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Sample records for adenylyl cyclase involved

  1. 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

  2. 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

  3. 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

  4. A HCO3−-dependent mechanism involving soluble adenylyl cyclase for the activation of Ca2+ currents in locus coeruleus neurons

    PubMed Central

    Imber, Ann N.; Santin, Joseph M.; Graham, Cathy D.; Putnam, Robert W.

    2014-01-01

    Hypercapnic acidosis activates Ca2+ channels and increases intracellular Ca2+ levels in neurons of the locus coeruleus (LC), a known chemosensitive region involved in respiratory control. We have also shown that large conductance Ca2+-activated K+ channels (BK), in conjunction with this pathway, limits the hypercapnic-induced increase in firing rate in LC neurons. Here, we present evidence that the Ca2+ current is activated by a HCO3−-sensitive pathway. The increase in HCO3− associated with hypercapnia activates HCO3−-sensitive adenylyl cyclase (sAC). This results in an increase in cAMP levels and activation of Ca2+ channels via cAMP-activated protein kinase A (PKA). We also show the presence of sAC in the cytoplasm of LC neurons, and that the cAMP analogue db-cAMP increases Ca2+i. Disrupting this pathway by decreasing HCO3− levels during acidification or inhibiting either sAC or PKA, but not transmembrane adenylyl cyclase (tmAC), can increase the magnitude of the firing rate response to hypercapnia in LC neurons from older neonates to the same extent as inhibition of BK channels. PMID:25092170

  5. 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

  6. 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

  7. 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

  8. 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

  9. Comparative involvement of cyclic nucleotide phosphodiesterases and adenylyl cyclase on adrenocorticotropin-induced increase of cyclic adenosine monophosphate in rat and human glomerulosa cells.

    PubMed

    Côté, M; Payet, M D; Rousseau, E; Guillon, G; Gallo-Payet, N

    1999-08-01

    The present study investigated the role and identity of cyclic nucleotide phosphodiesterases (PDEs) in the regulation of basal and ACTH-stimulated levels of intracellular cAMP in human and rat adrenal glomerulosa cells. Comparative dose-response curves indicated that maximal hormone-stimulated cAMP accumulation was 11- and 24-fold higher in human and rat cells, compared with cAMP production obtained in corresponding membranes, respectively. Similarly to 3-isobutyl-1-methyl-xanthine, 25 microM erythro-9-[2-hydroxy-3-nonyl]adenine (EHNA, a specific PDE2 inhibitor), caused a large increase in ACTH-stimulated cAMP accumulation; by contrast, it did not change cAMP production in membranes. Moreover, in membrane fractions, addition of 10 microM cGMP inhibited ACTH-induced cAMP production, an effect completely reversed by addition of 25 microM EHNA. These results indicate that PDE2 activity is involved in the regulation of cAMP accumulation induced by ACTH, and suggest that ACTH inhibits this activity. Indeed, time-course studies indicated that ACTH induced a rapid decrease in cGMP production, resulting in PDE2 inhibition, which in turn, contributed [with adenylyl cyclase (AC) activation] to an accumulation in cAMP for 15 min. Thereafter, cAMP content decreased, because of cAMP-stimulated PDE2, as confirmed by measurement of PDE activity that was activated by ACTH, but only after a 10-min incubation. Hence, we demonstrate that the ACTH-induced increase in intracellular cAMP is the result of a balance between activation of AC and direct modulation of PDE2 activity, an effect mediated by cGMP content. Although similar results were observed in both models, PDE2 involvement is more important in rat than in human adrenal glomerulosa cells, whereas AC is more stimulated in human than in rat glomerulosa cells. PMID:10433216

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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

  1. Cyclic nucleotide binding and structural changes in the isolated GAF domain of Anabaena adenylyl cyclase, CyaB2

    PubMed Central

    Badireddy, Suguna; Rajendran, Abinaya; Anand, Ganesh Srinivasan

    2015-01-01

    GAF domains are a large family of regulatory domains, and a subset are found associated with enzymes involved in cyclic nucleotide (cNMP) metabolism such as adenylyl cyclases and phosphodiesterases. CyaB2, an adenylyl cyclase from Anabaena, contains two GAF domains in tandem at the N-terminus and an adenylyl cyclase domain at the C-terminus. Cyclic AMP, but not cGMP, binding to the GAF domains of CyaB2 increases the activity of the cyclase domain leading to enhanced synthesis of cAMP. Here we show that the isolated GAFb domain of CyaB2 can bind both cAMP and cGMP, and enhanced specificity for cAMP is observed only when both the GAFa and the GAFb domains are present in tandem (GAFab domain). In silico docking and mutational analysis identified distinct residues important for interaction with either cAMP or cGMP in the GAFb domain. Structural changes associated with ligand binding to the GAF domains could not be detected by bioluminescence resonance energy transfer (BRET) experiments. However, amide hydrogen-deuterium exchange mass spectrometry (HDXMS) experiments provided insights into the structural basis for cAMP-induced allosteric regulation of the GAF domains, and differences in the changes induced by cAMP and cGMP binding to the GAF domain. Thus, our findings could allow the development of molecules that modulate the allosteric regulation by GAF domains present in pharmacologically relevant proteins. PMID:25922789

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. Mice Overexpressing Type 1 Adenylyl Cyclase Show Enhanced Spatial Memory Flexibility in the Absence of Intact Synaptic Long-Term Depression

    ERIC Educational Resources Information Center

    Zhang, Ming; Wang, Hongbing

    2013-01-01

    There is significant interest in understanding the contribution of intracellular signaling and synaptic substrates to memory flexibility, which involves new learning and suppression of obsolete memory. Here, we report that enhancement of Ca[superscript 2+]-stimulated cAMP signaling by overexpressing type 1 adenylyl cyclase (AC1) facilitated…

  7. 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

  8. 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

  9. 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

  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. 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

  12. 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

  13. 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

  14. Established and potential physiological roles of bicarbonate-sensing soluble adenylyl cyclase (sAC) in aquatic animals

    PubMed Central

    Tresguerres, Martin; Barott, Katie L.; Barron, Megan E.; Roa, Jinae N.

    2014-01-01

    Soluble adenylyl cyclase (sAC) is a recently recognized source of the signaling molecule cyclic AMP (cAMP) that is genetically and biochemically distinct from the classic G-protein-regulated transmembrane adenylyl cyclases (tmACs). Mammalian sAC is distributed throughout the cytoplasm and it may be present in the nucleus and inside mitochondria. sAC activity is directly stimulated by HCO3−, and sAC has been confirmed to be a HCO3− sensor in a variety of mammalian cell types. In addition, sAC can functionally associate with carbonic anhydrases to act as a de facto sensor of pH and CO2. The two catalytic domains of sAC are related to HCO3−-regulated adenylyl cyclases from cyanobacteria, suggesting the cAMP pathway is an evolutionarily conserved mechanism for sensing CO2 levels and/or acid/base conditions. Reports of sAC in aquatic animals are still limited but are rapidly accumulating. In shark gills, sAC senses blood alkalosis and triggers compensatory H+ absorption. In the intestine of bony fishes, sAC modulates NaCl and water absorption. And in sea urchin sperm, sAC may participate in the initiation of flagellar movement and in the acrosome reaction. Bioinformatics and RT-PCR results reveal that sAC orthologs are present in most animal phyla. This review summarizes the current knowledge on the physiological roles of sAC in aquatic animals and suggests additional functions in which sAC may be involved. PMID:24574382

  15. Established and potential physiological roles of bicarbonate-sensing soluble adenylyl cyclase (sAC) in aquatic animals.

    PubMed

    Tresguerres, Martin; Barott, Katie L; Barron, Megan E; Roa, Jinae N

    2014-03-01

    Soluble adenylyl cyclase (sAC) is a recently recognized source of the signaling molecule cyclic AMP (cAMP) that is genetically and biochemically distinct from the classic G-protein-regulated transmembrane adenylyl cyclases (tmACs). Mammalian sAC is distributed throughout the cytoplasm and it may be present in the nucleus and inside mitochondria. sAC activity is directly stimulated by HCO3(-), and sAC has been confirmed to be a HCO3(-) sensor in a variety of mammalian cell types. In addition, sAC can functionally associate with carbonic anhydrases to act as a de facto sensor of pH and CO2. The two catalytic domains of sAC are related to HCO3(-)-regulated adenylyl cyclases from cyanobacteria, suggesting the cAMP pathway is an evolutionarily conserved mechanism for sensing CO2 levels and/or acid/base conditions. Reports of sAC in aquatic animals are still limited but are rapidly accumulating. In shark gills, sAC senses blood alkalosis and triggers compensatory H(+) absorption. In the intestine of bony fishes, sAC modulates NaCl and water absorption. And in sea urchin sperm, sAC may participate in the initiation of flagellar movement and in the acrosome reaction. Bioinformatics and RT-PCR results reveal that sAC orthologs are present in most animal phyla. This review summarizes the current knowledge on the physiological roles of sAC in aquatic animals and suggests additional functions in which sAC may be involved. PMID:24574382

  16. Laboratory evolution of adenylyl cyclase independent learning in Drosophil and missing heritability

    PubMed Central

    Cressy, M.; Valente, D.; Altick, A; Kockenmeister, E.; Honegger, K.; Qin, H.; Mitra, P.P.; Dubnau, J

    2014-01-01

    Gene interactions are acknowledged to be a likely source of missing heritability in large-scale genetic studies of complex neurological phenotypes. However, involvement of rare variants, de novo mutations, genetic lesions that are not easily detected with commonly used methods and epigenetic factors also are possible explanations. We used a laboratory evolution study to investigate the modulatory effects of background genetic variation on the phenotypic effect size of a null mutation with known impact on olfactory learning. To accomplish this, we first established a population that contained variation at just 23 loci and used selection to evolve suppression of the learning defect seen with null mutations in the rutabaga adenylyl cyclase. We thus biased the system to favor relatively simplified outcomes by choosing a Mendelian trait and by restricting the genetic variation segregating in the population. This experimental design also assures that the causal effects are among the known 23 segregating loci. We observe a robust response to selection that requires the presence of the 23 variants. Analyses of the underlying genotypes showed that interactions between more than two loci are likely to be involved in explaining the selection response, with implications for the missing heritability problem. PMID:24888634

  17. Common mechanisms for calorie restriction and adenylyl cyclase type 5 knockout models of longevity.

    PubMed

    Yan, Lin; Park, Ji Yeon; Dillinger, Jean-Guillaume; De Lorenzo, Mariana S; Yuan, Chujun; Lai, Lo; Wang, Chunbo; Ho, David; Tian, Bin; Stanley, William C; Auwerx, Johan; Vatner, Dorothy E; Vatner, Stephen F

    2012-12-01

    Adenylyl cyclase type 5 knockout mice (AC5 KO) live longer and are stress resistant, similar to calorie restriction (CR). AC5 KO mice eat more, but actually weigh less and accumulate less fat compared with WT mice. CR applied to AC5 KO results in rapid decrease in body weight, metabolic deterioration, and death. These data suggest that despite restricted food intake in CR, but augmented food intake in AC5 KO, the two models affect longevity and metabolism similarly. To determine shared molecular mechanisms, mRNA expression was examined genome-wide for brain, heart, skeletal muscle, and liver. Significantly more genes were regulated commonly rather than oppositely in all the tissues in both models, indicating commonality between AC5 KO and CR. Gene ontology analysis identified many significantly regulated, tissue-specific pathways shared by the two models, including sensory perception in heart and brain, muscle function in skeletal muscle, and lipid metabolism in liver. Moreover, when comparing gene expression changes in the heart under stress, the glutathione regulatory pathway was consistently upregulated in the longevity models but downregulated with stress. In addition, AC5 and CR shared changes in genes and proteins involved in the regulation of longevity and stress resistance, including Sirt1, ApoD, and olfactory receptors in both young- and intermediate-age mice. Thus, the similarly regulated genes and pathways in AC5 KO and CR mice, particularly related to the metabolic phenotype, suggest a unified theory for longevity and stress resistance. PMID:23020244

  18. Metabolic Communication between Astrocytes and Neurons via Bicarbonate-Responsive Soluble Adenylyl Cyclase

    PubMed Central

    Choi, Hyun B.; Gordon, Grant R.J.; Zhou, Ning; Tai, Chao; Rungta, Ravi L.; Martinez, Jennifer; Milner, Teresa A.; Ryu, Jae K.; McLarnon, James G.; Tresguerres, Martin; Levin, Lonny R.; Buck, Jochen; MacVicar, Brian A.

