increased and adenylate energy charge (AEC = [ATP + 0.5ADP]/[ATP + ADP + AMP]) increased from 0.70 to 0.78. The temperature increase was associated with a decrease in FOL and Oi. We concluded that changes in nodule temperature alter the respiratory demand of the nodules for O2, resulting in a change in Oi and a shift in the balance between ATP consumption and ATP production within the nodule tissue. PMID:12228427

  12. Down-regulation of adenylate kinase 5 in temporal lobe epilepsy patients and rat model.

    PubMed

    Lai, Yujie; Hu, Xiaotong; Chen, Guojun; Wang, Xuefeng; Zhu, Binglin

    2016-07-15

    Adenylate kinase 5 (AK5) is one member of the AK family and plays a critical role in maintaining cellular homeostasis. Different from the other AKs, AK5 is almost exclusively expressed in the brain. However, its exact biological functions remain unclear. The aim of the present study is to explore the expression pattern of AK5 in patients with refractory epilepsy and in a chronic pilocarpine-induced epileptic rat model. Using Western blot, immunofluorescence and immunoprecipitation analysis, we found that AK5 protein was mainly expressed in neurons, demonstrated by colocalization with the dendritic marker, MAP2, which were similar to the corresponding controls. However, the expression of AK5 decreased remarkably in epileptic patients and experimental rats. Furthermore, immunoprecipitation analysis showed that the interaction of AK5 with copine VI (CPNE6, a brain specific protein) increased in epileptic patients and rat models. Our results are the first to indicate that the expression of AK5 in epileptic brain tissue may play important roles in epilepsy, especially refractory epilepsy. PMID:27288770

  13. Critical roles of the guanylyl cyclase B receptor in endochondral ossification and development of female reproductive organs

    PubMed Central

    Tamura, Naohisa; Doolittle, Lynda K.; Hammer, Robert E.; Shelton, John M.; Richardson, James A.; Garbers, David L.

    2004-01-01

    Guanylyl cyclase B is the receptor for a small peptide (C-type natriuretic peptide) produced locally in many different tissues. To unravel the functions of the receptor, we generated mice lacking guanylyl cyclase B through gene targeting. Expression of the receptor mRNA in tissues such as bone and female reproductive organs was evident, and significant phenotypes associated with each of these tissues were apparent in null mice. A dramatic impairment of endochondral ossification and an attenuation of longitudinal vertebra or limb-bone growth were seen in null animals. C-type natriuretic peptide-dependent increases of guanylyl cyclase B activity, but not basal enzyme activity, appeared to be required for the progression of endochondral ossification. Female mice were infertile, but male mice were not. This result was due to the failure of the female reproductive tract to develop. Thus, the guanylyl cyclase B receptor is critical for the development of both bone and female reproductive organs. PMID:15572448

  14. CFTR Inhibitors

    PubMed Central

    Verkman, Alan S.; Synder, David; Tradtrantip, Lukmanee; Thiagarajah, Jay R.; Anderson, Marc O.

    2014-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated Cl− channel whose major function is to facilitate epithelial fluid secretion. Loss-of-function mutations in CFTR cause the genetic disease cystic fibrosis. CFTR is required for transepithelial fluid transport in certain secretory diarrheas, such as cholera, and for cyst expansion in autosomal dominant polycystic kidney disease. High-throughput screening has yielded CFTR inhibitors of the thiazolidinone, glycine hydrazide and quinoxalinedione chemical classes. The glycine hydrazides target the extracellular CFTR pore, whereas the thiazolidinones and quinoxalinediones act at the cytoplasmic surface. These inhibitors have been widely used in cystic fibrosis research to study CFTR function at the cell and organ levels. The most potent CFTR inhibitor has IC50 of approximately 4 nM. Studies in animal models support the development of CFTR inhibitors for antisecretory therapy of enterotoxin-mediated diarrheas and polycystic kidney disease. PMID:23331030

  15. Receptor-type guanylate cyclase is required for carbon dioxide sensation by Caenorhabditis elegans.

    PubMed

    Hallem, Elissa A; Spencer, W Clay; McWhirter, Rebecca D; Zeller, Georg; Henz, Stefan R; Rätsch, Gunnar; Miller, David M; Horvitz, H Robert; Sternberg, Paul W; Ringstad, Niels

    2011-01-01

    CO(2) is both a critical regulator of animal physiology and an important sensory cue for many animals for host detection, food location, and mate finding. The free-living soil nematode Caenorhabditis elegans shows CO(2) avoidance behavior, which requires a pair of ciliated sensory neurons, the BAG neurons. Using in vivo calcium imaging, we show that CO(2) specifically activates the BAG neurons and that the CO(2)-sensing function of BAG neurons requires TAX-2/TAX-4 cyclic nucleotide-gated ion channels and the receptor-type guanylate cyclase GCY-9. Our results delineate a molecular pathway for CO(2) sensing and suggest that activation of a receptor-type guanylate cyclase is an evolutionarily conserved mechanism by which animals detect environmental CO(2). PMID:21173231

  16. Mechanistic Characterisation of Two Sesquiterpene Cyclases from the Plant Pathogenic Fungus Fusarium fujikuroi.

    PubMed

    Burkhardt, Immo; Siemon, Thomas; Henrot, Matthias; Studt, Lena; Rösler, Sarah; Tudzynski, Bettina; Christmann, Mathias; Dickschat, Jeroen S

    2016-07-18

    Two sesquiterpene cyclases from Fusarium fujikuroi were expressed in Escherichia coli and purified. The first enzyme was inactive because of a critical mutation, but activity was restored by sequence correction through site-directed mutagenesis. The mutated enzyme and two naturally functional homologues from other fusaria converted farnesyl diphosphate into guaia-6,10(14)-diene. The second enzyme produced eremophilene. The absolute configuration of guaia-6,10(14)-diene was elucidated by enantioselective synthesis, while that of eremophilene was evident from the sign of its optical rotation and is opposite to that in plants but the same as in Sorangium cellulosum. The mechanisms of both terpene cyclases were studied with various (13) C- and (2) H-labelled FPP isotopomers. PMID:27294564

  17. An Adenylyl Cyclase, CyaB, Acts as an Osmosensor in Myxococcus xanthus

    PubMed Central

    Kimura, Yoshio; Ohtani, Mika; Takegawa, Kaoru

    2005-01-01

    We have previously reported that a receptor-type adenylyl cyclase (CyaA) of Myxococcus xanthus undergoes an osmosensor mainly during spore germination (Y. Kimura et al., J. Bacteriol. 184:3578-3585, 2002). In the present study, we cloned another receptor-type adenylyl cyclase gene (cyaB) and characterized the function of the cyaB-encoded protein. Disruption of cyaB generates a mutant that showed growth retardation at high ionic (NaCl) or high nonionic (sucrose) osmolarity. When vegetative cells were stimulated with 0.15 M NaCl, the increases in intracellular cyclic AMP levels of cyaB mutant cells were lower than those of wild-type cells. Under nonionic osmostress, the cyaB mutant exhibited reduced spore germination; however, the germination rate of the cyaB mutant was significantly higher than that of the cyaA mutant. PMID:15866951

  18. Overexpression of the Type 1 Adenylyl Cyclase in the Forebrain Leads to Deficits of Behavioral Inhibition

    PubMed Central

    Cao, Hong; Saraf, Amit; Zweifel, Larry S.

    2015-01-01

    The type 1 adenylyl cyclase (AC1) is an activity-dependent, calcium-stimulated adenylyl cyclase expressed in the nervous system that is implicated in memory formation. We examined the locomotor activity, and impulsive and social behaviors of AC1+ mice, a transgenic mouse strain overexpressing AC1 in the forebrain. Here we report that AC1+ mice exhibit hyperactive behaviors and demonstrate increased impulsivity and reduced sociability. In contrast, AC1 and AC8 double knock-out mice are hypoactive, and exhibit increased sociability and reduced impulsivity. Interestingly, the hyperactivity of AC1+ mice can be corrected by valproate, a mood-stabilizing drug. These data indicate that increased expression of AC1 in the forebrain leads to deficits in behavioral inhibition. PMID:25568126

  19. Effects of 39 Compounds on Calmodulin-Regulated Adenylyl Cyclases AC1 and Bacillus anthracis Edema Factor

    PubMed Central

    Lübker, Carolin; Seifert, Roland

    2015-01-01

    Adenylyl cyclases (ACs) catalyze the conversion of ATP into the second messenger cAMP. Membranous AC1 (AC1) is involved in processes of memory and learning and in muscle pain. The AC toxin edema factor (EF) of Bacillus anthracis is involved in the development of anthrax. Both ACs are stimulated by the eukaryotic Ca2+-sensor calmodulin (CaM). The CaM-AC interaction could constitute a potential target to enhance or impair the AC activity of AC1 and EF to intervene in above (patho)physiological mechanisms. Thus, we analyzed the impact of 39 compounds including typical CaM-inhibitors, an anticonvulsant, an anticholinergic, antidepressants, antipsychotics and Ca2+-antagonists on CaM-stimulated catalytic activity of AC1 and EF. Compounds were tested at 10 μM, i.e., a concentration that can be reached therapeutically for certain antidepressants and antipsychotics. Calmidazolium chloride decreased CaM-stimulated AC1 activity moderately by about 30%. In contrast, CaM-stimulated EF activity was abrogated by calmidazolium chloride and additionally decreased by chlorpromazine, felodipine, penfluridol and trifluoperazine by about 20–40%. The activity of both ACs was decreased by calmidazolium chloride in the presence and absence of CaM. Thus, CaM-stimulated AC1 activity is more insensitive to inhibition by small molecules than CaM-stimulated EF activity. Inhibition of AC1 and EF by calmidazolium chloride is largely mediated via a CaM-independent allosteric mechanism. PMID:25946093

  20. Potential role of mitochondrial superoxide decreasing ferrochelatase and heme in coronary artery soluble guanylate cyclase depletion by angiotensin II.