    2013-01-01

    SUMMARY Astrocytes are proposed to participate in brain energy metabolism by supplying substrates to neurons from their glycogen stores and from glycolysis. However, the molecules involved in metabolic sensing and the molecular pathways responsible for metabolic coupling between different cell types in the brain are not fully understood. Here we show that a recently cloned bicarbonate (HCO3−) sensor, soluble adenylyl cyclase (sAC), is highly expressed in astrocytes and becomes activated in response to HCO3− entry via the electrogenic NaHCO3 cotransporter (NBC). Activated sAC increases intracellular cAMP levels, causing glycogen breakdown, enhanced glycolysis, and the release of lactate into the extracellular space, which is subsequently taken up by neurons for use as an energy substrate. This process is recruited over a broad physiological range of [K+]ext and also during aglycemic episodes, helping to maintain synaptic function. These data reveal a molecular pathway in astrocytes that is responsible for brain metabolic coupling to neurons. PMID:22998876

  19. 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

  20. Persistent Electrical Activity in Primary Nociceptors after Spinal Cord Injury Is Maintained by Scaffolded Adenylyl Cyclase and Protein Kinase A and Is Associated with Altered Adenylyl Cyclase Regulation

    PubMed Central

    Bavencoffe, Alexis; Li, Yong; Wu, Zizhen; Yang, Qing; Herrera, Juan; Kennedy, Eileen J.

    2016-01-01

    Little is known about intracellular signaling mechanisms that persistently excite neurons in pain pathways. Persistent spontaneous activity (SA) generated in the cell bodies of primary nociceptors within dorsal root ganglia (DRG) has been found to make major contributions to chronic pain in a rat model of spinal cord injury (SCI) (Bedi et al., 2010; Yang et al., 2014). The occurrence of SCI-induced SA in a large fraction of DRG neurons and the persistence of this SA long after dissociation of the neurons provide an opportunity to define intrinsic cell signaling mechanisms that chronically drive SA in pain pathways. The present study demonstrates that SCI-induced SA requires continuing activity of adenylyl cyclase (AC) and cAMP-dependent protein kinase (PKA), as well as a scaffolded complex containing AC5/6, A-kinase anchoring protein 150 (AKAP150), and PKA. SCI caused a small but significant increase in the expression of AKAP150 but not other AKAPs. DRG membranes isolated from SCI animals revealed a novel alteration in the regulation of AC. AC activity stimulated by Ca2+-calmodulin increased, while the inhibition of AC activity by Gαi showed an unexpected and dramatic decrease after SCI. Localized enhancement of the activity of AC within scaffolded complexes containing PKA is likely to contribute to chronic pathophysiological consequences of SCI, including pain, that are promoted by persistent hyperactivity in DRG neurons. SIGNIFICANCE STATEMENT Chronic neuropathic pain is a major clinical problem with poorly understood mechanisms and inadequate treatments. Recent findings indicate that chronic pain in a rat SCI model depends upon hyperactivity in dorsal root ganglia (DRG) neurons. Although cAMP signaling is involved in many forms of neural plasticity, including hypersensitivity of nociceptors in the presence of inflammatory mediators, our finding that continuing cAMP-PKA signaling is required for persistent SA months after SCI and long after isolation of

  1. 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

  2. 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

  3. Inhibition of atrial natriuretic peptide (ANP) C receptor expression by antisense oligodeoxynucleotides in A10 vascular smooth-muscle cells is associated with attenuation of ANP-C-receptor-mediated inhibition of adenylyl cyclase.

    PubMed Central

    Palaparti, A; Li, Y; Anand-Srivastava, M B

    2000-01-01

    Atrial natriuretic peptide (ANP) mediates a variety of physiological effects through its interaction with ANP-A, ANP-B or ANP-C receptors. However, controversies exist regarding the involvement of ANP-C receptor and adenylyl cyclase/cAMP signal-transduction systems to which these receptors are coupled in mediating these responses. In the present studies, we have employed an antisense approach to eliminate the ANP-C receptor and to examine the effect of this elimination on adenylyl cyclase inhibition. An 18-mer antisense phosphorothioate oligodeoxynucleotide (OH-2) targeted at the initiation codon of the ANP-C receptor was used to examine its effects on the expression of the ANP-C receptor and ANP-C-receptor-mediated inhibition of adenylyl cyclase in vascular smooth-muscle cells (A10). Treatment of the cells with antisense oligonucleotide resulted in complete attenuation of C-ANP(4-23) [des(Gln(18), Ser(19), Gln(20), Leu(21), Gly(22))ANP(4-23)-NH(2)]-mediated inhibition of adenylyl cyclase, whereas sense and missense oligomers did not affect the inhibition of adenylyl cyclase by C-ANP(4-23). In addition, the stimulatory effects of guanine nucleotides, isoproterenol, sodium fluoride and forskolin as well as the inhibitory effects of angiotensin II on adenylyl cyclase were not affected by antisense-oligonucleotide treatment. The attenuation of C-ANP(4-23)-mediated inhibition of adenylyl cyclase by antisense oligonucleotide was dose- and time-dependent. A complete attenuation of ANP-C-receptor-mediated inhibition of adenylyl cyclase was observed at 2.5 microM. In addition, treatment of the cells with antisense oligonucleotide and not with sense or missense oligomers resulted in the inhibition of the levels of ANP-C-receptor protein and mRNA as determined by immunoblotting and Northern blotting using antisera against the ANP-C receptor and a cDNA probe of the ANP-C receptor respectively. On the other hand, ANP-A/B-receptor-mediated increases in cGMP levels were not

  4. Adenylyl cyclase regulates heavy metal sensitivity, bikaverin production and plant tissue colonization in Fusarium proliferatum.

    PubMed

    Kohut, Gábor; Oláh, Brigitta; Adám, Attila L; García-Martínez, Jorge; Hornok, László

    2010-02-01

    A homologue of the adenylyl cyclase (AC) gene of Neurospora crassa, named Fpacy1 was cloned from the genomic library of Fusarium proliferatum ITEM 2287 by screening the library with a DNA fragment amplified by using PCR primers designed from conserved sequences of the catalytic domain of AC genes from other fungi. The deduced FPACY1 protein had 53-77% identity with the AC proteins of other fungi. DeltaFpacy1 mutants obtained by targeted gene disruption showed retarded vegetative growth, increased conidiation and delayed conidial germination. Colonization capability of the mutants, assessed on maize seedlings and tomato fruits also was adversely affected. In sexual crosses the AC mutants retained full male fertility, but their female fertility decreased significantly. Disruption of Fpacy1 abolished vegetative self-incompatibility, suggesting that the AC gene is involved in multiple developmental processes related to vegetative growth, as well as sexual and parasexual events. The elevated thermo- and H(2)O(2)-tolerance of the DeltaFpacy1 mutants was coupled to an increased sensitivity towards Cd and Cu, indicating that the cAMP signaling pathway may have both negative and positive regulatory roles on the stress response mechanisms of fungal cells. When grown under nitrogen limitation conditions, the DeltaFpacy1 mutants produced an average of approximately 274 microg g(-1) bikaverin, whereas only traces of this metabolite was detected in the wild type. This finding provides further evidence of the role of the cAMP-PKA pathway in regulating bikaverin production. PMID:20082366

  5. Gene Expression Profiles of Main Olfactory Epithelium in Adenylyl Cyclase 3 Knockout Mice

    PubMed Central

    Wang, Zhenshan; Zhou, Yanfen; Luo, Yingtao; Zhang, Jing; Zhai, Yunpeng; Yang, Dong; Zhang, Zhe; Li, Yongchao; Storm, Daniel R.; Ma, Runlin Z.

    2015-01-01

    Adenylyl Cyclase 3 (AC3) plays an important role in the olfactory sensation-signaling pathway in mice. AC3 deficiency leads to defects in olfaction. However, it is still unknown whether AC3 deficiency affects gene expression or olfactory signal transduction pathways within the main olfactory epithelium (MOE). In this study, gene microarrays were used to screen differentially expressed genes in MOE from AC3 knockout (AC3−/−) and wild-type (AC3+/+) mice. The differentially expressed genes identified were subjected to bioinformatic analysis and verified by qRT-PCR. Gene expression in the MOE from AC3−/− mice was significantly altered, compared to AC3+/+ mice. Of the 41266 gene probes, 3379 had greater than 2-fold fold change in expression levels between AC3−/− and AC3+/+ mice, accounting for 8% of the total gene probes. Of these genes, 1391 were up regulated, and 1988 were down regulated, including 425 olfactory receptor genes, 99 genes that are specifically expressed in the immature olfactory neurons, 305 genes that are specifically expressed in the mature olfactory neurons, and 155 genes that are involved in epigenetic regulation. Quantitative RT-PCR verification of the differentially expressed epigenetic regulation related genes, olfactory receptors, ion transporter related genes, neuron development and differentiation related genes, lipid metabolism and membrane protein transport etc. related genes showed that P75NTR, Hinfp, Gadd45b, and Tet3 were significantly up-regulated, while Olfr370, Olfr1414, Olfr1208, Golf, Faim2, Tsg101, Mapk10, Actl6b, H2BE, ATF5, Kirrrel2, OMP, Drd2 etc. were significantly down-regulated. In summary, AC3 may play a role in proximal olfactory signaling and play a role in the regulation of differentially expressed genes in mouse MOE. PMID:26633363

  6. Active-Site Structure of Class IV Adenylyl Cyclase and Transphyletic Mechanism

    SciTech Connect

    Gallagher, D.T.; Robinson, H.; Kim, S.-K.; Reddy, P. T.

    2011-01-21

    Adenylyl cyclases (ACs) belonging to three nonhomologous classes (II, III, and IV) have been structurally characterized, enabling a comparison of the mechanisms of cyclic adenosine 3',5'-monophosphate biosynthesis. We report the crystal structures of three active-site complexes for Yersinia pestis class IV AC (AC-IV)-two with substrate analogs and one with product. Mn{sup 2+} binds to all three phosphates, and to Glu12 and Glu136. Electropositive residues Lys14, Arg63, Lys76, Lys111, and Arg113 also form hydrogen bonds to phosphates. The conformation of the analogs is suitable for in-line nucleophilic attack by the ribose O3' on {alpha}-phosphate (distance {approx} 4 {angstrom}). In the product complex, a second Mn ion is observed to be coordinated to both ribose 2' oxygen and ribose 3' oxygen. Observation of both metal sites, together with kinetic measurements, provides strong support for a two-cation mechanism. Eleven active-site mutants were also made and kinetically characterized. These findings and comparisons with class II and class III enzymes enable a detailed transphyletic analysis of the AC mechanism. Consistent with its lack of coordination to purine, Y. pestis AC-IV cyclizes both ATP and GTP. As in other classes of AC, the ribose is loosely bound, and as in class III, no base appears to ionize the O3' nucleophile. Different syn/anti conformations suggest that the mechanism involves a conformational transition, and further evidence suggests a role for ribosyl pseudorotation. With resolutions of 1.6-1.7 {angstrom}, these are the most detailed active-site ligand complexes for any class of this ubiquitous signaling enzyme.

  7. Active-Site Structure of Class IV Adenylyl Cyclase and Transphyletic Mechanism

    SciTech Connect

    D Gallagher; S Kim; H Robinson; P Reddy

    2011-12-31

    Adenylyl cyclases (ACs) belonging to three nonhomologous classes (II, III, and IV) have been structurally characterized, enabling a comparison of the mechanisms of cyclic adenosine 3',5'-monophosphate biosynthesis. We report the crystal structures of three active-site complexes for Yersinia pestis class IV AC (AC-IV) - two with substrate analogs and one with product. Mn{sup 2+} binds to all three phosphates, and to Glu12 and Glu136. Electropositive residues Lys14, Arg63, Lys76, Lys111, and Arg113 also form hydrogen bonds to phosphates. The conformation of the analogs is suitable for in-line nucleophilic attack by the ribose O3' on {alpha}-phosphate (distance {approx} 4 {angstrom}). In the product complex, a second Mn ion is observed to be coordinated to both ribose 2' oxygen and ribose 3' oxygen. Observation of both metal sites, together with kinetic measurements, provides strong support for a two-cation mechanism. Eleven active-site mutants were also made and kinetically characterized. These findings and comparisons with class II and class III enzymes enable a detailed transphyletic analysis of the AC mechanism. Consistent with its lack of coordination to purine, Y. pestis AC-IV cyclizes both ATP and GTP. As in other classes of AC, the ribose is loosely bound, and as in class III, no base appears to ionize the O3' nucleophile. Different syn/anti conformations suggest that the mechanism involves a conformational transition, and further evidence suggests a role for ribosyl pseudorotation. With resolutions of 1.6-1.7 {angstrom}, these are the most detailed active-site ligand complexes for any class of this ubiquitous signaling enzyme.

  8. 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

  9. Phorbol ester-induced sensitisation of adenylyl cyclase type II is related to phosphorylation of threonine 1057.

    PubMed

    Böl, G F; Gros, C; Hülster, A; Bösel, A; Pfeuffer, T

    1997-08-18

    Following up the results from previous studies on chemical fragmentation of TPA-treated, [32P]phosphate labeled adenylyl cyclase type II (AC II) (Böl, G. F., Hülster, A., and Pfeuffer, T. in press) we have replaced serine 871 or threonine 1057 by alanine using site directed mutagenesis. Both mutants had unimpaired catalytic activity, however enhancement by phorbolester TPA was reduced by 60-80 % in the T1057A mutant, but not in the S871A mutant. The stimulation of adenylyl cyclase type II by betagamma subunits of heterotrimeric G-pro teins and that by PKC have been previously shown to be mutually exclusive (Zimmermann and Taussig (1996), J. Biol. Chem. 271, 27161-27166). This is in line with the present findings that AC II expressed in COS-1 cells was only barely stimulated (10%) by coexpressed betagamma-subunits in presence of TPA. Mutation of threonine 1057 to alanine however caused partial regain of betagamma-stimulation in the presence of TPA by 40%, as compared to that of WT adenylyl cyclase type II which was 70% in the absence of TPA. These data strongly implicate the importance of threonine 1057 as phosphate acceptor following PKC-mediated sensitisation of adenylyl cyclase type II. PMID:9268695

  10. 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

  11. Efficacy of inverse agonists in cells overexpressing a constitutively active β2-adrenoceptor and type II adenylyl cyclase

    PubMed Central

    Stevens, Patricia A; Milligan, Graeme

    1998-01-01

    Maximal stimulant output from the adenylyl cyclase cascade in neuroblastoma × glioma hybrid, NG108-15, cells is limited by the levels of expression of isoforms of adenylyl cyclase. Stable expression in these cells of a constitutively active mutant (CAM) version of the human β2-adrenoceptor resulted in higher basal adenylyl cyclase activity than following expression of the human wild type β2-adrenoceptor. Isoprenaline acted as a full agonist in membranes from both wild type and CAM β2-adrenoceptor expressing clones.Expression of type II adenylyl cyclase resulted in a substantially elevated capacity of isoprenaline to stimulate [3H]-forskolin binding, whereas in CAM β2-adrenoceptor expressing cells the basal high affinity [3H]-forskolin binding represented a markedly greater % of the maximal effect which could be produced by addition of isoprenaline, and the EC50 for isoprenaline was some 10 fold lower than in cells expressing the wild type β2-adrenoceptor.Further transfection of the CAM β2-adrenoceptor expressing cells with type II adenylyl cyclase greatly increased both absolute basal and agonist-stimulated levels of adenylyl cyclase activity.Betaxolol, ICI 118,551, sotalol and timolol acted as inverse agonists with varying degrees of efficacy, whereas propranolol functioned as a neutral antagonist and alprenolol as a partial agonist.Pretreatment of the CAM β2-adrenoceptor and type II adenylyl cyclase expressing clones with the irreversible alkylating agent BAAM (1 μM) did not reduce the efficacy of isoprenaline but eliminated efficacy from all the inverse agonist ligands. This effect was dependent upon the concentration of BAAM employed, with half-maximal effects being produced between 10 nM and 100 nM of the alkylating agent, which is similar to the concentrations required to prevent subsequent ligand access to some 50% of the CAM β2-adrenoceptor population.These data demonstrate that inverse agonist efficacy can be modulated by receptor

  12. Differential activation of yeast adenylyl cyclase by Ras1 and Ras2 depends on the conserved N terminus.

    PubMed

    Hurwitz, N; Segal, M; Marbach, I; Levitzki, A

    1995-11-21

    Although both Ras1 and Ras2 activate adenylyl cyclase in yeast, a number of differences can be observed regarding their function in the cAMP pathway. To explore the relative contribution of conserved and variable domains in determining these differences, chimeric RAS1-RAS2 or RAS2-RAS1 genes were constructed by swapping the sequences encoding the variable C-terminal domains. These constructs were expressed in a cdc25ts ras1 ras2 strain. Biochemical data show that the difference in efficacy of adenylyl cyclase activation between the two Ras proteins resides in the highly conserved N-terminal domain. This finding is supported by the observation that Ras2 delta, in which the C-terminal domain of Ras2 has been deleted, is a more potent activator of the yeast adenylyl cyclase than Ras1 delta, in which the C-terminal domain of Ras1 has been deleted. These observations suggest that amino acid residues other than the highly conserved residues of the effector domain within the N terminus may determine the efficiency of functional interaction with adenylyl cyclase. Similar levels of intracellular cAMP were found in Ras1, Ras1-Ras2, Ras1 delta, Ras2, and Ras2-Ras1 strains throughout the growth curve. This was found to result from the higher expression of Ras1 and Ras1-Ras2, which compensate for their lower efficacy in activating adenylyl cyclase. These results suggest that the difference between the Ras1 and the Ras2 phenotype is not due to their different efficacy in activating the cAMP pathway and that the divergent C-terminal domains are responsible for these differences, through interaction with other regulatory elements. PMID:7479926

  13. The YHS-Domain of an Adenylyl Cyclase from Mycobacterium phlei Is a Probable Copper-Sensor Module

    PubMed Central

    Linder, Jürgen Ulrich

    2015-01-01

    YHS-domains are small protein modules which have been proposed to bind transition-metal ions like the related TRASH-domains. They are found in a variety of enzymes including copper-transporting ATPases and adenylyl cyclases. Here we investigate a class IIIc adenylyl cyclase from Mycobacterium phlei which contains a C-terminal YHS-domain linked to the catalytic domain by a peptide of 8 amino acids. We expressed the isolated catalytic domain and the full-length enzyme in E. coli. The catalytic domain requires millimolar Mn2+ as a cofactor for efficient production of cAMP, is unaffected by low micromolar concentrations of Cu2+ and inhibited by concentrations higher than 10 μM. The full-length enzyme also requires Mn2+ in the absence of an activator. However, 1–10 μM Cu2+ stimulate the M. phlei adenylyl cyclase sixfold when assayed with Mn2+. With Mg2+ as the probable physiological cofactor of the adenylyl cyclase Cu2+ specifically switches the enzyme from an inactive to an active state. Other transition-metal ions do not elicit activity with Mg2+. We favor the view that the YHS-domain of M. phlei adenylyl cyclase acts as a sensor for copper ions and signals elevated levels of the transition-metal via cAMP. By analogy to TRASH-domains binding of Cu2+ probably occurs via one conserved aspartate and three conserved cysteine-residues in the YHS-domain. PMID:26512893

  14. Measles virus modulates human T-cell somatostatin receptors and their coupling to adenylyl cyclase.

    PubMed Central

    Krantic, S; Enjalbert, A; Rabourdin-Combe, C

    1997-01-01

    The possible role of immunomodulatory peptide somatostatin (SRIF) in measles virus (MV)-induced immunopathology was addressed by analysis of SRIF receptors and their coupling to adenylyl cyclase in mitogen-stimulated Jurkat T cells and human peripheral blood mononuclear cells (PBMC). SRIF-specific receptors were assayed in semipurified membrane preparations by using SRIF14 containing iodinated tyrosine at the first position in the amino acid chain ([125I]Tyr1) as a radioligand. A determination of receptor number by saturation of radioligand binding at equilibrium showed that in Jurkat cells, MV infection led to a dramatic decrease in the total receptor number. The virus-associated disappearance of one (Ki2 = 12 +/- 4 nM [mean +/- standard error of the mean [SEM

  15. Endothelial CD99 signals through soluble adenylyl cyclase and PKA to regulate leukocyte transendothelial migration

    PubMed Central

    Watson, Richard L.; Buck, Jochen; Levin, Lonny R.; Winger, Ryan C.; Wang, Jing; Arase, Hisashi

    2015-01-01

    CD99 is a critical regulator of leukocyte transendothelial migration (TEM). How CD99 signals during this process remains unknown. We show that during TEM, endothelial cell (EC) CD99 activates protein kinase A (PKA) via a signaling complex formed with the lysine-rich juxtamembrane cytoplasmic tail of CD99, the A-kinase anchoring protein ezrin, and soluble adenylyl cyclase (sAC). PKA then stimulates membrane trafficking from the lateral border recycling compartment to sites of TEM, facilitating the passage of leukocytes across the endothelium. Pharmacologic or genetic inhibition of EC sAC or PKA, like CD99 blockade, arrests neutrophils and monocytes partway through EC junctions, in vitro and in vivo, without affecting leukocyte adhesion or the expression of relevant cellular adhesion molecules. This is the first description of the CD99 signaling pathway in TEM as well as the first demonstration of a role for sAC in leukocyte TEM. PMID:26101266

  16. Phosphorylation of adenylyl cyclase III at serine1076 does not attenuate olfactory response in mice

    PubMed Central

    Cygnar, Katherine D; Collins, Sarah Ellen; Ferguson, Christopher H; Bodkin-Clarke, Chantal; Zhao, Haiqing

    2012-01-01

    Feedback inhibition of adenylyl cyclase III (ACIII) via Ca2+-induced phosphorylation has long been hypothesized to contribute to response termination and adaptation of olfactory sensory neurons (OSNs). To directly determine the functional significance of this feedback mechanism for olfaction in vivo, we genetically mutated serine1076 of ACIII, the only residue responsible for Ca2+-induced phosphorylation and inhibition of ACIII (Wei et al., 1996; Wei et al., 1998), to alanine in mice. Immunohistochemistry and Western blot analysis showed that the mutation affects neither the cilial localization nor the expression level of ACIII in OSNs. Electroolfactogram analysis showed no differences in the responses between wildtype and mutant mice to single-pulse odorant stimulations or in several stimulation paradigms for adaptation. These results suggest that phosphorylation of ACIII on serine1076 plays a far less important role in olfactory response attenuation than previously thought. PMID:23077041

  17. Phosphorylation of adenylyl cyclase III at serine1076 does not attenuate olfactory response in mice.

    PubMed

    Cygnar, Katherine D; Collins, Sarah Ellen; Ferguson, Christopher H; Bodkin-Clarke, Chantal; Zhao, Haiqing

    2012-10-17

    Feedback inhibition of adenylyl cyclase III (ACIII) via Ca(2+)-induced phosphorylation has long been hypothesized to contribute to response termination and adaptation of olfactory sensory neurons (OSNs). To directly determine the functional significance of this feedback mechanism for olfaction in vivo, we genetically mutated serine(1076) of ACIII, the only residue responsible for Ca(2+)-induced phosphorylation and inhibition of ACIII (Wei et al., 1996, 1998), to alanine in mice. Immunohistochemistry and Western blot analysis showed that the mutation affects neither the cilial localization nor the expression level of ACIII in OSNs. Electroolfactogram analysis showed no differences in the responses between wild-type and mutant mice to single-pulse odorant stimulations or in several stimulation paradigms for adaptation. These results suggest that phosphorylation of ACIII on serine(1076) plays a far less important role in olfactory response attenuation than previously thought. PMID:23077041

  18. Adenylyl cyclase 6 mediates loading-induced bone adaptation in vivo

    PubMed Central

    Lee, Kristen L.; Hoey, David A.; Spasic, Milos; Tang, Tong; Hammond, H. Kirk; Jacobs, Christopher R.

    2014-01-01

    Primary cilia are single, nonmotile, antenna-like structures extending from the apical membrane of most mammalian cells. They may mediate mechanotransduction, the conversion of external mechanical stimuli into biochemical intracellular signals. Previously we demonstrated that adenylyl cyclase 6 (AC6), a membrane-bound enzyme enriched in primary cilia of MLO-Y4 osteocyte-like cells, may play a role in a primary cilium-dependent mechanism of osteocyte mechanotransduction in vitro. In this study, we determined whether AC6 deletion impairs loading-induced bone formation in vivo. Skeletally mature mice with a global knockout of AC6 exhibited normal bone morphology and responded to osteogenic chemical stimuli similar to wild-type mice. Following ulnar loading over 3 consecutive days, bone formation parameters were assessed using dynamic histomorphometry. Mice lacking AC6 formed significantly less bone than control animals (41% lower bone formation rate). Furthermore, there was an attenuated flow-induced increase in COX-2 mRNA expression levels in primary bone cells isolated from AC6 knockout mice compared to controls (1.3±0.1- vs. 2.6±0.2-fold increase). Collectively, these data indicate that AC6 plays a role in loading-induced bone adaptation, and these findings are consistent with our previous studies implicating primary cilia and AC6 in a novel mechanism of osteocyte mechanotransduction.—Lee, K. L., Hoey, D. A., Spasic, M., Tang, T., Hammond, H. K., Jacobs, C. R. Adenylyl cyclase 6 mediates loading-induced bone adaptation in vivo. PMID:24277577

  19. Disruption of the type III adenylyl cyclase gene leads to peripheral and behavioral anosmia in transgenic mice.

    PubMed

    Wong, S T; Trinh, K; Hacker, B; Chan, G C; Lowe, G; Gaggar, A; Xia, Z; Gold, G H; Storm, D R

    2000-09-01

    Cyclic nucleotide-gated ion channels in olfactory sensory neurons (OSNs) are hypothesized to play a critical role in olfaction. However, it has not been demonstrated that the cAMP signaling is required for olfactory-based behavioral responses, and the contributions of specific adenylyl cyclases to olfaction have not been defined. Here, we report the presence of adenylyl cyclases 2, 3, and 4 in olfactory cilia. To evaluate the role of AC3 in olfactory responses, we disrupted the gene for AC3 in mice. Interestingly, electroolfactogram (EOG) responses stimulated by either cAMP- or inositol 1,4,5-triphosphate- (IP3-) inducing odorants were completely ablated in AC3 mutants, despite the presence of AC2 and AC4 in olfactory cilia. Furthermore, AC3 mutants failed several olfaction-based behavioral tests, indicating that AC3 and cAMP signaling are critical for olfactory-dependent behavior. PMID:11055432

  20. Role of the bicarbonate-responsive soluble adenylyl cyclase in pH sensing and metabolic regulation

    PubMed Central

    Chang, Jung-Chin; Oude-Elferink, Ronald P. J.