    PubMed

    Patel, Dhara; Alhawaj, Raed; Kelly, Melissa R; Accarino, John J O; Lakhkar, Anand; Gupte, Sachin A; Sun, Dong; Wolin, Michael S

    2016-06-01

    Oxidation of the soluble guanylate cyclase (sGC) heme promotes loss of regulation by nitric oxide (NO) and depletion of sGC. We hypothesized that angiotensin II (ANG II) stimulation of mitochondrial superoxide by its type 1 receptor could function as a potential inhibitor of heme biosynthesis by ferrochelatase, and this could decrease vascular responsiveness to NO by depleting sGC. These processes were investigated in a 24-h organoid culture model of bovine coronary arteries (BCA) with 0.1 μM ANG II. Treatment of BCA with ANG II increased mitochondrial superoxide, depleted mitochondrial superoxide dismutase (SOD2), ferrochelatase, and cytochrome oxidase subunit 4, and sGC, associated with impairment of relaxation to NO. These processes were attenuated by organoid culture with 8-bromo-cGMP and/or δ-aminolevulinic acid (a stimulator of sGC by protoporphyrin IX generation and heme biosynthesis). Organoid culture with Mito-TEMPOL, a scavenger of mitochondrial matrix superoxide, also attenuated ANG II-elicited ferrochelatase depletion and loss of relaxation to NO, whereas organoid culture with Tempol, an extramitochondrial scavenger of superoxide, attenuated the loss of relaxation to NO by ANG II, but not ferrochelatase depletion, suggesting cytosolic superoxide could be an initiating factor in the loss of sGC regulation by NO. The depletion of cytochrome oxidase subunit 4 and sGC (but not catalase) suggests that sGC expression may be very sensitive to depletion of heme caused by ANG II disrupting ferrochelatase activity by increasing mitochondrial superoxide. In addition, cGMP-dependent activation of protein kinase G appears to attenuate these ANG II-stimulated processes through both preventing SOD2 depletion and increases in mitochondrial and extramitochondrial superoxide. PMID:27037373

  1. Preconditioning with soluble guanylate cyclase activation prevents postischemic inflammation and reduces nitrate tolerance in heme oxygenase-1 knockout mice.

    PubMed

    Wang, Walter Z; Jones, Allan W; Wang, Meifang; Durante, William; Korthuis, Ronald J

    2013-08-15

    Previously we have shown that, unlike wild-type mice (WT), heme oxygenase-1 knockout (HO-1-/-) mice developed nitrate tolerance and were not protected from inflammation caused by ischemia-reperfusion (I/R) when preconditioned with a H2S donor. We hypothesized that stimulation (with BAY 41-2272) or activation (with BAY 60-2770) of soluble guanylate cyclase (sGC) would precondition HO-1-/- mice against an inflammatory effect of I/R and increase arterial nitrate responses. Intravital fluorescence microscopy was used to visualize leukocyte rolling and adhesion to postcapillary venules of the small intestine in anesthetized mice. Relaxation to ACh and BAY compounds was measured on superior mesenteric arteries isolated after I/R protocols. Preconditioning with either BAY compound 10 min (early phase) or 24 h (late phase) before I/R reduced postischemic leukocyte rolling and adhesion to sham control levels and increased superior mesenteric artery responses to ACh, sodium nitroprusside, and BAY 41-2272 in WT and HO-1-/- mice. Late-phase preconditioning with BAY 60-2770 was maintained in HO-1-/- and endothelial nitric oxide synthase knockout mice pretreated with an inhibitor (dl-propargylglycine) of enzymatically produced H2S. Pretreatment with BAY compounds also prevented the I/R increase in small intestinal TNF-α. We speculate that increasing sGC activity and related PKG acts downstream to H2S and disrupts signaling processes triggered by I/R in part by maintaining low cellular Ca²⁺. In addition, BAY preconditioning did not increase sGC levels, yet increased the response to agents that act on reduced heme-containing sGC. Collectively these actions would contribute to increased nitrate sensitivity and vascular function. PMID:23771693

  2. Gastrointestinal pain: unraveling a novel endogenous pathway through uroguanylin/guanylate cyclase-C/cGMP activation.

    PubMed

    Silos-Santiago, Inmaculada; Hannig, Gerhard; Eutamene, Helene; Ustinova, Elena E; Bernier, Sylvie G; Ge, Pei; Graul, Christopher; Jacobson, Sarah; Jin, Hong; Liong, Elaine; Kessler, Marco M; Reza, Tammi; Rivers, Samuel; Shea, Courtney; Tchernychev, Boris; Bryant, Alexander P; Kurtz, Caroline B; Bueno, Lionel; Pezzone, Michael A; Currie, Mark G

    2013-09-01

    The natural hormone uroguanylin regulates intestinal fluid homeostasis and bowel function through activation of guanylate cyclase-C (GC-C), resulting in increased intracellular cyclic guanosine-3',5'-monophosphate (cGMP). We report the effects of uroguanylin-mediated activation of the GC-C/cGMP pathway in vitro on extracellular cGMP transport and in vivo in rat models of inflammation- and stress-induced visceral hypersensitivity. In vitro exposure of intestinal Caco-2 cells to uroguanylin stimulated bidirectional, active extracellular transport of cGMP into luminal and basolateral spaces. cGMP transport was significantly and concentration dependently decreased by probenecid, an inhibitor of cGMP efflux pumps. In ex vivo Ussing chamber assays, uroguanylin stimulated cGMP secretion from the basolateral side of rat colonic epithelium into the submucosal space. In a rat model of trinitrobenzene sulfonic acid (TNBS)-induced visceral hypersensitivity, orally administered uroguanylin increased colonic thresholds required to elicit abdominal contractions in response to colorectal distension (CRD). Oral administration of cGMP mimicked the antihyperalgesic effects of uroguanylin, significantly decreasing TNBS- and restraint stress-induced visceromotor response to graded CRD in rats. The antihyperalgesic effects of cGMP were not associated with increased colonic spasmolytic activity, but were linked to significantly decreased firing rates of TNBS-sensitized colonic afferents in rats in response to mechanical stimuli. In conclusion, these data suggest that the continuous activation of the GC-C/cGMP pathway along the intestinal tract by the endogenous hormones guanylin and uroguanylin results in significant reduction of gastrointestinal pain. Extracellular cGMP produced on activation of GC-C is the primary mediator in this process via modulation of sensory afferent activity. PMID:23748116

  3. Control of the Diadenylate Cyclase CdaS in Bacillus subtilis

    PubMed Central

    Mehne, Felix M. P.; Schröder-Tittmann, Kathrin; Eijlander, Robyn T.; Herzberg, Christina; Hewitt, Lorraine; Kaever, Volkhard; Lewis, Richard J.; Kuipers, Oscar P.; Tittmann, Kai; Stülke, Jörg

    2014-01-01

    The Gram-positive bacterium Bacillus subtilis encodes three diadenylate cyclases that synthesize the essential signaling nucleotide cyclic di-AMP. The activities of the vegetative enzymes DisA and CdaA are controlled by protein-protein interactions with their conserved partner proteins. Here, we have analyzed the regulation of the unique sporulation-specific diadenylate cyclase CdaS. Very low expression of CdaS as the single diadenylate cyclase resulted in the appearance of spontaneous suppressor mutations. Several of these mutations in the cdaS gene affected the N-terminal domain of CdaS. The corresponding CdaS mutant proteins exhibited a significantly increased enzymatic activity. The N-terminal domain of CdaS consists of two α-helices and is attached to the C-terminal catalytically active diadenylate cyclase (DAC) domain. Deletion of the first or both helices resulted also in strongly increased activity indicating that the N-terminal domain serves to limit the enzyme activity of the DAC domain. The structure of YojJ, a protein highly similar to CdaS, indicates that the protein forms hexamers that are incompatible with enzymatic activity of the DAC domains. In contrast, the mutations and the deletions of the N-terminal domain result in conformational changes that lead to highly increased enzymatic activity. Although the full-length CdaS protein was found to form hexamers, a truncated version with a deletion of the first N-terminal helix formed dimers with high enzyme activity. To assess the role of CdaS in sporulation, we assayed the germination of wild type and cdaS mutant spores. The results indicate that cyclic di-AMP formed by CdaS is required for efficient germination. PMID:24939848

  4. An improved technique for the rapid chemical characterisation of bacterial terpene cyclases.

    PubMed

    Dickschat, Jeroen S; Pahirulzaman, Khomaizon A K; Rabe, Patrick; Klapschinski, Tim A

    2014-04-14

    A derivative of the pET28c(+) expression vector was constructed. It contains a yeast replication system (2μ origin of replication) and a yeast selectable marker (URA3), and can be used for gene cloning in yeast by efficient homologous recombination, and for heterologous expression in E. coli. The vector was used for the expression and chemical characterisation of three bacterial terpene cyclases. PMID:24573945

  5. Elevation of lutein content in tomato: a biochemical tug-of-war between lycopene cyclases.

    PubMed

    Giorio, Giovanni; Yildirim, Arzu; Stigliani, Adriana Lucia; D'Ambrosio, Caterina

    2013-11-01

    Lutein is becoming increasingly important in preventive medicine due to its possible role in maintaining good vision and in preventing age-related maculopathy. Average daily lutein intake in developed countries is often below suggested daily consumption levels, and lutein supplementation could be beneficial. Lutein is also valuable in the food and feed industries and is emerging in nutraceutical and pharmaceutical markets. Currently, lutein is obtained at high cost from marigold petals, and synthesis alternatives are thus desirable. Tomato constitutes a promising starting system for production as it naturally accumulates high levels of lycopene. To develop tomato for lutein synthesis, the tomato Red Setter cultivar was transformed with the tomato lycopene ε-cyclase-encoding gene under the control of a constitutive promoter, and the HighDelta (HD) line, characterised by elevated lutein and δ-carotene content in ripe fruits, was selected. HD was crossed to the transgenic HC line and to RS(B) with the aim of converting all residual fruit δ-carotene to lutein. Fruits of both crosses were enriched in lutein and presented unusual carotenoid profiles. The unique genetic background of the crosses used in this study permitted an unprecedented analysis of the role and regulation of the lycopene cyclase enzymes in tomato. A new defined biochemical index, the relative cyclase activity ratio, was used to discern post-transcriptional regulation of cyclases, and will help in the study of carotenoid biosynthesis in photosynthetic plant species and particularly in those, like tomato, that have been domesticated for the production of food, feed or useful by-products. PMID:24141052

  6. Pharmacological analysis of feeding in a caterpillar: different transduction pathways for umami and saccharin?

    NASA Astrophysics Data System (ADS)

    Pszczolkowski, Maciej A.; Durden, Kevin; Marquis, Juleah; Ramaswamy, Sonny B.; Brown, John J.

    2009-05-01

    Neonate larvae of codling moth, Cydia pomonella (L.), modify their behavior in the presence of saccharin, monosodium glutamate (MSG), or L(+)-2-amino-4-phosphonobutyric acid (L-AP4) by commencing their feeding earlier. Previously published pharmacological analysis demonstrated that phagostimulatory effects of MSG and L-AP4 (which elicit umami taste sensation in humans) are reversed by adenylate cyclase activator and phosphodiesterase inhibitor. In this study, by measuring the time needed to start ingestion of foliage treated with mixtures of phagostimulants and signal transduction modulators, we show that phagostimulatory effects of l-aspartate (the third hallmark umami substance) are also abolished by both adenylate cyclase activator and phosphodiesterase inhibitor, but not by phospholipase C inhibitor. However, stimulatory effects of hemicalcium saccharin were affected only by phospholipase C inhibitor. The results suggest that codling moth neonates use different transduction pathways for perception of hemicalcium saccharin and umami.

  7. Differential contribution of the guanylyl cyclase-cyclic GMP-protein kinase G pathway to the proliferation of neural stem cells stimulated by nitric oxide.

    PubMed

    Carreira, Bruno P; Morte, Maria Inês; Lourenço, Ana Sofia; Santos, Ana Isabel; Inácio, Angela; Ambrósio, António F; Carvalho, Caetana M; Araújo, Inês M

    2013-01-01

    Nitric oxide (NO) is an important inflammatory mediator involved in the initial boost in the proliferation of neural stem cells following brain injury. However, the mechanisms underlying the proliferative effect of NO are still unclear. The aim of this work was to investigate whether cyclic GMP (cGMP) and the cGMP-dependent kinase (PKG) are involved in the proliferative effect triggered by NO in neural stem cells. For this purpose, cultures of neural stem cells isolated from the mouse subventricular zone (SVZ) were used. We observed that long-term exposure to the NO donor (24 h), NOC-18, increased the proliferation of SVZ cells in a cGMP-dependent manner, since the guanylate cyclase inhibitor, ODQ, prevented cell proliferation. Similarly to NOC-18, the cGMP analogue, 8-Br-cGMP, also increased cell proliferation. Interestingly, shorter exposures to NO (6 h) increased cell proliferation in a cGMP-independent manner via the ERK/MAP kinase pathway. The selective inhibitor of PKG, KT5823, prevented the proliferative effect induced by NO at 24 h but not at 6 h. In conclusion, the proliferative effect of NO is initially mediated by the ERK/MAPK pathway, and at later stages by the GC/cGMP/PKG pathway. Thus, our work shows that NO induces neural stem cell proliferation by targeting these two pathways in a biphasic manner. PMID:22378242

  8. Identification of photoactivated adenylyl cyclases in Naegleria australiensis and BLUF-containing protein in Naegleria fowleri.

    PubMed

    Yasukawa, Hiro; Sato, Aya; Kita, Ayaka; Kodaira, Ken-Ichi; Iseki, Mineo; Takahashi, Tetsuo; Shibusawa, Mami; Watanabe, Masakatsu; Yagita, Kenji

    2013-01-01

    Complete genome sequencing of Naegleria gruberi has revealed that the organism encodes polypeptides similar to photoactivated adenylyl cyclases (PACs). Screening in the N. australiensis genome showed that the organism also encodes polypeptides similar to PACs. Each of the Naegleria proteins consists of a "sensors of blue-light using FAD" domain (BLUF domain) and an adenylyl cyclase domain (AC domain). PAC activity of the Naegleria proteins was assayed by comparing sensitivities of Escherichia coli cells heterologously expressing the proteins to antibiotics in a dark condition and a blue light-irradiated condition. Antibiotics used in the assays were fosfomycin and fosmidomycin. E. coli cells expressing the Naegleria proteins showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light, indicating that the proteins functioned as PACs in the bacterial cells. Analysis of the N. fowleri genome revealed that the organism encodes a protein bearing an amino acid sequence similar to that of BLUF. A plasmid expressing a chimeric protein consisting of the BLUF-like sequence found in N. fowleri and the adenylyl cyclase domain of N. gruberi PAC was constructed to determine whether the BLUF-like sequence functioned as a sensor of blue light. E. coli cells expressing a chimeric protein showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light. These experimental results indicated that the sequence similar to the BLUF domain found in N. fowleri functioned as a sensor of blue light. PMID:24201148

  9. The Presence of Two Cyclase Thioesterases Expands the Conformational Freedom of the Cyclic Peptide Occidiofungin

    PubMed Central

    Ravichandran, Akshaya; Gu, Ganyu; Escano, Jerome; Lu, Shi-En; Smith, Leif

    2014-01-01

    Occidiofungin is a cyclic nonribosomally synthesized antifungal peptide with submicromolar activity produced by Gram-negative bacterium Burkholderia contaminans. The biosynthetic gene cluster was confirmed to contain two cyclase thioesterases. NMR analysis revealed that the presence of both thioesterases is used to increase the conformational repertoire of the cyclic peptide. The loss of the OcfN cyclic thioesterase by mutagenesis results in a reduction of conformational variants and an appreciable decrease in bioactivity against Candida species. Presumably, the presence of both asparagine and β-hydroxyasparagine variants coordinate the enzymatic function of both of the cyclase thioesterases. OcfN has presumably evolved to be part of the biosynthetic gene cluster due to its ability to produce structural variants that enhance antifungal activity against some fungi. The enhancement of the antifungal activity from the incorporation of an additional cyclase thioesterase into the biosynthetic gene cluster of occidiofungin supports the need to explore new conformational variants of other therapeutic or potentially therapeutic cyclic peptides. PMID:23394257

  10. Sensitization of olfactory guanylyl cyclase to a specific imprinted odorant in coho salmon.

    PubMed

    Dittman, A H; Quinn, T P; Nevitt, G A; Hacker, B; Storm, D R

    1997-08-01

    The role of cGMP in olfactory signaling is not fully understood, but it is believed to play a modulatory role in intracellular signaling in vertebrate olfactory receptor neurons (ORNs). Here, we present evidence that cGMP in ORNs may play an important role in recognition of biologically relevant odors and olfactory learning. Specifically, we investigated the cellular mechanisms underlying olfactory imprinting in salmon. Salmon learn odors associated with their natal site as juveniles and later use these odors to guide their homing migration. This imprinting is believed to involve sensitization of the peripheral olfactory system to specific homestream odorants. We imprinted juvenile salmon to the odorant beta-phenylethyl alcohol (PEA) and examined the sensitivity of olfactory adenylyl and guanylyl cyclases to PEA during development. Stimulation of guanylyl cyclase activity by PEA was significantly greater in olfactory cilia isolated from PEA-imprinted salmon compared with PEA-naive fish only at the time of the homing migration, 2 years after PEA exposure. These results suggest that sensitization of olfactory guanylyl cyclase may play an important role in olfactory imprinting by salmon. PMID:9292727

  11. Sll0254 (CrtLdiox) Is a Bifunctional Lycopene Cyclase/Dioxygenase in Cyanobacteria Producing Myxoxanthophyll

    PubMed Central

    Mohamed, Hatem E.; Vermaas, Wim F. J.

    2006-01-01

    Upon depletion of Sll0254 in Synechocystis sp. strain PCC 6803, cyclized carotenoids were replaced by linear, relatively hydrophilic carotenoids, and the amount of the two photosystems decreased greatly. Full segregants of the sll0254 deletion in Synechocystis were not obtained, implying that this gene is essential for survival, most likely to allow normal cell division. The N-terminal half of Sll0254 has limited similarity to the family of lycopene cyclases, has an additional dehydrogenase motif near the N terminus, and is followed by a Rieske 2Fe-2S center sequence signature. To test whether Sll0254 serves as a lycopene cyclase in Synechocystis, the corresponding gene was expressed in Escherichia coli strains that can produce lycopene or neurosporene. In the presence of Sll0254 these linear carotenoids were converted into cyclized, relatively hydrophilic pigments, with masses consistent with the introduction of two hydroxyl groups and with spectra indicative of only small changes in the number of conjugated double bonds. This suggests that Sll0254 catalyzes formation of oxygenated, cyclized carotenoids. We interpret the appearance of the hydroxyl groups in the carotenoids to be due to dioxygenase activity involving the Rieske 2Fe-2S center and the additional dehydrogenase domain. This dioxygenase activity is required in the myxoxanthophyll biosynthesis pathway, after or concomitant with cyclization on the other end of the molecule. We interpret Sll0254 to be a dual-function enzyme with both lycopene cyclase and dioxygenase activity and have named it CrtLdiox. PMID:16621828

  12. Central role of soluble adenylyl cyclase and cAMP in sperm physiology

    PubMed Central

    Buffone, Mariano G.; Wertheimer, Eva V.; Visconti, Pablo E.; Krapf, Dario

    2014-01-01

    Cyclic adenosine 3′,5′-monophosphate (cAMP), the first second messenger to be described, plays a central role in cell signaling in a wide variety of cell types. Over the last decades, a wide body of literature addressed the different roles of cAMP in cell physiology, mainly in response to neurotransmitters and hormones. cAMP is synthesized by a wide variety of adenylyl cylases that can generally be grouped in two types: transmembrane adenylyl cyclase and soluble adenylyl cyclases. In particular, several aspects of sperm physiology are regulated by cAMP produced by a single atypical adenylyl cyclase (Adcy10, aka sAC, SACY). The signature that identifies sAC among other ACs, is their direct stimulation by bicarbonate. The essential nature of cAMP in sperm function has been demonstrated using gain of function as well as loss of function approaches. This review unifies state of the art knowledge of the role of cAMP and those enzymes involved in cAMP signaling pathways required for the acquisition of fertilizing capacity of mammalian sperm. PMID:25066614