    2014-01-01

    The evolutionarily conserved soluble adenylyl cyclase (sAC, adcy10) was recently identified as a unique source of cAMP in the cytoplasm and the nucleus. Its activity is regulated by bicarbonate and fine-tuned by calcium. As such, and in conjunction with carbonic anhydrase (CA), sAC constitutes an HCO−3/CO−2/pH sensor. In both alpha-intercalated cells of the collecting duct and the clear cells of the epididymis, sAC is expressed at significant level and involved in pH homeostasis via apical recruitment of vacuolar H+-ATPase (VHA) in a PKA-dependent manner. In addition to maintenance of pH homeostasis, sAC is also involved in metabolic regulation such as coupling of Krebs cycle to oxidative phosphorylation via bicarbonate/CO2 sensing. Additionally, sAC also regulates CFTR channel and plays an important role in regulation of barrier function and apoptosis. These observations suggest that sAC, via bicarbonate-sensing, plays an important role in maintaining homeostatic status of cells against fluctuations in their microenvironment. PMID:24575049

  1. Adenylyl cyclase 6 mediates loading-induced bone adaptation in vivo.

    PubMed

    Lee, Kristen L; Hoey, David A; Spasic, Milos; Tang, Tong; Hammond, H Kirk; Jacobs, Christopher R

    2014-03-01

    Primary cilia are single, nonmotile, antenna-like structures extending from the apical membrane of most mammalian cells. They may mediate mechanotransduction, the conversion of external mechanical stimuli into biochemical intracellular signals. Previously we demonstrated that adenylyl cyclase 6 (AC6), a membrane-bound enzyme enriched in primary cilia of MLO-Y4 osteocyte-like cells, may play a role in a primary cilium-dependent mechanism of osteocyte mechanotransduction in vitro. In this study, we determined whether AC6 deletion impairs loading-induced bone formation in vivo. Skeletally mature mice with a global knockout of AC6 exhibited normal bone morphology and responded to osteogenic chemical stimuli similar to wild-type mice. Following ulnar loading over 3 consecutive days, bone formation parameters were assessed using dynamic histomorphometry. Mice lacking AC6 formed significantly less bone than control animals (41% lower bone formation rate). Furthermore, there was an attenuated flow-induced increase in COX-2 mRNA expression levels in primary bone cells isolated from AC6 knockout mice compared to controls (1.3±0.1- vs. 2.6±0.2-fold increase). Collectively, these data indicate that AC6 plays a role in loading-induced bone adaptation, and these findings are consistent with our previous studies implicating primary cilia and AC6 in a novel mechanism of osteocyte mechanotransduction. PMID:24277577

  2. 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

  3. Adenylyl cyclase localization to the uropod of aggregating Dictyostelium cells requires RacC.

    PubMed

    Wang, C; Jung, D; Cao, Z; Chung, C Y

    2015-09-25

    The localization of adenylyl cyclase A (ACA) to uropod of cells is required for the stream formation during Dictyostelium development. RacC is a Dictyostelium orthologue of Cdc42. We identified a streaming defect of racC(-) cells as they are clearly less polarized and form smaller and fragmented streams. ACA-YFP is mainly associated with intracellular vesicular structures, but not with the plasma membrane in racC(-) cells. racC(-) cells have a slightly higher number of vesicles than Ax3 cells, suggesting that the defect of ACA trafficking is not simply due to the lack of vesicle formation. While the ACA-YFP vesicles traveled with an average velocity of 9.1 μm/min in Ax3 cells, a slow and diffusional movement without direction with an average velocity of 4 μm/min was maintained in racC(-) cells. Images acquired by using total internal reflection fluorescence (TIRF) microscopy and fluorescence recovery after photobleaching (FRAP) analysis revealed that a significantly decreased number of ACA-YFP vesicles appeared near the cell membrane, indicating a defect in ACA-YFP vesicle trafficking. These results suggest an important role of RacC in the rapid and directional movements of ACA vesicles on microtubules to the plasma membrane, especially to the back of polarized cell. PMID:26315268

  4. Structure of the Class IV Adenylyl Cyclase Reveals a Novel Fold

    SciTech Connect

    Gallagher,D.; Smith, N.; Kim, S.; Heroux, A.; Robinson, H.; Reddy, P.

    2006-01-01

    The crystal structure of the class IV adenylyl cyclase (AC) from Yersinia pestis (Yp) is reported at 1.9 {angstrom} resolution. The class IV AC fold is distinct from the previously described folds for class II and class III ACs. The dimeric AC-IV folds into an antiparallel eight-stranded barrel whose connectivity has been seen in only three previous structures: yeast RNA triphosphatase and two proteins of unknown function from Pyrococcus furiosus and Vibrio parahaemolyticus. Eight highly conserved ionic residues E10, E12, K14, R63, K76, K111, D126, and E136 lie in the barrel core and form the likely binding sites for substrate and divalent cations. A phosphate ion is observed bound to R63, K76, K111, and R113 near the center of the conserved cluster. Unlike the AC-II and AC-III active sites that utilize two-Asp motifs for cation binding, the AC-IV active site is relatively enriched in glutamate and features an ExE motif as its most conserved element. Homologs of Y. pestis AC-IV, including human thiamine triphosphatase, span the three kingdoms of life and delineate an ancient family of phosphonucleotide processing enzymes.

  5. Dominant regulation of interendothelial cell gap formation by calcium-inhibited type 6 adenylyl cyclase

    PubMed Central

    Cioffi, Donna L.; Moore, Timothy M.; Schaack, Jerry; Creighton, Judy R.; Cooper, Dermot M.F.; Stevens, Troy

    2002-01-01

    Acute transitions in cytosolic calcium ([Ca2+]i) through store-operated calcium entry channels catalyze interendothelial cell gap formation that increases permeability. However, the rise in [Ca2+]i only disrupts barrier function in the absence of a rise in cAMP. Discovery that type 6 adenylyl cyclase (AC6; EC 4.6.6.1) is inhibited by calcium entry through store-operated calcium entry pathways provided a plausible explanation for how inflammatory [Ca2+]i mediators may decrease cAMP necessary for endothelial cell gap formation. [Ca2+]i mediators only modestly decrease global cAMP concentrations and thus, to date, the physiological role of AC6 is unresolved. Present studies used an adenoviral construct that expresses the calcium-stimulated AC8 to convert normal calcium inhibition into stimulation of cAMP, within physiologically relevant concentration ranges. Thrombin stimulated a dose-dependent [Ca2+]i rise in both pulmonary artery (PAECs) and microvascular (PMVEC) endothelial cells, and promoted intercellular gap formation in both cell types. In PAECs, gap formation was progressive over 2 h, whereas in PMVECs, gap formation was rapid (within 10 min) and gaps resealed within 2 h. Expression of AC8 resulted in a modest calcium stimulation of cAMP, which virtually abolished thrombin-induced gap formation in PMVECs. Findings provide the first direct evidence that calcium inhibition of AC6 is essential for endothelial gap formation. PMID:12082084

  6. Photoactivated adenylyl cyclase (PAC) reveals novel mechanisms underlying cAMP-dependent axonal morphogenesis

    PubMed Central

    Zhou, Zhiwen; Tanaka, Kenji F.; Matsunaga, Shigeru; Iseki, Mineo; Watanabe, Masakatsu; Matsuki, Norio; Ikegaya, Yuji; Koyama, Ryuta

    2016-01-01

    Spatiotemporal regulation of axonal branching and elongation is essential in the development of refined neural circuits. cAMP is a key regulator of axonal growth; however, whether and how intracellular cAMP regulates axonal branching and elongation remain unclear, mainly because tools to spatiotemporally manipulate intracellular cAMP levels have been lacking. To overcome this issue, we utilized photoactivated adenylyl cyclase (PAC), which produces cAMP in response to blue-light exposure. In primary cultures of dentate granule cells transfected with PAC, short-term elevation of intracellular cAMP levels induced axonal branching but not elongation, whereas long-term cAMP elevation induced both axonal branching and elongation. The temporal dynamics of intracellular cAMP levels regulated axonal branching and elongation through the activation of protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), respectively. Thus, using PAC, our study for the first time reveals that temporal cAMP dynamics could regulate axonal branching and elongation via different signaling pathways. PMID:26795422

  7. Disruption of Epac1 protects the heart from adenylyl cyclase type 5-mediated cardiac dysfunction.

    PubMed

    Cai, Wenqian; Fujita, Takayuki; Hidaka, Yuko; Jin, Huiling; Suita, Kenji; Prajapati, Rajesh; Liang, Chen; Umemura, Masanari; Yokoyama, Utako; Sato, Motohiko; Okumura, Satoshi; Ishikawa, Yoshihiro

    2016-06-17

    Type 5 adenylyl cyclase (AC5) plays an important role in the development of chronic catecholamine stress-induced heart failure and arrhythmia in mice. Epac (exchange protein activated by cAMP), which is directly activated by cAMP independent of protein kinase A, has been recently identified as a novel mediator of cAMP signaling in the heart. However, the role of Epac in AC5-mediated cardiac dysfunction and arrhythmias remains poorly understood. We therefore generated AC5 transgenic mice (AC5TG) with selective disruption of the Epac1 gene (AC5TG-Epac1KO), and compared their phenotypes with those of AC5TG after chronic isoproterenol (ISO) infusion. Decreased cardiac function as well as increased susceptibility to pacing-induced atrial fibrillation (AF) in response to ISO were significantly attenuated in AC5TG-Epac1KO mice, compared to AC5TG mice. Increased cardiac apoptosis and cardiac fibrosis were also concomitantly attenuated in AC5TG-Epac1KO mice compared to AC5TG mice. These findings indicate that Epac1 plays an important role in AC5-mediated cardiac dysfunction and AF susceptibility. PMID:27117748

  8. 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

  9. Activation of the adenylyl cyclase/cyclic AMP/protein kinase A pathway in endothelial cells exposed to cyclic strain

    NASA Technical Reports Server (NTRS)

    Cohen, C. R.; Mills, I.; Du, W.; Kamal, K.; Sumpio, B. E.

    1997-01-01

    The aim of this study was to assess the involvement of the adenylyl cyclase/cyclic AMP/protein kinase A pathway (AC) in endothelial cells (EC) exposed to different levels of mechanical strain. Bovine aortic EC were seeded to confluence on flexible membrane-bottom wells. The membranes were deformed with either 150 mm Hg (average 10% strain) or 37.5 mm Hg (average 6% strain) vacuum at 60 cycles per minute (0.5 s strain; 0.5 s relaxation) for 0-60 min. The results demonstrate that at 10% average strain (but not 6% average strain) there was a 1.5- to 2.2-fold increase in AC, cAMP, and PKA activity by 15 min when compared to unstretched controls. Further studies revealed an increase in cAMP response element binding protein in EC subjected to the 10% average strain (but not 6% average strain). These data support the hypothesis that cyclic strain activates the AC/cAMP/PKA signal transduction pathway in EC which may occur by exceeding a strain threshold and suggest that cyclic strain may stimulate the expression of genes containing cAMP-responsive promoter elements.

  10. Association of adenylyl cyclase 6 rs3730070 polymorphism and hemolytic level in patients with sickle cell anemia.

    PubMed

    Cita, Kizzy-Clara; Ferdinand, Séverine; Connes, Philippe; Brudey, Laura; Tressières, Benoit; Etienne-Julan, Maryse; Lemonne, Nathalie; Tarer, Vanessa; Elion, Jacques; Romana, Marc

    2016-05-01

    A recent study suggested that adenosine signaling pathway could promote hemolysis in patients with sickle cell anemia (SCA). This signaling pathway involves several gene coding enzymes for which variants have been described. In this study, we analyzed the genotype-phenotype relationships between functional polymorphisms or polymorphisms associated with altered expression of adenosine pathway genes, namely adenosine deaminase (ada; rs73598374), adenosine A2b receptor (adora2b; rs7208480), adenylyl cyclase6 (adcy6; rs3730071, rs3730070, rs7300155), and hemolytic rate in SCA patients. One hundred and fifty SCA patients were genotyped for adcy6, ada, and adora2b variants as well as alpha-globin gene, a genetic factor known to modulate hemolytic rate. Hematological and biochemical data were obtained at steady-state. Lactate dehydrogenase, aspartate aminotransferase, reticulocytes and total bilirubin were used to calculate a hemolytic index. Genotype-phenotype relationships were investigated using parametric tests and multivariate analysis. SCA patients carrying at least one allele of adcy6 rs3730070-G exhibited lower hemolytic rate than non-carriers in univariate analysis (p=0.006). The presence of adcy6 rs3730070-G variant was associated with a decreased hemolytic rate in adjusted model for age and alpha-thalassemia (p=0.032). Our results support a protective effect of adcy6 rs3730070-G variant on hemolysis in SCA patients. PMID:27067484

  11. Type VI adenylyl cyclase negatively regulates GluN2B-mediated LTD and spatial reversal learning

    PubMed Central

    Chang, Ching-Pang; Lee, Cheng-Ta; Hou, Wen-Hsien; Lin, Meng-Syuan; Lai, Hsing-Lin; Chien, Chen-Li; Chang, Chen; Cheng, Pei-Lin; Lien, Cheng-Chang; Chern, Yijuang