  13. Evidence for an essential histidine residue in 4S-limonene synthase and other terpene cyclases.

    PubMed

    Rajaonarivony, J I; Gershenzon, J; Miyazaki, J; Croteau, R

    1992-11-15

    (4S)-Limonene synthase, isolated from glandular trichome secretory cell preparations of Mentha x piperita (peppermint) leaves, catalyzes the metal ion-dependent cyclization of geranyl pyrophosphate, via 3S-linalyl pyrophosphate, to (-)-(4S)-limonene as the principal product. Treatment of this terpene cyclase with the histidine-directed reagent diethyl pyrocarbonate at a concentration of 0.25 mM resulted in 50% loss of enzyme activity, and this activity could be completely restored by treatment of the preparation with 5 mM hydroxylamine. Inhibition with diethyl pyrocarbonate was distinguished from inhibition with thiol-directed reagents by protection studies with histidine and cysteine carried out at varying pH. Inactivation of the cyclase by dye-sensitized photooxidation in the presence of rose bengal gave further indication of the presence of a readily modified histidine residue. Protection of the enzyme against inhibition with diethyl pyrocarbonate was afforded by the substrate geranyl pyrophosphate in the presence of Mn2+, and by the sulfonium ion analog of the linalyl carbocation intermediate of the reaction in the presence of inorganic pyrophosphate plus Mn2+, suggesting that an essential histidine residue is located at or near the active site. Similar studies on the inhibition of other monoterpene and sesquiterpene cyclases with diethyl pyrocarbonate suggest that a histidine residue (or residues) may play an important role in catalysis by this class of enzymes. PMID:1444454

  14. Microarray evidence of glutaminyl cyclase gene expression in melanoma: implications for tumor antigen specific immunotherapy

    PubMed Central

    Gillis, John Stuart

    2006-01-01

    Background In recent years encouraging progress has been made in developing vaccine treatments for cancer, particularly with melanoma. However, the overall rate of clinically significant results has remained low. The present research used microarray datasets from previous investigations to examine gene expression patterns in cancer cell lines with the goal of better understanding the tumor microenvironment. Methods Principal Components Analyses with Promax rotational transformations were carried out with 90 cancer cell lines from 3 microarray datasets, which had been made available on the internet as supplementary information from prior publications. Results In each of the analyses a well defined melanoma component was identified that contained a gene coding for the enzyme, glutaminyl cyclase, which was as highly expressed as genes from a variety of well established biomarkers for melanoma, such as MAGE-3 and MART-1, which have frequently been used in clinical trials of melanoma vaccines. Conclusion Since glutaminyl cyclase converts glutamine and glutamic acid into a pyroglutamic form, it may interfere with the tumor destructive process of vaccines using peptides having glutamine or glutamic acid at their N-terminals. Finding ways of inhibiting the activity of glutaminyl cyclase in the tumor microenvironment may help to increase the effectiveness of some melanoma vaccines. PMID:16820060

  15. Crystallization and preliminary X-ray diffraction studies of the glutaminyl cyclase from Carica papaya latex

    SciTech Connect

    Azarkan, Mohamed; Clantin, Bernard; Bompard, Coralie; Belrhali, Hassan; Baeyens-Volant, Danielle; Looze, Yvan; Wintjens, René

    2005-01-01

    The glutaminyl cyclase isolated from C. papaya latex has been crystallized using the hanging-drop method. Diffraction data have been collected at ESRF beamline BM14 and processed to 1.7 Å resolution. In living systems, the intramolecular cyclization of N-terminal glutamine residues is accomplished by glutaminyl cyclase enzymes (EC 2.3.2.5). While in mammals these enzymes are involved in the synthesis of hormonal and neurotransmitter peptides, the physiological role played by the corresponding plant enzymes still remains to be unravelled. Papaya glutaminyl cyclase (PQC), a 33 kDa enzyme found in the latex of the tropical tree Carica papaya, displays an exceptional resistance to chemical and thermal denaturation as well as to proteolysis. In order to elucidate its enzymatic mechanism and to gain insights into the structural determinants underlying its remarkable stability, PQC was isolated from papaya latex, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 62.82, b = 81.23, c = 108.17 Å and two molecules per asymmetric unit. Diffraction data have been collected at ESRF beamline BM14 and processed to a resolution of 1.7 Å.

  16. Eubacterial Diterpene Cyclase Genes Essential for Production of the Isoprenoid Antibiotic Terpentecin

    PubMed Central

    Dairi, Tohru; Hamano, Yoshimitsu; Kuzuyama, Tomohisa; Itoh, Nobuya; Furihata, Kazuo; Seto, Haruo

    2001-01-01

    A gene cluster containing the mevalonate pathway genes (open reading frame 2 [ORF2] to ORF7) for the formation of isopentenyl diphosphate and a geranylgeranyl diphosphate (GGDP) synthase gene (ORF1) had previously been cloned from Streptomyces griseolosporeus strain MF730-N6, a diterpenoid antibiotic, terpentecin (TP) producer (Y. Hamano, T. Dairi, M. Yamamoto, T. Kawasaki, K Kaneda, T. Kuzuyama, N. Itoh, and H. Seto, Biosci. Biotech. Biochem. 65:1627–1635, 2001). Sequence analysis in the upstream region of the cluster revealed seven new ORFs, ORF8 to ORF14, which were suggested to encode TP biosynthetic genes. We constructed two mutants, in which ORF11 and ORF12, which encode a protein showing similarities to eukaryotic diterpene cyclases (DCs) and a eubacterial pentalenene synthase, respectively, were inactivated by gene disruptions. The mutants produced no TP, confirming that these cyclase genes are essential for the production of TP. The two cyclase genes were also expressed in Streptomyces lividans together with the GGDP synthase gene under the control of the ermE* constitutive promoter. The transformant produced a novel cyclic diterpenoid, ent-clerod-3,13(16),14-triene (terpentetriene), which has the same basic skeleton as TP. The two enzymes, each of which was overproduced in Escherichia coli and purified to homogeneity, converted GGDP into terpentetriene. To the best of our knowledge, this is the first report of a eubacterial DC. PMID:11567009

  17. Dcsbis (PA2771) from Pseudomonas aeruginosa is a highly active diguanylate cyclase with unique activity regulation.

    PubMed

    Chen, Ying; Liu, Shiheng; Liu, Cuilan; Huang, Yan; Chi, Kaikai; Su, Tiantian; Zhu, Deyu; Peng, Jin; Xia, Zhijie; He, Jing; Xu, Sujuan; Hu, Wei; Gu, Lichuan

    2016-01-01

    C-di-GMP (3',5' -Cyclic diguanylic acid) is an important second messenger in bacteria that influences virulence, motility, biofilm formation, and cell division. The level of c-di-GMP in cells is controlled by diguanyl cyclases (DGCs) and phosphodiesterases (PDEs). Here, we report the biochemical functions and crystal structure of the potential diguanylase Dcsbis (PA2771, a diguanylate cyclase with a self-blocked I-site) from Pseudomonas aeruginosa PAO1. The full-length Dcsbis protein contains an N-terminal GAF domain and a C-terminal GGDEF domain. We showed that Dcsbis tightly coordinates cell motility without markedly affecting biofilm formation and is a diguanylate cyclase with a catalytic activity much higher than those of many other DGCs. Unexpectedly, we found that a peptide loop (protecting loop) extending from the GAF domain occupies the conserved inhibition site, thereby largely relieving the product-inhibition effect. A large hydrophobic pocket was observed in the GAF domain, thus suggesting that an unknown upstream signaling molecule may bind to the GAF domain, moving the protecting loop from the I-site and thereby turning off the enzymatic activity. PMID:27388857

  18. An Adenylyl Cyclase, CyaA, of Myxococcus xanthus Functions in Signal Transduction during Osmotic Stress

    PubMed Central

    Kimura, Yoshio; Mishima, Yukako; Nakano, Hiromi; Takegawa, Kaoru

    2002-01-01

    An adenylyl cyclase gene (cyaA) present upstream of an osmosensor protein gene (mokA) was isolated from Myxococcus xanthus. cyaA encoded a polypeptide of 843 amino acids with a predicted molecular mass of 91,187 Da. The predicted cyaA gene product had structural similarity to the receptor-type adenylyl cyclases that are composed of an amino-terminal sensor domain and a carboxy-terminal catalytic domain of adenylyl cyclase. In reverse transcriptase PCR experiments, the transcript of the cyaA gene was detected mainly during development and spore germination. A cyaA mutant, generated by gene disruption, showed normal growth, development, and germination. However, a cyaA mutant placed under conditions of ionic (NaCl) or nonionic (sucrose) osmostress exhibited a marked reduction in spore formation and spore germination. When wild-type and cyaA mutant cells at developmental stages were stimulated with 0.2 M NaCl or sucrose, the mutant cells increased cyclic AMP accumulation at levels similar to those of the wild-type cells. In contrast, the mutant cells during spore germination had mainly lost the ability to respond to high-ionic osmolarity. In vegetative cells, the cyaA mutant responded normally to osmotic stress. These results suggested that M. xanthus CyaA functions mainly as an ionic osmosensor during spore germination and that CyaA is also required for osmotic tolerance in fruiting formation and sporulation. PMID:12057952

  19. Evolutionary Divergence of Sedoheptulose 7-phosphate Cyclases Leads to Several Distinct Cyclic Products

    PubMed Central

    Asamizu, Shumpei; Xie, Pengfei; Brumsted, Corey J.; Flatt, Patricia M.; Mahmud, Taifo

    2012-01-01

    Sedoheptulose 7-phosphate cyclases are enzymes that utilize the pentose phosphate pathway intermediate, sedoheptulose 7-phosphate, to generate cyclic precursors of many bioactive natural products, such as the antidiabetic drug acarbose, the crop protectant validamycin, and the natural sunscreens mycosporine-like amino acids. These proteins are phylogenetically related to the dehydroquinate (DHQ) synthases from the shikimate pathway, and are part of the more recently recognized superfamily of sugar phosphate cyclases, which includes DHQ synthases, aminoDHQ synthases and 2-deoxy-scyllo-inosose synthases. Through genome mining and biochemical studies, we identified yet another subset of DHQS-like proteins in the actinomycete Actinosynnema mirum and the myxobacterium Stigmatella aurantiaca DW4/3–1. These enzymes catalyze the conversion of sedoheptulose 7-phosphate to 2-epi-valiolone, which is predicted to be an alternative precursor for aminocyclitol biosynthesis. Comparative bioinformatics and biochemical analyses of these proteins with 2-epi-5-epi-valiolone synthases (EEVS) and desmethyl-4-deoxygadusol synthases (DDGS) provided further insights into their genetic diversity, conserved amino acid sequences, and plausible catalytic mechanisms. The results further highlight the uniquely diverse DHQS-like sugar phosphate cyclases, which may provide new tools for chemoenzymatic, stereospecific synthesis of various cyclic molecules. PMID:22741921

  20. Dcsbis (PA2771) from Pseudomonas aeruginosa is a highly active diguanylate cyclase with unique activity regulation

    PubMed Central

    Chen, Ying; Liu, Shiheng; Liu, Cuilan; Huang, Yan; Chi, Kaikai; Su, Tiantian; Zhu, Deyu; Peng, Jin; Xia, Zhijie; He, Jing; Xu, Sujuan; Hu, Wei; Gu, Lichuan

    2016-01-01

    C-di-GMP (3’,5’ -Cyclic diguanylic acid) is an important second messenger in bacteria that influences virulence, motility, biofilm formation, and cell division. The level of c-di-GMP in cells is controlled by diguanyl cyclases (DGCs) and phosphodiesterases (PDEs). Here, we report the biochemical functions and crystal structure of the potential diguanylase Dcsbis (PA2771, a diguanylate cyclase with a self-blocked I-site) from Pseudomonas aeruginosa PAO1. The full-length Dcsbis protein contains an N-terminal GAF domain and a C-terminal GGDEF domain. We showed that Dcsbis tightly coordinates cell motility without markedly affecting biofilm formation and is a diguanylate cyclase with a catalytic activity much higher than those of many other DGCs. Unexpectedly, we found that a peptide loop (protecting loop) extending from the GAF domain occupies the conserved inhibition site, thereby largely relieving the product-inhibition effect. A large hydrophobic pocket was observed in the GAF domain, thus suggesting that an unknown upstream signaling molecule may bind to the GAF domain, moving the protecting loop from the I-site and thereby turning off the enzymatic activity. PMID:27388857

  1. HAMP domain-mediated signal transduction probed with a mycobacterial adenylyl cyclase as a reporter.

    PubMed

    Mondéjar, Laura García; Lupas, Andrei; Schultz, Anita; Schultz, Joachim E

    2012-01-01

    HAMP domains, ∼55 amino acid motifs first identified in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases, operate as signal mediators in two-component signal transduction proteins. A bioinformatics study identified a coevolving signal-accepting network of 10 amino acids in membrane-delimited HAMP proteins. To probe the functionality of this network we used a HAMP containing mycobacterial adenylyl cyclase, Rv3645, as a reporter enzyme in which the membrane anchor was substituted by the Escherichia coli chemotaxis receptor for serine (Tsr receptor) and the HAMP domain alternately with that from the protein Af1503 of the archaeon Archaeoglobus fulgidus or the Tsr receptor. In a construct with the Tsr-HAMP, cyclase activity was inhibited by serine, whereas in a construct with the HAMP domain from A. fulgidus, enzyme activity was not responsive to serine. Amino acids of the signal-accepting network were mutually swapped between both HAMP domains, and serine signaling was examined. The data biochemically tentatively established the functionality of the signal-accepting network. Based on a two-state gearbox model of rotation in HAMP domain-mediated signal propagation, we characterized the interaction between permanent and transient core residues in a coiled coil HAMP structure. The data are compatible with HAMP rotation in signal propagation but do not exclude alternative models for HAMP signaling. Finally, we present data indicating that the connector, which links the α-helices of HAMP domains, plays an important structural role in HAMP function. PMID:22094466

  2. Two members of a widely expressed subfamily of hormone-stimulated adenylyl cyclases.

    PubMed Central

    Premont, R T; Chen, J; Ma, H W; Ponnapalli, M; Iyengar, R

    1992-01-01

    cDNA encoding a hormone- and guanine nucleotide-stimulated adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] (type 6) from rat liver and kidney has been cloned and expressed. This enzyme is stimulated by forskolin, guanosine 5'-[gamma-thio]triphosphate, and isoproterenol plus GTP but is not stimulated by beta gamma subunits of guanine nucleotide-binding proteins. A second form (type 5), which is 75% similar to type 6, has also been cloned. Both types 5 and 6 cDNAs have multiple messages. PCR-based detection of the mRNA for the type 5 and 6 enzymes indicates that both are widely distributed. Homology analyses indicate at least four distinct subfamilies of guanine nucleotide stimulatory protein-regulated adenylyl cyclases. Types 5 and 6 enzymes define one distinct subfamily of mammalian adenylyl cyclases. Diversity of one guanine nucleotide-binding protein-regulated effector may allow different modes of regulation of cell-surface signal transmission. Images PMID:1409703

  3. Alignment-Free Methods for the Detection and Specificity Prediction of Adenylation Domains.

    PubMed

    Agüero-Chapin, Guillermin; Pérez-Machado, Gisselle; Sánchez-Rodríguez, Aminael; Santos, Miguel Machado; Antunes, Agostinho

    2016-01-01

    Identifying adenylation domains (A-domains) and their substrate specificity can aid the detection of nonribosomal peptide synthetases (NRPS) at genome/proteome level and allow inferring the structure of oligopeptides with relevant biological activities. However, that is challenging task due to the high sequence diversity of A-domains (~10-40 % of amino acid identity) and their selectivity for 50 different natural/unnatural amino acids. Altogether these characteristics make their detection and the prediction of their substrate specificity a real challenge when using traditional sequence alignment methods, e.g., BLAST searches. In this chapter we describe two workflows based on alignment-free methods intended for the identification and substrate specificity prediction of A-domains. To identify A-domains we introduce a graphical-numerical method, implemented in TI2BioP version 2.0 (topological indices to biopolymers), which in a first step uses protein four-color maps to represent A-domains. In a second step, simple topological indices (TIs), called spectral moments, are derived from the graphical representations of known A-domains (positive dataset) and of unrelated but well-characterized sequences (negative set). Spectral moments are then used as input predictors for statistical classification techniques to build alignment-free models. Finally, the resulting alignment-free models can be used to explore entire proteomes for unannotated A-domains. In addition, this graphical-numerical methodology works as a sequence-search method that can be ensemble with homology-based tools to deeply explore the A-domain signature and cope with the diversity of this class (Aguero-Chapin et al., PLoS One 8(7):e65926, 2013). The second workflow for the prediction of A-domain's substrate specificity is based on alignment-free models constructed by transductive support vector machines (TSVMs) that incorporate information of uncharacterized A-domains. The construction of the models was

  4. Mapping the Dynamics Landscape of Conformational Transitions in Enzyme: The Adenylate Kinase Case

    PubMed Central

    Li, Dechang; Liu, Ming S.; Ji, Baohua

    2015-01-01

    Conformational transition describes the essential dynamics and mechanism of enzymes in pursuing their various functions. The fundamental and practical challenge to researchers is to quantitatively describe the roles of large-scale dynamic transitions for regulating the catalytic processes. In this study, we tackled this challenge by exploring the pathways and free energy landscape of conformational changes in adenylate kinase (AdK), a key ubiquitous enzyme for cellular energy homeostasis. Using explicit long-timescale (up to microseconds) molecular dynamics and bias-exchange metadynamics simulations, we determined at the atomistic level the intermediate conformational states and mapped the transition pathways of AdK in the presence and absence of ligands. There is clearly chronological operation of the functional domains of AdK. Specifically in the ligand-free AdK, there is no significant energy barrier in the free energy landscape separating the open and closed states. Instead there are multiple intermediate conformational states, which facilitate the rapid transitions of AdK. In the ligand-bound AdK, the closed conformation is energetically most favored with a large energy barrier to open it up, and the conformational population prefers to shift to the closed form coupled with transitions. The results suggest a perspective for a hybrid of conformational selection and induced fit operations of ligand binding to AdK. These observations, depicted in the most comprehensive and quantitative way to date, to our knowledge, emphasize the underlying intrinsic dynamics of AdK and reveal the sophisticated conformational transitions of AdK in fulfilling its enzymatic functions. The developed methodology can also apply to other proteins and biomolecular systems. PMID:26244746

  5. Vascular natriuretic peptide receptor-linked particulate guanylate cyclases are modulated by nitric oxide–cyclic GMP signalling

    PubMed Central

    Madhani, Melanie; Scotland, Ramona S; MacAllister, Raymond J; Hobbs, Adrian J

    2003-01-01

    The sensitivity of the particulate guanylate cyclase–cyclic guanosine-3′,5′-monophosphate (cGMP) system to atrial (ANP) and C-type (CNP) natriuretic peptides was investigated in aortae and mesenteric small arteries from wild-type (WT) and endothelial nitric oxide synthase (eNOS) knockout (KO) mice. ANP and CNP produced concentration-dependent relaxations of mouse aorta that were significantly attenuated by the natriuretic peptide receptor (NPR)-A/B antagonist HS-142-1 (10−5 M). Both ANP and CNP were more potent in aortae from eNOS KO mice compared to WT. The potency of ANP and CNP in aortae from WT animals was increased in the presence of the NOS inhibitor, NG-nitro-L-arginine (3 × 10−4 M) and the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolol[4,3,a]quinoxalin-1-one (5 × 10−6 M). In contrast, the potency of ANP and CNP in aortae from eNOS KO animals was reduced following pretreatment of tissues with supramaximal concentrations of the NO-donor, glyceryl trinitrate (3 × 10−5 M, 30 min) or ANP (10−7 M, 30 min). Responses to acetylcholine in aortae from WT mice (dependent on the release of endothelium-derived NO) were significantly reduced following pretreatment of tissues with GTN (3 × 10−5 M, 30 min) and ANP (10−7 M, 30 min). CNP and the NO-donor, spermine-NONOate caused concentration-dependent relaxations of mesenteric small arteries from WT animals that were significantly increased in eNOS KO mice compared to WT. ANP was unable to significantly relax mesenteric arteries from WT or eNOS KO animals. In conclusion, both NPR-A- and NPR-B-linked pGC pathways are modulated by NO–cGMP in murine aorta and mesenteric small arteries and crossdesensitisation occurs between NPR subtypes. The biological activity of endothelium-derived NO is also influenced by the ambient concentration of NO and natriuretic peptides. Such an autoregulatory pathway may represent an important physiological homeostatic mechanism and link the paracrine activity

  6. Free energy of hydrolysis of tyrosyl adenylate and its binding to wild-type and engineered mutant tyrosyl-tRNA synthetases

    SciTech Connect

    Wells, T.N.C.; Ho, C.K.; Fersht, A.R.