    2016-01-01

    The calcium-sensitive type VI adenylyl cyclase (AC6) is a membrane-bound adenylyl cyclase (AC) that converts ATP to cAMP under stimulation. It is a calcium-inhibited AC and integrates negative inputs from Ca2+ and multiple other signals to regulate the intracellular cAMP level. In the present study, we demonstrate that AC6 functions upstream of CREB and negatively controls neuronal plasticity in the hippocampus. Genetic removal of AC6 leads to cyclase-independent and N-terminus of AC6 (AC6N)-dependent elevation of CREB expression, and enhances the expression of GluN2B-containing NMDA receptors in hippocampal neurons. Consequently, GluN2B-dependent calcium signaling and excitatory postsynaptic current, long-term depression, and spatial reversal learning are enhanced in the hippocampus of AC6−/− mice without altering the gross anatomy of the brain. Together, our results suggest that AC6 negatively regulates neuronal plasticity by modulating the levels of CREB and GluN2B in the hippocampus. PMID:26932446

  12. 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

  13. Distinct Mechanisms of Calmodulin Binding and Regulation of Adenylyl Cyclases 1 and 8

    PubMed Central

    2012-01-01

    Calmodulin (CaM), by mediating the stimulation of the activity of two adenylyl cyclases (ACs), plays a key role in integrating the cAMP and Ca2+ signaling systems. These ACs, AC1 and AC8, by decoding discrete Ca2+ signals can contribute to fine-tuning intracellular cAMP dynamics, particularly in neurons where they predominate. CaM comprises an α-helical linker separating two globular regions at the N-terminus and the C-terminus that each bind two Ca2+ ions. These two lobes have differing affinities for Ca2+, and they can interact with target proteins independently. This study explores previous indications that the two lobes of CaM can regulate AC1 and AC8 differently and thereby yield different responses to cellular transitions in [Ca2+]i. We first compared by glutathione S-transferase pull-down assays and offline nanoelectrospray ionization mass spectrometry the interaction of CaM and Ca2+-binding deficient mutants of CaM with the internal CaM binding domain (CaMBD) of AC1 and the two terminal CaMBDs of AC8. We then examined the influence of these three CaMBDs on Ca2+ binding by native and mutated CaM in stopped-flow experiments to quantify their interactions. The three CaMBDs show quite distinct interactions with the two lobes of CaM. These findings establish the critical kinetic differences between the mechanisms of Ca2+-CaM activation of AC1 and AC8, which may underpin their different physiological roles. PMID:22971080

  14. Biological Activity of the Alternative Promoters of the Dictyostelium discoideum Adenylyl Cyclase A Gene

    PubMed Central

    Rodriguez-Centeno, Javier; Sastre, Leandro

    2016-01-01

    Amoebae of the Dictyostelium discoideum species form multicellular fruiting bodies upon starvation. Cyclic adenosine monophosphate (cAMP) is used as intercellular signalling molecule in cell-aggregation, cell differentiation and morphogenesis. This molecule is synthesized by three adenylyl cyclases, one of which, ACA, is required for cell aggregation. The gene coding for ACA (acaA) is transcribed from three different promoters that are active at different developmental stages. Promoter 1 is active during cell-aggregation, promoters 2 and 3 are active in prespore and prestalk tip cells at subsequent developmental stages. The biological relevance of acaA expression from each of the promoters has been studied in this article. The acaA gene was expressed in acaA-mutant cells, that do not aggregate, under control of each of the three acaA promoters. acaA expression under promoter 1 control induced cell aggregation although subsequent development was delayed, very small fruiting bodies were formed and cell differentiation genes were expressed at very low levels. Promoter 2-driven acaA expression induced the formation of small aggregates and small fruiting bodies were formed at the same time as in wild-type strains and differentiation genes were also expressed at lower levels. Expression of acaA from promoter 3 induced aggregates and fruiting bodies formation and their size and the expression of differentiation genes were more similar to that of wild-type cells. Expression of acaA from promoters 1 and 2 in AX4 cells also produced smaller structures. In conclusion, the expression of acaA under control of the aggregation-specific Promoter 1 is able to induce cell aggregation in acaA-mutant strains. Expression from promoters 2 and 3 also recovered aggregation and development although promoter 3 induced a more complete recovery of fruiting body formation. PMID:26840347

  15. Biological Activity of the Alternative Promoters of the Dictyostelium discoideum Adenylyl Cyclase A Gene.

    PubMed

    Rodriguez-Centeno, Javier; Sastre, Leandro

    2016-01-01

    Amoebae of the Dictyostelium discoideum species form multicellular fruiting bodies upon starvation. Cyclic adenosine monophosphate (cAMP) is used as intercellular signalling molecule in cell-aggregation, cell differentiation and morphogenesis. This molecule is synthesized by three adenylyl cyclases, one of which, ACA, is required for cell aggregation. The gene coding for ACA (acaA) is transcribed from three different promoters that are active at different developmental stages. Promoter 1 is active during cell-aggregation, promoters 2 and 3 are active in prespore and prestalk tip cells at subsequent developmental stages. The biological relevance of acaA expression from each of the promoters has been studied in this article. The acaA gene was expressed in acaA-mutant cells, that do not aggregate, under control of each of the three acaA promoters. acaA expression under promoter 1 control induced cell aggregation although subsequent development was delayed, very small fruiting bodies were formed and cell differentiation genes were expressed at very low levels. Promoter 2-driven acaA expression induced the formation of small aggregates and small fruiting bodies were formed at the same time as in wild-type strains and differentiation genes were also expressed at lower levels. Expression of acaA from promoter 3 induced aggregates and fruiting bodies formation and their size and the expression of differentiation genes were more similar to that of wild-type cells. Expression of acaA from promoters 1 and 2 in AX4 cells also produced smaller structures. In conclusion, the expression of acaA under control of the aggregation-specific Promoter 1 is able to induce cell aggregation in acaA-mutant strains. Expression from promoters 2 and 3 also recovered aggregation and development although promoter 3 induced a more complete recovery of fruiting body formation. PMID:26840347

  16. 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

  17. 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

  18. 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

  19. CO2/HCO3−- and Calcium-regulated Soluble Adenylyl Cyclase as a Physiological ATP Sensor*

    PubMed Central

    Zippin, Jonathan H.; Chen, Yanqiu; Straub, Susanne G.; Hess, Kenneth C.; Diaz, Ana; Lee, Dana; Tso, Patrick; Holz, George G.; Sharp, Geoffrey W. G.; Levin, Lonny R.; Buck, Jochen

    2013-01-01

    The second messenger molecule cAMP is integral for many physiological processes. In mammalian cells, cAMP can be generated from hormone- and G protein-regulated transmembrane adenylyl cyclases or via the widely expressed and structurally and biochemically distinct enzyme soluble adenylyl cyclase (sAC). sAC activity is uniquely stimulated by bicarbonate ions, and in cells, sAC functions as a physiological carbon dioxide, bicarbonate, and pH sensor. sAC activity is also stimulated by calcium, and its affinity for its substrate ATP suggests that it may be sensitive to physiologically relevant fluctuations in intracellular ATP. We demonstrate here that sAC can function as a cellular ATP sensor. In cells, sAC-generated cAMP reflects alterations in intracellular ATP that do not affect transmembrane AC-generated cAMP. In β cells of the pancreas, glucose metabolism generates ATP, which corresponds to an increase in cAMP, and we show here that sAC is responsible for an ATP-dependent cAMP increase. Glucose metabolism also elicits insulin secretion, and we further show that sAC is necessary for normal glucose-stimulated insulin secretion in vitro and in vivo. PMID:24100033

  20. 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

  1. 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

  2. An adenylyl cyclase signaling pathway predicts direct dopaminergic input to vestibular hair cells.

    PubMed

    Drescher, M J; Cho, W J; Folbe, A J; Selvakumar, D; Kewson, D T; Abu-Hamdan, M D; Oh, C K; Ramakrishnan, N A; Hatfield, J S; Khan, K M; Anne, S; Harpool, E C; Drescher, D G

    2010-12-29

    Adenylyl cyclase (AC) signaling pathways have been identified in a model hair cell preparation from the trout saccule, for which the hair cell is the only intact cell type. The use of degenerate primers targeting cDNA sequence conserved across AC isoforms, and reverse transcription-polymerase chain reaction (RT-PCR), coupled with cloning of amplification products, indicated expression of AC9, AC7 and AC5/6, with cloning efficiencies of 11:5:2. AC9 and AC5/6 are inhibited by Ca(2+), the former in conjunction with calcineurin, and message for calcineurin has also been identified in the trout saccular hair cell layer. AC7 is independent of Ca(2+). Given the lack of detection of calcium/calmodulin-activated isoforms previously suggested to mediate AC activation in the absence of Gαs in mammalian cochlear hair cells, the issue of hair-cell Gαs mRNA expression was re-examined in the teleost vestibular hair cell model. Two full-length coding sequences were obtained for Gαs/olf in the vestibular type II-like hair cells of the trout saccule. Two messages for Gαi have also been detected in the hair cell layer, one with homology to Gαi1 and the second with homology to Gαi3 of higher vertebrates. Both Gαs/olf protein and Gαi1/Gαi3 protein were immunolocalized to stereocilia and to the base of the hair cell, the latter consistent with sites of efferent input. Although a signaling event coupling to Gαs/olf and Gαi1/Gαi3 in the stereocilia is currently unknown, signaling with Gαs/olf, Gαi3, and AC5/6 at the base of the hair cell would be consistent with transduction pathways activated by dopaminergic efferent input. mRNA for dopamine receptors D1A4 and five forms of dopamine D2 were found to be expressed in the teleost saccular hair cell layer, representing information on vestibular hair cell expression not directly available for higher vertebrates. Dopamine D1A receptor would couple to Gαolf and activation of AC5/6. Co-expression with dopamine D2 receptor, which

  3. High adenylyl cyclase activity and in vivo cAMP fluctuations in corals suggest central physiological role.

    PubMed

    Barott, K L; Helman, Y; Haramaty, L; Barron, M E; Hess, K C; Buck, J; Levin, L R; Tresguerres, M

    2013-01-01

    Corals are an ecologically and evolutionarily significant group, providing the framework for coral reef biodiversity while representing one of the most basal of metazoan phyla. However, little is known about fundamental signaling pathways in corals. Here we investigate the dynamics of cAMP, a conserved signaling molecule that can regulate virtually every physiological process. Bioinformatics revealed corals have both transmembrane and soluble adenylyl cyclases (AC). Endogenous cAMP levels in live corals followed a potential diel cycle, as they were higher during the day compared to the middle of the night. Coral homogenates exhibited some of the highest cAMP production rates ever to be recorded in any organism; this activity was inhibited by calcium ions and stimulated by bicarbonate. In contrast, zooxanthellae or mucus had >1000-fold lower AC activity. These results suggest that cAMP is an important regulator of coral physiology, especially in response to light, acid/base disturbances and inorganic carbon levels. PMID:23459251

  4. Modulation of β-Adrenergic Receptor Signaling in Heart Failure and Longevity: Targeting Adenylyl Cyclase Type 5

    PubMed Central

    Ho, David; Yan, Lin; Iwatsubo, Kousaku; Vatner, Dorothy E.; Vatner, Stephen F.