    1986-10-21

    The equilibrium constant for the formation of tyrosyl adenylate and pyrophosphate from ATP and tyrosine in solution has been measured by applying the Haldane relationship to wild-type and three mutant tyrosyl-tRNA synthetases from Bacillus stearothermophilus. The formation constant (=(Tyr-AMP)(PP/sub i/)/(ATP)(Tyr)) at pH 7.78, 25/sup 0/C, and 10 mM MgCl/sub 2/ (3.5 +/- 0.5) x 10/sup -7/. This corresponds to a free energy of hydrolysis of tyrosyl adenylate at pH 7.0 and 25/sup 0/C of -16.7 kcal mol/sup -1/. All necessary rate constants had been determined previously for the calculations apart from the dissociation constant of tyrosyl adenylate from its enzyme-bound complex. This was measured by taking advantage of the 100-fold difference in hydrolysis rates of the tyrosyl adenylate when sequestered by the enzyme and when free in solution. These are technically difficult measurements because the dissociation constants are so low and the complexes unstable. The task was simplified by using mutants prepared by site-directed mutagenesis. These were designed to have different rate and equilibrium constants for dissociation of tyrosyl adenylate from the enzyme-bound complexes. The dissociation constants were in the range (3.5-38) x 10/sup -12/ M, with that for wild type at 13 x 10/sup -12/ M. The four enzymes all gave consistent data for the formation constant of tyrosyl adenylate in solution. This not only improves the reliability of the measurement but also provides confirmation of the reliability of the measured kinetic constants for the series of enzymes.

  7. Nitric oxide and guanylate cyclase signalling are differentially involved in gonadotrophin (LH) release responses to two endogenous GnRHs from goldfish pituitary cells.

    PubMed

    Meints, A N; Pemberton, J G; Chang, J P

    2012-08-01

    Nitric oxide synthase (NOS) immunoreactivity is present in goldfish gonadotrophs. The present study investigated whether two native goldfish gonadotrophin-releasing hormones (GnRHs), salmon (s)GnRH and chicken (c)GnRH-II, use NOS/nitric oxide (NO) and soluble guanylate cyclase (sGC)/cyclic (c)GMP/protein kinase G (PKG) signalling to stimulate maturational gonadotrophin [teleost gonadotrophin-II, luteinising hormone (LH)] release. In cell column perifusion experiments with dispersed goldfish pituitary cells, the application of three NOS inhibitors (aminoguanidine hemisulphate, 1400W and 7-nitroindazole) and two NO scavengers [2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) and rutin hydrate] reduced sGnRH-elicited, but not cGnRH-II-induced, LH increases. The NO donor sodium nitroprusside (SNP) increased NO production in goldfish pituitary cells in static incubation. SNP-stimulated LH release in column perifusion was attenuated by PTIO and the sGC inhibitor 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-oneon (ODQ), and additive to responses elicited by cGnRH-II, but not sGnRH. ODQ and the PKG inhibitor KT5823 decreased sGnRH- and cGnRH-II-stimulated LH release. Similarly, the LH response to dibutyryl cGMP was reduced by KT5823. These results indicate that, although only sGnRH uses the NOS/NO pathway to stimulate LH release, both GnRHs utilise sGC/PKG to increase LH secretion. PMID:22487215

  8. Modelling the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase on adenylate kinase.

    PubMed Central

    Bertrand, L; Vertommen, D; Depiereux, E; Hue, L; Rider, M H; Feytmans, E

    1997-01-01

    Simultaneous multiple alignment of available sequences of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase revealed several segments of conserved residues in the 2-kinase domain. The sequence of the kinase domain was also compared with proteins of known three-dimensional structure. No similarity was found between the kinase domain of 6-phosphofructo-2-kinase and 6-phosphofructo-1-kinase. This questions the modelling of the 2-kinase domain on bacterial 6-phosphofructo-1-kinase that has previously been proposed [Bazan, Fletterick and Pilkis (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646]. However, sequence similarities were found between the 2-kinase domain and several nucleotide-binding proteins, the most similar being adenylate kinase. A structural model of the 2-kinase domain based on adenylate kinase is proposed. It accommodates all the results of site-directed mutagenesis studies carried out to date on residues in the 2-kinase domain. It also allows residues potentially involved in catalysis and/or substrate binding to be predicted. PMID:9032445

  9. Signal-transduction protein P(II) from Synechococcus elongatus PCC 7942 senses low adenylate energy charge in vitro.

    PubMed

    Fokina, Oleksandra; Herrmann, Christina; Forchhammer, Karl

    2011-11-15

    P(II) proteins belong to a family of highly conserved signal-transduction proteins that occurs widely in bacteria, archaea and plants. They respond to the central metabolites ATP, ADP and 2-OG (2-oxoglutarate), and control enzymes, transcription factors and transport proteins involved in nitrogen metabolism. In the present study, we examined the effect of ADP on in vitro P(II)-signalling properties for the cyanobacterium Synechococcus elongatus, a model for oxygenic phototrophic organisms. Different ADP/ATP ratios strongly affected the properties of P(II) signalling. Increasing ADP antagonized the binding of 2-OG and directly affected the interactions of P(II) with its target proteins. The resulting P(II)-signalling properties indicate that, in mixtures of ADP and ATP, P(II) trimers are occupied by mixtures of adenylate nucleotides. Binding and kinetic activation of NAGK (N-acetyl-L-glutamate kinase), the controlling enzyme of arginine biosynthesis, by P(II) was weakened by ADP, but relief from arginine inhibition remained unaffected. On the other hand, ADP enhanced the binding of P(II) to PipX, a co-activator of the transcription factor NtcA and, furthermore, antagonized the inhibitory effect of 2-OG on P(II)-PipX interaction. These results indicate that S. elongatus P(II) directly senses the adenylate energy charge, resulting in target-dependent differential modification of the P(II)-signalling properties. PMID:21774788

  10. Cocrystal Structures of Primed Side-Extending α-Ketoamide Inhibitors Reveal Novel Calpain-Inhibitor Aromatic Interactions

    SciTech Connect

    Qian,J.; Cuerrier, D.; Davies, P.; Li, Z.; Powers, J.; Campbell, R.

    2008-01-01

    Calpains are intracellular cysteine proteases that catalyze the cleavage of target proteins in response to Ca2+ signaling. When Ca2+ homeostasis is disrupted, calpain overactivation causes unregulated proteolysis, which can contribute to diseases such as postischemic injury and cataract formation. Potent calpain inhibitors exist, but of these many cross-react with other cysteine proteases and will need modification to specifically target calpain. Here, we present crystal structures of rat calpain 1 protease core ({mu}I-II) bound to two a-ketoamide-based calpain inhibitors containing adenyl and piperazyl primed-side extensions. An unexpected aromatic-stacking interaction is observed between the primed-side adenine moiety and the Trp298 side chain. This interaction increased the potency of the inhibitor toward {mu}I-II and heterodimeric m-calpain. Moreover, stacking orients the adenine such that it can be used as a scaffold for designing novel primed-side address regions, which could be incorporated into future inhibitors to enhance their calpain specificity.

  11. Regulation and therapeutic targeting of peptide-activated receptor guanylyl cyclases

    PubMed Central

    Potter, Lincoln R.

    2016-01-01

    Cyclic GMP is a ubiquitous second messenger that regulates a wide array of physiologic processes such as blood pressure, long bone growth, intestinal fluid secretion, phototransduction and lipolysis. Soluble and single-membrane-spanning enzymes called guanylyl cyclases (GC) synthesize cGMP. In humans, the latter group consists of GC-A, GC-B, GC-C, GC-E and GC-F, which are also known as NPR-A, NPR-B, StaR, Ret1-GC and Ret2-GC, respectively. Membrane GCs are activated by peptide ligands such as atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), C-type natriuretic peptide (CNP), guanylin, uroguanylin, heat stable enterotoxin and GC-activating proteins. Nesiritide and carperitide are clinically approved peptide-based drugs that activate GC-A. CD-NP is an experimental heart failure drug that primarily activates GC-B but also activates GC-A at high concentrations and is resistant to degradation. Inactivating mutations in GC-B cause acromesomelic dysplasia type Maroteaux dwarfism and chromosomal mutations that increase CNP concentrations are associated with Marfanoid-like skeletal overgrowth. Pump-based CNP infusions increase skeletal growth in a mouse model of the most common type of human dwarfism, which supports CNP/GC-B-based therapies for short stature diseases. Linaclotide is a peptide activator of GC-C that stimulates intestinal motility and is in late-stage clinical trials for the treatment of chronic constipation. This review discusses the discovery of cGMP, guanylyl cyclases, the general characteristics and therapeutic applications of GC-A, GC-B and GC-C, and emphasizes the regulation of transmembrane guanylyl cyclases by phosphorylation and ATP. PMID:21185863

  12. Structure, signaling mechanism and regulation of the natriuretic peptide receptor guanylate cyclase.

    SciTech Connect

    Misono, K. S.; Philo, J. S.; Arakawa, T.; Ogata, C. M.; Qiu, Y.; Ogawa, H.; Young, H. S.