    2011-01-01

    Despite remarkable advances in therapy, heart failure remains a leading cause of morbidity and mortality. Although enhanced β-adrenergic receptor stimulation is part of normal physiologic adaptation to either the increase in physiologic demand or decrease in cardiac function, chronic β-adrenergic stimulation has been associated with increased mortality and morbidity in both animal models and humans. For example, overexpression of cardiac Gsα or β-adrenergic receptors in transgenic mice results in enhanced cardiac function in young animals, but with prolonged overstimulation of this pathway, cardiomyopathy develops in these mice as they age. Similarly, chronic sympathomimetic amine therapy increases morbidity and mortality in patients with heart failure. Conversely, the use of β-blockade has proven to be of benefit and is currently part of the standard of care for heart failure. It is conceivable that interrupting distal mechanisms in the β-adrenergic receptor-G protein-adenylyl cyclase pathway may also provide targets for future therapeutic modalities for heart failure. Interestingly, there are two major isoforms of adenylyl cyclase (AC) in the heart (type 5 and type 6), which may exert opposite effects on the heart, i.e., cardiac overexpression of AC6 appears to be protective, whereas disruption of type 5 AC prolongs longevity and protects against cardiac stress. The goal of this review is to summarize the paradigm shift in the treatment of heart failure over the past 50 years from administering sympathomimetic amine agonists to administering β-adrenergic receptor antagonists, and to explore the basis for a novel therapy of inhibiting type 5 AC. PMID:20658186

  5. Expression, purification, crystallization and preliminary X-ray diffraction analysis of a mammalian type 10 adenylyl cyclase

    PubMed Central

    Kleinboelting, Silke; van den Heuvel, Joop; Kambach, Christian; Weyand, Michael; Leipelt, Martina; Steegborn, Clemens

    2014-01-01

    The second messenger cAMP is synthesized in mammals by ten differently regulated adenylyl cyclases (AC1–10). These ACs are grouped into nucleotidyl cyclase class III based on homologies in their catalytic domains. The catalytic domain of AC10 is unique, however, in being activated through direct interaction with calcium and bicarbonate. Here, the production, crystallization and X-ray diffraction analysis of the catalytic domain of human AC10 are described as a basis for structural studies of regulator binding sites and mechanisms. The recombinant protein had high specific AC activity, and crystals of AC10 in space group P63 diffracted to ∼2.0 Å resolution on a synchrotron beamline. A complete diffraction data set revealed unit-cell parameters a = b = 99.65, c = 98.04 Å, indicating one AC10 catalytic domain per asymmetric unit, and confirmed that the obtained crystals are suitable for structure solution and mechanistic studies. PMID:24699740

  6. Fluorogenic Green-Inside Red-Outside (GIRO) Labeling Approach Reveals Adenylyl Cyclase-Dependent Control of BKα Surface Expression

    PubMed Central

    2015-01-01

    The regulation of surface levels of protein is critical for proper cell function and influences properties including cell adhesion, ion channel contributions to current flux, and the sensitivity of surface receptors to ligands. Here we demonstrate a two-color labeling system in live cells using a single fluorogen activating peptide (FAP) based fusion tag, which enables the rapid and simultaneous quantification of surface and internal proteins. In the nervous system, BK channels can regulate neural excitability and neurotransmitter release, and the surface trafficking of BK channels can be modulated by signaling cascades and assembly with accessory proteins. Using this labeling approach, we examine the dynamics of BK channel surface expression in HEK293 cells. Surface pools of the pore-forming BKα subunit were stable, exhibiting a plasma membrane half-life of >10 h. Long-term activation of adenylyl cyclase by forskolin reduced BKα surface levels by 30%, an effect that could not be attributed to increased bulk endocytosis of plasma membrane proteins. This labeling approach is compatible with microscopic imaging and flow cytometry, providing a solid platform for examining protein trafficking in living cells. PMID:26301573

  7. 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

  8. Genetic Ablation of Type III Adenylyl Cyclase Exerts Region-Specific Effects on Cilia Architecture in the Mouse Nose

    PubMed Central

    Challis, Rosemary C.; Tian, Huikai; Yin, Wenbin; Ma, Minghong

    2016-01-01

    We recently reported that olfactory sensory neurons in the dorsal zone of the mouse olfactory epithelium exhibit drastic location-dependent differences in cilia length. Furthermore, genetic ablation of type III adenylyl cyclase (ACIII), a key olfactory signaling protein and ubiquitous marker for primary cilia, disrupts the cilia length pattern and results in considerably shorter cilia, independent of odor-induced activity. Given the significant impact of ACIII on cilia length in the dorsal zone, we sought to further investigate the relationship between cilia length and ACIII level in various regions throughout the mouse olfactory epithelium. We employed whole-mount immunohistochemical staining to examine olfactory cilia morphology in phosphodiesterase (PDE) 1C-/-;PDE4A-/- (simplified as PDEs-/- hereafter) and ACIII-/- mice in which ACIII levels are reduced and ablated, respectively. As expected, PDEs-/- animals exhibit dramatically shorter cilia in the dorsal zone (i.e., where the cilia pattern is found), similar to our previous observation in ACIII-/- mice. Remarkably, in a region not included in our previous study, ACIII-/- animals (but not PDEs-/- mice) have dramatically elongated, comet-shaped cilia, as opposed to characteristic star-shaped olfactory cilia. Here, we reveal that genetic ablation of ACIII has drastic, location-dependent effects on cilia architecture in the mouse nose. These results add a new dimension to our current understanding of olfactory cilia structure and regional organization of the olfactory epithelium. Together, these findings have significant implications for both cilia and sensory biology. PMID:26942602

  9. 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

  10. Adenylyl cyclase-associated protein-1/CAP1 as a biological target substrate of gelatinase B/MMP-9

    SciTech Connect

    Cauwe, Benedicte; Martens, Erik; Van den Steen, Philippe E.; Proost, Paul; Van Aelst, Ilse; Blockmans, Daniel; Opdenakker, Ghislain

    2008-09-10

    Matrix metalloproteinases (MMPs) are classically associated with the turnover of secreted structural and functional proteins. Although MMPs have been shown to process also a kaleidoscope of membrane-associated substrates, little is known about the processing of intracellular proteins by MMPs. Physiological and pathological cell apoptosis, necrosis and tumor lysis by chemotherapy, radiotherapy or immunological cytotoxicity, are examples of conditions in which an overload of intracellular proteins becomes accessible to the action of MMPs. We used a model system of dying human myelomonocytic cells to study the processing of intracellular protein substrates by gelatinase B/MMP-9 in vitro. Adenylyl cyclase-associated protein-1 or CAP1 was identified as a novel and most efficient substrate of gelatinase B/MMP-9. The presence of CAP1 in the extracellular milieu in vivo was documented by analysis of urine of patients with systemic autoimmune diseases. Whereas no active MMP-9 could be detected in urines of healthy controls, all urine samples of patients with clinical parameters of renal failure contained activated MMP-9 and/or MMP-2. In addition, in some of these patients indications of CAP1 cleavage are observed, implying CAP1 degradation in vivo. The high turnover rate of CAP1 by MMP-9, comparable to that of gelatin as the natural extracellular substrate of this enzyme, may be critical to prevent pathological conditions associated with considerable cytolysis.

  11. 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

  12. High-throughput FACS-based mutant screen identifies a gain-of-function allele of the Fusarium graminearum adenylyl cyclase causing deoxynivalenol over-production.

    PubMed

    Blum, Ailisa; Benfield, Aurélie H; Stiller, Jiri; Kazan, Kemal; Batley, Jacqueline; Gardiner, Donald M

    2016-05-01

    Fusarium head blight and crown rot, caused by the fungal plant pathogen Fusarium graminearum, impose a major threat to global wheat production. During the infection, plants are contaminated with mycotoxins such as deoxynivalenol (DON), which can be toxic for humans and animals. In addition, DON is a major virulence factor during wheat infection. However, it is not fully understood how DON production is regulated in F. graminearum. In order to identify regulators of DON production, a high-throughput mutant screen using Fluorescence Activated Cell Sorting (FACS) of a mutagenised TRI5-GFP reporter strain was established and a mutant over-producing DON under repressive conditions identified. A gain-of-function mutation in the F. graminearum adenylyl cyclase (FAC1), which is a known positive regulator of DON production, was identified as the cause of this phenotype through genome sequencing and segregation analysis. Our results show that the high-throughput mutant screening procedure developed here can be applied for identification of fungal proteins involved in diverse processes. PMID:26932301

  13. 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

  14. Effect of overexpressed adenylyl cyclase VI on β1- and β2-adrenoceptor responses in adult rat ventricular myocytes

    PubMed Central

    Stark, Joalice C C; Haydock, Stephen F; Foo, Roger; Brown, Morris J; Harding, Sian E

    2004-01-01

    Adenylyl cyclase VI (ACVI) is one of the most abundantly expressed β adrenergic receptor (βAR)-coupled cyclases responsible for cyclic AMP (cAMP) production within the mammalian myocardium. We investigated the role of ACVI in the regulation of cardiomyocyte contractility and whether it is functionally coupled with β1 adrenergic receptor (β1AR). Recombinant adenoviruses were generated for ACVI and for antisense to ACVI (AS). Adult rat ventricular myocytes were transfected with ACVI virus, AS or both (SAS). Adenovirus for green fluorescent protein (GFP) served as control. Myocyte contraction amplitudes (% shortening) and relaxation times (R50) were analysed. ACVI function was determined using cAMP assays. ACVI-transfected cells demonstrated a strong 139 kDa ACVI protein band compared to controls. ACVI myocytes had higher steady-state intracellular cAMP levels than GFP myocytes when unstimulated (GFP vs ACVI=6.60±0.98 vs 14.2±2.1 fmol cAMP/viable cell, n=4, P<0.05) and in the presence of 1 μM isoprenaline or 10 μM forskolin. ACVI myocytes had increased basal contraction (% shortening: GFP vs ACVI: 1.90±1.36 vs 3.91±2.29, P<0.0001) and decreased basal R50 (GFP vs ACVI: 62.6±24.2 ms (n=50) vs 45.0±17.2 ms (n=248), P<0.0001). ACVI myocyte responses were increased for forskolin (Emax: GFP=6.70±1.59 (n=6); ACVI=9.06±0.69 (n=14), P<0.01) but not isoprenaline. ACVI myocyte responses were increased (Emax: GFP vs ACVI=3.16±0.77 vs 5.10±0.60, P<0.0001) to xamoterol (a partial β1AR-selective agonist) under β2AR blockade (+50 nM ICI 118, 551). AS decreased both control and ACVI-stimulated xamoterol responses (Emax: AS=2.59±1.42, SAS=1.38±0.5). ACVI response was not mimicked by IBMX. Conversely, response through β2 adrenergic receptor (β2AR) was decreased in ACVI myocytes. In conclusion, ACVI overexpression constitutively increases myocyte contraction amplitudes by raising cAMP levels. Native ACVI did not contribute to basal cAMP production or contraction

  15. Pituitary adenylyl cyclase-activating polypeptide is an intrinsic regulator of Treg abundance and protects against experimental autoimmune encephalomyelitis.

    PubMed

    Tan, Yossan-Var; Abad, Catalina; Lopez, Robert; Dong, Hongmei; Liu, Shen; Lee, Alice; Gomariz, Rosa P; Leceta, Javier; Waschek, James A

    2009-02-10

    Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a widely expressed neuropeptide originally discovered in the hypothalamus. It closely resembles vasoactive intestinal peptide (VIP), a neuropeptide well known to inhibit macrophage activity, promote Th2-type responses, and enhance regulatory T cell (Treg) production. Recent studies have shown that administration of PACAP, like VIP, can attenuate dramatically the clinical and pathological features of murine models of autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis. However, specific roles (if any) of endogenous VIP and PACAP in the protection against autoimmune diseases have not been explored. Here, we subjected PACAP-deficient mice to myelin oligodendrocyte glycoprotein (MOG(35-55))-induced EAE. MOG immunization of PACAP-deficient mice triggered heightened clinical and pathological manifestations of EAE compared to wild-type mice. The increased sensitivity was accompanied by enhanced mRNA expression of proinflammatory cytokines (TNFalpha, IL-6, IFN-gamma, IL-12p35, IL-23p19, and IL-17), chemokines (MCP-1/CCL2, MIP-1alpha/CCL3, and RANTES/CCL5), and chemotactic factor receptors (CCR1, CCR2, and CCR5), but downregulation of the anti-inflammatory cytokines (IL-4, IL-10, and TGF-beta) in the spinal cord. Moreover, the abundance of CD4(+)CD25(+)FoxP3(+) Tregs in lymph nodes and levels of FoxP3 mRNA in the spinal cord were also diminished. The reduction in Tregs was associated with increased proliferation and decreased TGF-beta secretion in lymph node cultures stimulated with MOG. These results demonstrate that endogenous PACAP provides protection in EAE and identify PACAP as an intrinsic regulator of Treg abundance after inflammation. PMID:19190179

  16. β3GnT2 Maintains Adenylyl Cyclase-3 Signaling and Axon Guidance Molecule Expression in the Olfactory Epithelium

    PubMed Central

    Faden, Ashley A.; Knott, Thomas K.

    2011-01-01

    In the olfactory epithelium (OE), odorant receptor stimulation generates cAMP signals that function in both odor detection and the regulation of axon guidance molecule expression. The enzyme that synthesizes cAMP, adenylyl cyclase 3 (AC3), is coexpressed in olfactory sensory neurons (OSNs) with poly-N-acetyllactosamine (PLN) oligosaccharides determined by the glycosyltransferase β3GnT2. The loss of either enzyme results in similar defects in olfactory bulb (OB) innervation and OSN survival, suggesting that glycosylation may be important for AC3 function. We show here that AC3 is extensively modified with N-linked PLN, which is essential for AC3 activity and localization. On Western blots, AC3 from the wild-type OE migrates diffusely as a heavily glycosylated 200 kDa band that interacts with the PLN-binding lectin LEA. AC3 from the β3GnT2−/− OE loses these PLN modifications, migrating instead as a 140 kDa glycoprotein. Furthermore, basal and forskolin-stimulated cAMP production is reduced 80–90% in the β3GnT2−/− OE. Although AC3 traffics normally to null OSN cilia, it is absent from axon projections that aberrantly target the OB. The cAMP-dependent guidance receptor neuropilin-1 is also lost from β3GnT2−/− OSNs and axons, while semaphorin-3A ligand expression is upregulated. In addition, kirrel2, a mosaically expressed adhesion molecule that functions in axon sorting, is absent from β3GnT2−/− OB projections. These results demonstrate that PLN glycans are essential in OSNs for proper AC3 localization and function. We propose that the loss of cAMP-dependent guidance cues is also a critical factor in the severe axon guidance defects observed in β3GnT2−/− mice. PMID:21525298

  17. Pituitary adenylyl cyclase-activating peptide: A pivotal modulator of glutamatergic regulation of the suprachiasmatic circadian clock

    PubMed Central

    Chen, Dong; Buchanan, Gordon F.; Ding, Jian M.; Hannibal, Jens; Gillette, Martha U.