    2011-06-01

    Atrial natriuretic peptide (ANP) and the homologous B-type natriuretic peptide are cardiac hormones that dilate blood vessels and stimulate natriuresis and diuresis, thereby lowering blood pressure and blood volume. ANP and B-type natriuretic peptide counterbalance the actions of the renin-angiotensin-aldosterone and neurohormonal systems, and play a central role in cardiovascular regulation. These activities are mediated by natriuretic peptide receptor-A (NPRA), a single transmembrane segment, guanylyl cyclase (GC)-linked receptor that occurs as a homodimer. Here, we present an overview of the structure, possible chloride-mediated regulation and signaling mechanism of NPRA and other receptor GCs. Earlier, we determined the crystal structures of the NPRA extracellular domain with and without bound ANP. Their structural comparison has revealed a novel ANP-induced rotation mechanism occurring in the juxtamembrane region that apparently triggers transmembrane signal transduction. More recently, the crystal structures of the dimerized catalytic domain of green algae GC Cyg12 and that of cyanobacterium GC Cya2 have been reported. These structures closely resemble that of the adenylyl cyclase catalytic domain, consisting of a C1 and C2 subdomain heterodimer. Adenylyl cyclase is activated by binding of G{sub s}{alpha} to C2 and the ensuing 7{sup o} rotation of C1 around an axis parallel to the central cleft, thereby inducing the heterodimer to adopt a catalytically active conformation. We speculate that, in NPRA, the ANP-induced rotation of the juxtamembrane domains, transmitted across the transmembrane helices, may induce a similar rotation in each of the dimerized GC catalytic domains, leading to the stimulation of the GC catalytic activity.

  13. Structure of a Sedoheptulose 7-Phosphate Cyclase: ValA from Streptomyces hygroscopicus

    PubMed Central

    2015-01-01

    Sedoheptulose 7-phosphate cyclases (SH7PCs) encompass three enzymes involved in producing the core cyclitol structures of pseudoglycosides and similar bioactive natural products. One such enzyme is ValA from Streptomyces hygroscopicus subsp. jinggangensis 5008, which makes 2-epi-5-epi-valiolone as part of the biosynthesis of the agricultural antifungal agent validamycin A. We present, as the first SH7PC structure, the 2.1 Å resolution crystal structure of ValA in complex with NAD+ and Zn2+ cofactors. ValA has a fold and active site organization resembling those of the sugar phosphate cyclase dehydroquinate synthase (DHQS) and contains two notable, previously unrecognized interactions between NAD+ and Asp side chains conserved in all sugar phosphate cyclases that may influence catalysis. Because the domains of ValA adopt a nearly closed conformation even though no sugar substrate is present, comparisons with a ligand-bound DHQS provide a model for aspects of substrate binding. One striking active site difference is a loop that adopts a distinct conformation as a result of an Asp → Asn change with respect to DHQS and alters the identity and orientation of a key Arg residue. This and other active site differences in ValA are mostly localized to areas where the ValA substrate differs from that of DHQS. Sequence comparisons with a second SH7PC making a product with distinct stereochemistry lead us to postulate that the product stereochemistry of a given SH7PC is not the result of events taking place during catalysis but is accomplished by selective binding of either the α or β pyranose anomer of the substrate. PMID:24832673

  14. Multifunctional oxidosqualene cyclases and cytochrome P450 involved in the biosynthesis of apple fruit triterpenic acids.

    PubMed

    Andre, Christelle M; Legay, Sylvain; Deleruelle, Amélie; Nieuwenhuizen, Niels; Punter, Matthew; Brendolise, Cyril; Cooney, Janine M; Lateur, Marc; Hausman, Jean-François; Larondelle, Yvan; Laing, William A

    2016-09-01

    Apple (Malus × domestica) accumulates bioactive ursane-, oleanane-, and lupane-type triterpenes in its fruit cuticle, but their biosynthetic pathway is still poorly understood. We used a homology-based approach to identify and functionally characterize two new oxidosqualene cyclases (MdOSC4 and MdOSC5) and one cytochrome P450 (CYP716A175). The gene expression patterns of these enzymes and of previously described oxidosqualene cyclases were further studied in 20 apple cultivars with contrasting triterpene profiles. MdOSC4 encodes a multifunctional oxidosqualene cyclase producing an oleanane-type triterpene, putatively identified as germanicol, as well as β-amyrin and lupeol, in the proportion 82 : 14 : 4. MdOSC5 cyclizes 2,3-oxidosqualene into lupeol and β-amyrin at a ratio of 95 : 5. CYP716A175 catalyses the C-28 oxidation of α-amyrin, β-amyrin, lupeol and germanicol, producing ursolic acid, oleanolic acid, betulinic acid, and putatively morolic acid. The gene expression of MdOSC1 was linked to the concentrations of ursolic and oleanolic acid, whereas the expression of MdOSC5 was correlated with the concentrations of betulinic acid and its caffeate derivatives. Two new multifuntional triterpene synthases as well as a multifunctional triterpene C-28 oxidase were identified in Malus × domestica. This study also suggests that MdOSC1 and MdOSC5 are key genes in apple fruit triterpene biosynthesis. PMID:27214242

  15. [Proteasome inhibitor].

    PubMed

    Yagi, Hideo

    2014-06-01

    The ubiquitin-proteasome system plays an essential role in degradation of eukaryotic intracellular protein, including cell cycle regulation, cell growth and proliferation, and survival. Cancer cells generally have higher level of proteasome activity compared with normal cells, suggesting proteasome inhibition could be therapeutic target in oncology. Bortezomib, the first proteasome inhibitor introduced into the clinic, is approved for the treatment of patients with multiple myeloma (MM). Although it was approved as single agent in the relapsed setting, bortezomib is now predominantly used in combination with conventional and novel targeted agents because bortezomib has demonstrated additive and synergistic activity in preclinical studies. Recently, several second-generation proteasome inhibitors, such as carfilzomib and MLN9708, have been developed and entered into clinical trials. These agents were investigated in frontline MM in combination with lenalidomide and low-dose dexamethasone. These studies demonstrated positive efficacy and safety, and it is expected that they will be approved in near future. PMID:25016815

  16. Structural and functional characterization of the rod outer segment membrane guanylate cyclase.

    PubMed Central

    Goraczniak, R M; Duda, T; Sitaramayya, A; Sharma, R K

    1994-01-01

    In the vertebrate photoreceptor cell, rod outer segment (ROS) is the site of visual signal-transduction process, and a pivotal molecule that regulates this process is cyclic GMP. Cyclic GMP controls the cationic conductance into the ROS, and light causes a decrease in the conductance by activating hydrolysis of the cyclic nucleotide. The identity of the granylate cyclase (ROS-GC) that synthesizes this pool of cyclic GMP is unknown. We now report the cloning, expression and functional characterization of a DNA from bovine retina that encodes ROS-GC. Images Figure 2 Figure 5 PMID:7916565

  17. Enzymatic 13C Labeling and Multidimensional NMR Analysis of Miltiradiene Synthesized by Bifunctional Diterpene Cyclase in Selaginella moellendorffii*

    PubMed Central

    Sugai, Yoshinori; Ueno, Yohei; Hayashi, Ken-ichiro; Oogami, Shingo; Toyomasu, Tomonobu; Matsumoto, Sadamu; Natsume, Masahiro; Nozaki, Hiroshi; Kawaide, Hiroshi

    2011-01-01

    Diterpenes show diverse chemical structures and various physiological roles. The diversity of diterpene is primarily established by diterpene cyclases that catalyze a cyclization reaction to form the carbon skeleton of cyclic diterpene. Diterpene cyclases are divided into two types, monofunctional and bifunctional cyclases. Bifunctional diterpene cyclases (BDTCs) are involved in hormone and defense compound biosyntheses in bryophytes and gymnosperms, respectively. The BDTCs catalyze the successive two-step type-B (protonation-initiated cyclization) and type-A (ionization-initiated cyclization) reactions of geranylgeranyl diphosphate (GGDP). We found that the genome of a lycophyte, Selaginella moellendorffii, contains six BDTC genes with the majority being uncharacterized. The cDNA from S. moellendorffii encoding a BDTC-like enzyme, miltiradiene synthase (SmMDS), was cloned. The recombinant SmMDS converted GGDP to a diterpene hydrocarbon product with a molecular mass of 272 Da. Mutation in the type-B active motif of SmMDS abolished the cyclase activity, whereas (+)-copalyl diphosphate, the reaction intermediate from the conversion of GGDP to the hydrocarbon product, rescued the cyclase activity of the mutant to form a diterpene hydrocarbon. Another mutant lacking type-A activity accumulated copalyl diphosphate as the reaction intermediate. When the diterpene hydrocarbon was enzymatically synthesized from [U-13C6]mevalonate, all carbons were labeled with 13C stable isotope (>99%). The fully 13C-labeled product was subjected to 13C-13C COSY NMR spectroscopic analyses. The direct carbon-carbon connectivities observed in the multidimensional NMR spectra demonstrated that the hydrocarbon product by SmMDS is miltiradiene, a putative biosynthetic precursor of tanshinone identified from the Chinese medicinal herb Salvia miltiorrhiza. Hence, SmMDS functions as a bifunctional miltiradiene synthase in S. moellendorffii. In this study, we demonstrate that one-dimensional and

  18. Novel inhibitors of Anthrax edema factor

    PubMed Central

    Chen, Deliang; Misra, Milind; Sower, Laurie; Peterson, Johnny W.; Kellogg, Glen E.; Schein, Catherine H.

    2008-01-01

    Several pathogenic bacteria produce adenylyl cyclase toxins, such as the edema factor (EF) of Bacillus anthracis. These disturb cellular metabolism by catalyzing production of excessive amounts of the regulatory molecule cAMP. Here, a structure-based method, where a 3D- pharmacophore that fit the active site of EF was constructed from fragments, was used to identify non-nucleotide inhibitors of EF. A library of small molecule fragments was docked to the EF- active site in existing crystal structures and those with the highest HINT scores were assembled into a 3D-pharmacophore. About 10,000 compounds, from over 2.7 million compounds in the ZINC database, had a similar molecular framework. These were ranked according to their docking scores, using methodology that was shown to achieve maximum accuracy (i.e., how well the docked position matched the experimentally determined site for ATP analogues in crystal structures of the complex). Finally, 19 diverse compounds with the best AutoDock binding/docking scores were assayed in a cell based assay for their ability to reduce cAMP secretion induced by EF. Four of the test compounds, from different structural groups, inhibited in the low micromolar range. One of these has a core structure common to phosphatase inhibitors previously identified by high-throughput assays of a diversity library. Thus, the fragment based pharmacophore identified a small number of diverse compounds for assay, and greatly enhanced the selection process of advanced lead compounds for combinatorial design. PMID:18620864

  19. Inhibition of glutaminyl cyclase ameliorates amyloid pathology in an animal model of Alzheimer's disease via the modulation of γ-secretase activity.

    PubMed

    Song, Hyundong; Chang, Yu Jin; Moon, Minho; Park, Sarah Kyua; Tran, Phuong-Thao; Hoang, Van-Hai; Lee, Jeewoo; Mook-Jung, Inhee

    2015-01-01

    Alzheimer's disease is the most prevalent neurodegenerative disorder, characterized by neurofibrillary tangles, senile plaques, and neuron loss. Amyloid beta peptides are generated from amyloid beta precursor protein by consecutive catalysis by β and γ-secretases. Diversely modified forms of A have been N3pE-42 Aβ has received considerable attention as one of the major constituents of the senile plaques of AD brains due to its higher aggregation velocity, stability, and hydrophobicity compared to the full-length A. A previous study suggested that is catalyzed by glutaminyl cyclase (QC) following limited proteolysis of Aβ at the N-terminus. Here, we reveal that decreasing the QC activity via application of a QC inhibitor modulates-γ-secretase activity, resulting in diminished plaque formation as well as reduced N3pE 42 Aβ aggregates in the subiculum of the 5XFAD mouse model of AD. This study suggests a possible novel mechanism by which QC regulates Aβ formation , namely modulation of γ-secretase activity. PMID:25114069

  20. Structural analysis of the diadenylate cyclase reaction of DNA-integrity scanning protein A (DisA) and its inhibition by 3'-dATP.

    PubMed

    Müller, Martina; Deimling, Tobias; Hopfner, Karl-Peter; Witte, Gregor

    2015-08-01

    The identification of the essential bacterial second messenger cyclic-di-AMP (c-di-AMP) synthesized by the DNA-integrity scanning protein A (DisA) has opened up a new and emerging field in bacterial signalling. To further analyse the diadenylate cyclase (DAC) reaction catalysed by the DAC domains of DisA, we crystallized Thermotoga maritima DisA in the presence of different ATP analogues and metal ions to identify the metal-binding site and trap the enzyme in pre- and post-reaction states. Through structural and biochemical assays we identified important residues essential for the reaction in the active site of the DAC domains. Our structures resolve the metal-binding site and thus explain the activation of ATP for the DAC reaction. Moreover, we were able to identify a potent inhibitor of the DAC domain. Based on the available structures and homology to annotated DAC domains we propose a common mechanism for c-di-AMP synthesis by DAC domains in c-di-AMP-producing species and a possible approach for its effective inhibition. PMID:26014055

  1. Activation of IKK/NF-κB provokes renal inflammatory responses in guanylyl cyclase/natriuretic peptide receptor-A gene-knockout mice

    PubMed Central

    Das, Subhankar; Periyasamy, Ramu

    2012-01-01

    The present study was aimed at determining the consequences of the disruption of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene (Npr1) on proinflammatory responses of nuclear factor kappa B, inhibitory kappa B kinase, and inhibitory kappa B alpha (NF-κB, IKK, IκBα) in the kidneys of mutant mice. The results showed that the disruption of Npr1 enhanced the renal NF-κB binding activity by 3.8-fold in 0-copy (−/−) mice compared with 2-copy (+/+) mice. In parallel, IKK activity and IκBα protein phosphorylation were increased by 8- and 11-fold, respectively, in the kidneys of 0-copy mice compared with wild-type mice. Interestingly, IκBα was reduced by 80% and the expression of proinflammatory cytokines and renal fibrosis were significantly enhanced in 0-copy mice than 2-copy mice. Treatment of 0-copy mice with NF-κB inhibitors andrographolide, pyrrolidine dithiocarbamate, and etanercept showed a substantial reduction in renal fibrosis, attenuation of proinflammatory cytokines gene expression, and significantly reduced IKK activity and IkBα phosphorylation. These findings indicate that the systemic disruption of Npr1 activates the renal NF-κB pathways in 0-copy mice, which transactivates the expression of various proinflammatory cytokines to initiate renal remodeling; however, inhibition of NF-κB pathway repairs the abnormal renal pathology in mutant mice. PMID:22318993

  2. Involvement of H1 and H2 receptors and soluble guanylate cyclase in histamine-induced relaxation of rat mesenteric collecting lymphatics

    PubMed Central

    Kurtz, Kristine H.; Moor, Andrea N.; Souza-Smith, Flavia M.; Breslin, Jerome W.

    2014-01-01

    Objective This study investigated the roles of the H1 and H2 histamine receptors, nitric oxide (NO) synthase, and soluble guanylate (sGC) cyclase in histamine-induced modulation of rat mesenteric collecting lymphatic pumping. Methods Isolated rat mesenteric collecting lymphatics were treated with 1–100 μM histamine. Histamine receptors were blocked with either the H1 antagonist mepyramine or the H2 antagonist cimetidine. The role of NO/sGC signaling was tested using the arginine analog L-NAME, the sGC inhibitor ODQ, and sodium nitroprusside (SNP) as a positive control. Results Histamine applied at 100 μM decreased tone and contraction frequency (CF) of isolated rat mesenteric collecting lymphatics. Pharmacologic blockade of either H1 or H2 histamine receptors significantly inhibited the response to histamine. Pretreatment with ODQ, but not L-NAME, completely inhibited the histamine-induced decrease in tone. ODQ pretreatment also significantly inhibited SNP-induced lymphatic relaxation. Conclusions H1 and H2 histamine receptors are both involved in histamine-induced relaxation of rat mesenteric collecting lymphatics. NO synthesis does not appear to contribute to the histamine-induced response. However, sGC is critical for the histamine-induced decrease in tone and contributes to the drop in CF. PMID:24702851

  3. The vasorelaxant effect of 8(17),12E,14-labdatrien-18-oic acid involves stimulation of adenylyl cyclase and cAMP/PKA pathway: Evidences by pharmacological and molecular docking studies.

    PubMed

    Ribeiro, Luciano A A; Alencar Filho, Edilson B; Coelho, Maisa C; Silva, Bagnólia A

    2015-10-01

    The relaxant effect of 8(17),12E,14-labdatrien-18-oic acid (LBD) was investigated on isolated aortic rings and compared with forskolin (FSK), a standard and potent activator of adenylyl cyclase (AC) with relaxing effect. The presence of potassium channel blockers, such as glibenclamide (ATP-blocker), apamin (SKCa-blocker), charybdotoxin (BKCa-blocker) did not significantly affect either the LBD or FSK concentration-response curves. However, in the presence of 4-aminopyridine (KV-blocker), the relaxant effect for both diterpenes was significantly attenuated, with reduction of its relative potencies. Moreover, the relaxation induced by 8-Br-cAMP, an analog of cAMP, was also significantly attenuated in the same conditions, i.e., in the presence of 4-aminopyridine. The presence of aminophylline, a nonselective phosphodiesterase inhibitor, caused a significant increasing in the potency for both LBD and FSK. On the other hand, the presence of Rp-cAMPS, a selective PKA-inhibitor, significantly attenuated the relaxant effect of LBD. In this work, in the same experimental conditions, both labdane-type diterpenes presented remarkably similar results; FSK, however, presented a higher potency (100-fold) than LBD. Thus, the hypothesis that LBD could be a novel AC-activator emerged. To assess that hypothesis, computational molecular docking studies were performed. Crystallographic structure of adenylyl cyclase/forskolin complex (1AB8) was obtained from RSCB Protein Data Bank and used to compare the modes of interaction of the native ligand and LBD. The computational data shows many similarities between LBD and FSK concerning the interaction with the regulatory site of AC. Taken together, the results presented here pointed to LBD as a novel AC-activator. PMID:26144373

  4. Regulation of sesquiterpene cyclase gene expression. Characterization of an elicitor- and pathogen-inducible promoter.

    PubMed Central

    Yin, S; Mei, L; Newman, J; Back, K; Chappell, J

    1997-01-01

    The promoter for a tobacco (Nicotiana tabacum) sesquiterpene cyclase gene, a key regulatory step in sesquiterpene phytoalexin biosynthesis, has been analyzed. The EAS4 promoter was fused to the beta-glucuronidase (GUS) reporter gene, and the temporal and spatial expression patterns of GUS activity were examined in stably transformed plants and in transient expression assays using electroporated protoplasts of tobacco. No GUS activity was observed in any tissues under normal growth conditions. A low level of GUS activity was detected in wounded leaf, root, and stem tissues, whereas a much higher level was observed when these tissues were challenged with elicitors or microbial pathogens. The GUS expression pattern directed by the EAS4 promoter was identical to the induction patterns observed for the endogenous sesquiterpene cyclase genes. Neither exogenous salicylic acid nor methyl jasmonate induced GUS expression; and H2O2 induced GUS expression to only a limited extent. Although the EAS4 promoter contains cis-sequences resembling previously identified transcriptional control motifs, other cis-sequences important for quantitative and qualitative gene expression were identified by deletion and gain-of-function analyses. The EAS4 promoter differs from previously described pathogen-/elicitor-inducible promoters because it only supports inducible gene expression and directs unique spatial expression patterns. PMID:9342864

  5. Phosphorylation-Independent Regulation of the Diguanylate Cyclase WspR

    PubMed Central

    De, Nabanita; Pirruccello, Michelle; Krasteva, Petya Violinova; Bae, Narae; Raghavan, Rahul Veera; Sondermann, Holger

    2008-01-01

    Environmental signals that trigger bacterial pathogenesis and biofilm formation are mediated by changes in the level of cyclic dimeric guanosine monophosphate (c-di-GMP), a unique eubacterial second messenger. Tight regulation of cellular c-di-GMP concentration is governed by diguanylate cyclases and phosphodiesterases, which are responsible for its production and degradation, respectively. Here, we present the crystal structure of the diguanylate cyclase WspR, a conserved GGDEF domain-containing response regulator in Gram-negative bacteria, bound to c-di-GMP at an inhibitory site. Biochemical analyses revealed that feedback regulation involves the formation of at least three distinct oligomeric states. By switching from an active to a product-inhibited dimer via a tetrameric assembly, WspR utilizes a novel mechanism for modulation of its activity through oligomerization. Moreover, our data suggest that these enzymes can be activated by phosphodiesterases. Thus, in addition to the canonical pathways via phosphorylation of the regulatory domains, both product and enzyme concentration contribute to the coordination of c-di-GMP signal