    1999-01-01

    The circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus organizes behavioral rhythms, such as the sleep–wake cycle, on a near 24-h time base and synchronizes them to environmental day and night. Light information is transmitted to the SCN by direct retinal projections via the retinohypothalamic tract (RHT). Both glutamate (Glu) and pituitary adenylyl cyclase-activating peptide (PACAP) are localized within the RHT. Whereas Glu is an established mediator of light entrainment, the role of PACAP is unknown. To understand the functional significance of this colocalization, we assessed the effects of nocturnal Glu and PACAP on phasing of the circadian rhythm of neuronal firing in slices of rat SCN. When coadministered, PACAP blocked the phase advance normally induced by Glu during late night. Surprisingly, blocking PACAP neurotransmission, with either PACAP6–38, a specific PACAP receptor antagonist, or anti-PACAP antibodies, augmented the Glu-induced phase advance. Blocking PACAP in vivo also potentiated the light-induced phase advance of the rhythm of hamster wheel-running activity. Conversely, PACAP enhanced the Glu-induced delay in the early night, whereas PACAP6–38 inhibited it. These results reveal that PACAP is a significant component of the Glu-mediated light-entrainment pathway. When Glu activates the system, PACAP receptor-mediated processes can provide gain control that generates graded phase shifts. The relative strengths of the Glu and PACAP signals together may encode the amplitude of adaptive circadian behavioral responses to the natural range of intensities of nocturnal light. PMID:10557344

  18. Type 3 Adenylyl Cyclase and Somatostatin Receptor 3 Expression Persists in Aged Rat Neocortical and Hippocampal Neuronal Cilia

    PubMed Central

    Guadiana, Sarah M.; Parker, Alexander K.; Filho, Gileno F.; Sequeira, Ashton; Semple-Rowland, Susan; Shaw, Gerry; Mandel, Ronald J.; Foster, Thomas C.; Kumar, Ashok; Sarkisian, Matthew R.

    2016-01-01

    The primary cilia of forebrain neurons assemble around birth and become enriched with neuromodulatory receptors. Our understanding of the permanence of these structures and their associated signaling pathways in the aging brain is poor, but they are worthy of investigation because disruptions in neuronal cilia signaling have been implicated in changes in learning and memory, depression-like symptoms, and sleep anomalies. Here, we asked whether neurons in aged forebrain retain primary cilia and whether the staining characteristics of aged cilia for type 3 adenylyl cyclase (ACIII), somatostatin receptor 3 (SSTR3), and pericentrin resemble those of cilia in younger forebrain. To test this, we analyzed immunostained sections of forebrain tissues taken from young and aged male Fischer 344 (F344) and F344 × Brown Norway (F344 × BN) rats. Analyses of ACIII and SSTR3 in young and aged cortices of both strains of rats revealed that the staining patterns in the neocortex and hippocampus were comparable. Virtually every NeuN positive cell examined possessed an ACIII positive cilium. The lengths of ACIII positive cilia in neocortex were similar between young and aged for both strains, whereas in F344 × BN hippocampus, the cilia lengths increased with age in CA1 and CA3, but not in dentate gyrus (DG). Additionally, the percentages of ACIII positive cilia that were also SSTR3 positive did not differ between young and aged tissues in either strain. We also found that pericentrin, a protein that localizes to the basal bodies of neuronal cilia and functions in primary cilia assembly, persisted in aged cortical neurons of both rat strains. Collectively, our data show that neurons in aged rat forebrain possess primary cilia and that these cilia, like those present in younger brain, continue to localize ACIII, SSTR3, and pericentrin. Further studies will be required to determine if the function and signaling pathways regulated by cilia are similar in aged compared to young brain

  19. delta-Opioid receptors are more efficiently coupled to adenylyl cyclase than to L-type Ca(2+) channels in transfected rat pituitary cells.

    PubMed

    Prather, P L; Song, L; Piros, E T; Law, P Y; Hales, T G

    2000-11-01

    Opioid receptors often couple to multiple effectors within the same cell. To examine potential mechanisms that contribute to the specificity by which delta-receptors couple to distinct intracellular effectors, we stably transfected rat pituitary GH(3) cells with cDNAs encoding for delta-opioid receptors. In cells transfected with a relatively low delta-receptor density of 0.55 pmol/mg of protein (GH(3)DOR), activation of delta-receptors produced inhibition of adenylyl cyclase activity but was unable to alter L-type Ca(2+) current. In contrast, activation of delta-receptors in a clone that contained a higher density of delta-receptors (2.45 pmol/mg of protein) and was also coexpressed with mu-opioid receptors (GH(3)MORDOR), resulted in not only the expected inhibition of adenylyl cyclase activity but also produced inhibition of L-type Ca(2+) current. The purpose of the present study was to determine whether these observations resulted from differences in delta-opioid receptor density between clones or interaction between delta- and mu-opioid receptors to allow the activation of different G proteins and signaling to Ca(2+) channels. Using the delta-opioid receptor alkylating agent SUPERFIT, reduction of available delta-opioid receptors in GH(3)MORDOR cells to a density similar to that of delta-opioid receptors in the GH(3)DOR clone resulted in abolishment of coupling to Ca(2+) channels, but not to adenylyl cyclase. Furthermore, although significantly greater amounts of all G proteins were activated by delta-opioid receptors in GH(3)MORDOR cells, delta-opioid receptor activation in GH(3)DOR cells resulted in coupling to the identical pattern of G proteins seen in GH(3)MORDOR cells. These findings suggest that different threshold densities of delta-opioid receptors are required to activate critical amounts of G proteins needed to produce coupling to specific effectors and that delta-opioid receptors couple more efficiently to adenylyl cyclase than to L-type Ca(2

  20. Some sweet and bitter tastants stimulate inhibitory pathway of adenylyl cyclase via melatonin and alpha 2-adrenergic receptors in Xenopus laevis melanophores.

    PubMed

    Zubare-Samuelov, Meirav; Peri, Irena; Tal, Michael; Tarshish, Mark; Spielman, Andrew I; Naim, Michael

    2003-11-01

    The sweeteners saccharin, D-tryptophan, and neohesperidin dihydrochalcone (NHD) and the bitter tastant cyclo(Leu-Trp) stimulated concentration-dependent pigment aggregation in a Xenopus laevis melanophore cell line similar to melatonin. Like melatonin, these tastants inhibited (by 45-92%) cAMP formation in melanophores; pertussis toxin pretreatment almost completely abolished the tastant-induced cAMP inhibition, suggesting the involvement of the inhibitory pathway (Gi) of adenylyl cyclase. The presence of luzindole (melatonin receptor antagonist) almost completely abolished the inhibition of cAMP formation induced by saccharin, D-tryptophan, and cyclo(Leu-Trp) but only slightly affected the inhibitory effect of NHD. In contrast, the presence of an alpha2-adrenergic receptor antagonist, yohimbine, almost completely abolished the inhibition of cAMP formation induced by NHD but had only a minor effect on that induced by the other tastants. Thus saccharin, D-tryptophan, and cyclo(Leu-Trp) are melatonin receptor agonists whereas NHD is an alpha2-adrenergic receptor agonist, but both pathways lead to the same transduction output and cellular response. Formation of D-myo-inositol 1,4,5-trisphosphate (IP3) in melanophores was reduced (15-58%, no concentration dependence) by saccharin, D-tryptophan, and cyclo(Leu-Trp) stimulation but increased by NHD stimulation. Tastant stimulation did not affect cGMP. Although some of the above tastants were found to be membrane permeant, their direct activation of downstream transduction components in this experimental system is questionable. MT1 and MT2 melatonin receptor mRNAs were identified in rat circumvallate papilla taste buds and nonsensory epithelium, suggesting the occurrence of MT1 and MT2 receptors in these tissues. Melatonin stimulation reduced the cellular content of cAMP in taste cells, which may or may not be related to taste sensation. PMID:12839835

  1. Critical periods for chlorpyrifos-induced developmental neurotoxicity: alterations in adenylyl cyclase signaling in adult rat brain regions after gestational or neonatal exposure.

    PubMed Central

    Meyer, Armando; Seidler, Frederic J; Aldridge, Justin E; Tate, Charlotte A; Cousins, Mandy M; Slotkin, Theodore A

    2004-01-01

    Developmental exposure to chlorpyrifos (CPF) alters the function of a wide variety of neural systems. In the present study we evaluated the effects in adulthood of CPF exposure of rats during different developmental windows, using the adenylyl cyclase (AC) signaling cascade, which mediates the cellular responses to numerous neurotransmitters. Animals were exposed on gestational days (GD) 9-12 or 17-20 or on postnatal days (PN) 1-4 or 11-14 and assessed at PN60. In addition to basal AC activity, we evaluated the responses to direct AC stimulants (forskolin, Mn2+) and to isoproterenol, which activates signaling through ss-adrenoceptors coupled to stimulatory G-proteins. CPF exposure in any of the four periods elicited significant changes in AC signaling in a wide variety of brain regions in adulthood. In general, GD9-12 was the least sensitive stage, requiring doses above the threshold for impaired maternal weight gain, whereas effects were obtained at subtoxic doses for all other regimens. Most of the effects were heterologous, involving signaling elements downstream from the receptors, and thus shared by multiple stimulants; superimposed on this basic pattern, there were also selective alterations in receptor-mediated responses, in G-protein function, and in AC expression and subtypes. Exposures conducted at GD17-20 and later all produced sex-selective alterations. These results suggest that developmental exposure to CPF elicits long-lasting alterations in cell-signaling cascades that are shared by multiple neurotransmitter and hormonal inputs; the resultant abnormalities of synaptic communication are thus likely to occur in widespread neural circuits and their corresponding behaviors. PMID:14998743

  2. Separate Elements within a Single IQ-like Motif in Adenylyl Cyclase Type 8 Impart Ca2+/Calmodulin Binding and Autoinhibition*

    PubMed Central

    MacDougall, David A.; Wachten, Sebastian; Ciruela, Antonio; Sinz, Andrea; Cooper, Dermot M. F.

    2009-01-01

    The ubiquitous Ca2+-sensing protein calmodulin (CaM) fulfills its numerous signaling functions through a wide range of modular binding and activation mechanisms. By activating adenylyl cyclases (ACs) 1 and 8, Ca2+ acting via calmodulin impacts on the signaling of the other major cellular second messenger cAMP. In possessing two CaM-binding domains, a 1-5-8-14 motif at the N terminus and an IQ-like motif (IQlm) at the C terminus, AC8 offers particularly sophisticated regulatory possibilities. The IQlm has remained unexplored beyond the suggestion that it bound CaM, and the larger C2b region of which it is part was involved in the relief of autoinhibition of AC8. Here we attempt to distinguish the function of individual residues of the IQlm. From a complementary approach of in vitro and cell population AC activity assays, as well as CaM binding, we propose that the IQlm alone, and not the majority of the C2b, imparts CaM binding and autoinhibitory functions. Moreover, this duality of function is spatially separated and depends on amino acid side-chain character. Accordingly, residues critical for CaM binding are positively charged and clustered toward the C terminus, and those essential for the maintenance of autoinhibition are hydrophobic and more N-terminal. Secondary structure prediction of the IQlm supports this separation, with an ideally placed break in the α-helical nature of the sequence. We additionally find that the N and C termini of AC8 interact, which is an association specifically abrogated by fully Ca2+-bound, but not Ca2+-free, CaM. These data support a sophisticated activation mechanism of AC8 by CaM, in which the duality of the IQlm function is critical. PMID:19305019

  3. Pivotal role for aspartate-80 in the regulation of dopamine D2 receptor affinity for drugs and inhibition of adenylyl cyclase.

    PubMed

    Neve, K A; Cox, B A; Henningsen, R A; Spanoyannis, A; Neve, R L

    1991-06-01

    An aspartate residue corresponding to aspartate-80 of dopamine D2 receptors is strictly conserved among receptors that couple to guanine nucleotide-binding proteins. Mutation of this residue alters the function of several classes of neurotransmitter receptors. Dopamine D2 receptors couple to the guanine nucleotide-binding protein Gi to inhibit adenylyl cyclase (ATP-pyrophosphate-lyase, cyclizing; EC 4.6.1.1). Like other Gi-coupled receptors, the binding of agonists and some antagonists to D2 receptors is sensitive to pH and sodium. In the present report, we demonstrate that substitution of an alanine or glutamate residue for aspartate-80 severely impairs inhibition of adenylyl cyclase by D2 receptors and also abolishes or decreases the regulation of the affinity of D2 receptors for agonists and substituted benzamide antagonists by sodium and pH. Our data support the hypothesis that the conformation of D2 receptors is maintained by interactions of monovalent cations with aspartate-80. The regulation of D2 receptors by this interaction has important consequences for the affinity of D2 receptors for ligands and for signal transduction by D2 receptors. PMID:1828858

  4. Adenylyl cyclase AC8 directly controls its micro-environment by recruiting the actin cytoskeleton in a cholesterol-rich milieu

    PubMed Central

    Ayling, Laura J.; Briddon, Stephen J.; Halls, Michelle L.; Hammond, Gerald R. V.; Vaca, Luis; Pacheco, Jonathan; Hill, Stephen J.; Cooper, Dermot M. F.

    2012-01-01

    The central and pervasive influence of cAMP on cellular functions underscores the value of stringent control of the organization of adenylyl cyclases (ACs) in the plasma membrane. Biochemical data suggest that ACs reside in membrane rafts and could compartmentalize intermediary scaffolding proteins and associated regulatory elements. However, little is known about the organization or regulation of the dynamic behaviour of ACs in a cellular context. The present study examines these issues, using confocal image analysis of various AC8 constructs, combined with fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. These studies reveal that AC8, through its N-terminus, enhances the cortical actin signal at the plasma membrane; an interaction that was confirmed by GST pull-down and immunoprecipitation experiments. AC8 also associates dynamically with lipid rafts; the direct association of AC8 with sterols was confirmed in Förster resonance energy transfer experiments. Disruption of the actin cytoskeleton and lipid rafts indicates that AC8 tracks along the cytoskeleton in a cholesterol-enriched domain, and the cAMP that it produces contributes to sculpting the actin cytoskeleton. Thus, an adenylyl cyclase is shown not just to act as a scaffold, but also to actively orchestrate its own micro-environment, by associating with the cytoskeleton and controlling the association by producing cAMP, to yield a highly organized signalling hub. PMID:22399809

  5. Identification of a human cDNA encoding a protein that is structurally and functionally related to the yeast adenylyl cyclase-associated CAP proteins

    SciTech Connect

    Matviw, Yu, G.; Young, D. )

    1992-11-01

    The adenylyl cyclases of both Saccharomyces cerevisiae and Schizosaccharomyces pombe are associated with related proteins named CAP. In S. cerevisiae, CAP is required for cellular responses mediated by the RAS/cyclic AMP pathway. Both yeast CAPs appear to be bifunctional proteins: The N-terminal domains are required for the proper function of adenylyl cyclase, while loss of the C-terminal domains results in morphological and nutritional defects that appear to be unrelated to the cAMP pathways. Expression of either yeast CAP in the heterologous yeast suppresses phenotypes associated with loss of the C-terminal domain of the endogenous CAP but does not suppress loss of the N-terminal domain. On the basis of the homology between the two yeast CAP proteins, we have designed degenerate oligonucleotides that we used to detect, by the polymerase chain reaction method, a human cDNA fragment encoding a CAP-related peptide. Using the polymerase chain reaction fragment as a probe, we isolated a human cDNA clone encoding a 475-amino-acid protein that is homologous to the yeast CAP proteins. Expressions of the human CAP protein in S. cerevisiae suppresses the phenotypes associated with loss of the C-terminal domain of CAP but does not suppress phenotypes associated with loss of the N-terminal domain. Thus, CAP proteins have been structurally and, to some extent, functionally conserved in evolution between yeasts and mammals. 42 refs., 5 figs.

  6. An Improved Targeted cAMP Sensor to Study the Regulation of Adenylyl Cyclase 8 by Ca2+ Entry through Voltage-Gated Channels

    PubMed Central

    Everett, Katy L.; Cooper, Dermot M. F.

    2013-01-01

    Here we describe an improved sensor with reduced pH sensitivity tethered to adenylyl cyclase (AC) 8. The sensor was used to study cAMP dynamics in the AC8 microdomain of MIN6 cells, a pancreatic β-cell line. In these cells, AC8 was activated by Ca2+ entry through L-type voltage-gated channels following depolarisation. This activation could be reconstituted in HEK293 cells co-expressing AC8 and either the α1C or α1D subunit of L-type voltage-gated Ca2+ channels. The development of this improved sensor opens the door to the study of cAMP microdomains in excitable cells that have previously been challenging due to the sensitivity of fluorescent proteins to pH changes. PMID:24086669

  7. Differentiation of photocycle characteristics of flavin-binding BLUF domains of α- and β-subunits of photoactivated adenylyl cyclase of Euglena gracilis.

    PubMed

    Ito, Shinji; Murakami, Akio; Iseki, Mineo; Takahashi, Tetsuo; Higashi, Shoichi; Watanabe, Masakatsu

    2010-10-28

    Photoactivated adenylyl cyclase (PAC), an FAD-containing photoreceptor of Euglena gracilis, appears to be a heterotetrameric structure composed of 2 homologous subunits (PACα and PACβ), each with a pair of BLUF domains (F1 and F2). PAC promotes blue light-induced activation of adenylyl cyclase. In our previous report, we demonstrated that a recombinant version of the PACαF2 domain displays blue light-induced photocycle similar to those of prokaryotic BLUFs (Ito et al., Photochem. Photobiol. Sci., 2005, 4, 762-769). Here, we further examine the recombinant PACβF2 domain, which like PACαF2 exhibits a blue light-induced photocycle. The estimated quantum efficiency for the phototransformation of PACβF2 was 0.06-0.08, and the half-life for dark relaxation was 3-6 s while the corresponding values for the PACαF2 were 0.28-0.32 and 34-44 s. The remarkable differences between PACαF2 and PACβF2 may be related to the sensitivity of the photoactivation. In PACαF2, amino acid position 556, which is equivalent to Trp104 in the BLUF domain of the purple bacterial AppA protein, is occupied by a Leu residue, while in PACβF2 the equivalent BLUF domain site is conserved as Trp560. Amino acid substitution at this site in PACβF2-Trp560Leu markedly increased the estimated quantum efficiency (0.23) and accelerated the half-life of the dark-relaxation (2 s). These results indicate that Trp560 in PACβF2 plays a main role in suppressing the quantum efficiency. PMID:20842310

  8. [Peptide 612-627 of thyrotropin receptor and its modified derivatives as the regulators of adenylyl cyclase in the rat thyroid gland].

    PubMed

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

    2014-01-01

    The regulation of the specific activity of the thyroid gland is carried by thyroid-stimulating hormone (TSH) through TSH receptor (TSHR). This receptor is coupled to different types of G-proteins, including the G(s)-proteins, through which TSH stimulates the enzyme adenylyl cyclase (AC). As the application of TSH in medicine is limited, the development of selective regulators of TSHR with agonistic and antagonistic activity is carried out. One of the approaches to their creation is to develop the peptides corresponding to functionally important regions of TSHR which are located in its intracellular loops (ICL) and are involved in the binding and activation of G-proteins. We have synthesized peptide corresponding to the C-terminal region 612-627 of the third ICL of TSHR and its derivatives modified by palmitic acid residue (at the N- or the C-terminus) or by polylysine dendrimer (at the N-terminus), and studied their effect on the basal and TSH-stimulated AC activity in the membrane fraction isolated from the rat thyroid. The most active was peptide 612-627-K(Pal)A modified by palmitate at the C-terminus, where in TSHR the hydrophobic transmembrane region is located. At the micromolar concentrations the peptide increased AC activity and reduced the AC stimulating effect of TSH. The action of the 612-627-K(Pal)A has been directed onto TSHR homologous to it, as indicated by the following facts: 1) the inhibition of G(s)-protein, the downstream component of AC system, by treating the membranes with cholera toxin led to the blocking of peptide AC effect, 2) this effect was not detected in the tissues where no TSHR, 3) the peptide did not significantly affect the AC stimulating effects of hormones acting via other receptors. The unmodified peptide and the peptide with N-terminal dendrimer are far behind the 612-627-K(Pal)A in their ability to activate AC in the thyroid, while the peptide modified by palmitate at the N-terminus was inactive. At the same time, the peptide

  9. 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

  10. [A change of hormonal regulation of adenylyl cyclase in the epididymal adipose tissue of rats with experimental models of diabetes mellitus].

    PubMed

    Derkach, K V; Chistyakova, O V; Shpakov, A O

    2014-01-01

    One of the key causes of diabetes mellitus (DM) and its complications are hormonal disturbances in functioning of hormonal signaling systems, including the adenylyl cyclase signaling system (ACSS). The goal of this work was to study the functional state and hormonal sensitivity of ACSS in the epididymal adipose tissue of male rats in the 7-month model of mild type 1 DM (DM1), in the 18-month neonatal model of type 2 DM (DM2), and in the taken for comparison model of the 30-day acute DM1. It is shown for the first time that in adipocytes from the epididymal fat of rats with the studied DM models the basal AC activity and its stimulation by forskolin were decreased, which indicates a weakening of the catalytic function of the enzyme adenylyl cyclase (AC). Stimulation of AC by guanine nucleotides in DM changed to the lesser extent, which speaks in favor of preservation of functions of heterotrimeric G(s)-proteins in the epididymal fat. In rats with DM1 the sensitivity of AC of adipocytes to agonists of β-adrenergic receptors (β-AR), activators of lipolysis, remained practically unchanged, while in animals with DM2 the AC stimulating effects of β-AR-agonists were reduced or completely blocked, like in the case of β3-AR-agonist BRL-37344 and CL-316243. In adipocytes of rats with DM1 the AC inhibitory effect of N6-cyclopentyladenosine, agonist of type 1 adenosine receptors (Aden1R), an inhibitor of lipolysis, was attenuated, whe- reas in DM2 this effect was completely preserved. Thus, in the epididymal adipose tissue of rats with DM1 the antilipolytic AC cascades including Aden1R were decreased and the stimulation of AC by β-AR-agonists was preserved, whereas in rats with DM2 the β-AR-mediated AC cascades activating lipolysis were reduced, but Aden1R-mediated AC cascades inhibiting lipolysis did not change. The changes of hormonal regulation of ACSS in adipocytes from the epididymal fat lead to disturbances of the metabolic status of animal with DM1 and DM2 and

  11. Sensing Positive versus Negative Reward Signals through Adenylyl Cyclase-Coupled GPCRs in Direct and Indirect Pathway Striatal Medium Spiny Neurons

    PubMed Central

    Nair, Anu G.; Eriksson, Olivia; Vincent, Pierre

    2015-01-01

    Transient changes in striatal dopamine (DA) concentration are considered to encode a reward prediction error (RPE) in reinforcement learning tasks. Often, a phasic DA change occurs concomitantly with a dip in striatal acetylcholine (ACh), whereas other neuromodulators, such as adenosine (Adn), change slowly. There are abundant adenylyl cyclase (AC) coupled GPCRs for these neuromodulators in striatal medium spiny neurons (MSNs), which play important roles in plasticity. However, little is known about the interaction between these neuromodulators via GPCRs. The interaction between these transient neuromodulator changes and the effect on cAMP/PKA signaling via Golf- and Gi/o-coupled GPCR are studied here using quantitative kinetic modeling. The simulations suggest that, under basal conditions, cAMP/PKA signaling could be significantly inhibited in D1R+ MSNs via ACh/M4R/Gi/o and an ACh dip is required to gate a subset of D1R/Golf-dependent PKA activation. Furthermore, the interaction between ACh dip and DA peak, via D1R and M4R, is synergistic. In a similar fashion, PKA signaling in D2+ MSNs is under basal inhibition via D2R/Gi/o and a DA dip leads to a PKA increase by disinhibiting A2aR/Golf, but D2+ MSNs could also respond to the DA peak via other intracellular pathways. This study highlights the similarity between the two types of MSNs in terms of high basal AC inhibition by Gi/o and the importance of interactions between Gi/o and Golf signaling, but at the same time predicts differences between them with regard to the sign of RPE responsible for PKA activation. SIGNIFICANCE STATEMENT Dopamine transients are considered to carry reward-related signal in reinforcement learning. An increase in dopamine concentration is associated with an unexpected reward or salient stimuli, whereas a decrease is produced by omission of an expected reward. Often dopamine transients are accompanied by other neuromodulatory signals, such as acetylcholine and adenosine. We highlight the

  12. The Adenylyl Cyclase Plays a Regulatory Role in the Morphogenetic Switch from Vegetative to Pathogenic Lifestyle of Fusarium graminearum on Wheat

    PubMed Central

    Bormann, Jörg; Boenisch, Marike Johanne; Brückner, Elena; Firat, Demet; Schäfer, Wilhelm

    2014-01-01

    Cyclic 3′,5′-adenosine monophosphate (cAMP) is a nucleotide derived from adenosine triphosphate that acts as a second messenger throughout all kingdoms. Intracellular cAMP levels are synthesized by a membrane-bound protein, the adenylyl cyclase. In order to analyze the function of this gene and the importance of cAMP in the life cycle of the cereal pathogen Fusarium graminearum, the adenylyl cyclase gene (FGSG_01234) was deleted by gene replacement (ΔFgac1). The ΔFgac1 mutant displayed a drastically reduced growth on agar medium which could be rescued by a cAMP analogon. Furthermore, the ΔFgac1 mutant was unable to produce perithecia on detached wheat nodes. However, artificial conditions like carrot agar allowed perithecia development. Pathogenicity towards wheat was drastically reduced in ΔFgac1 compared to the wild type. Point-inoculated spikelets showed only small lesions but no typical head blight disease symptoms. Fluorescence microscopy using dsRed-expressing strains revealed that the ΔFgac1 strain was unable to develop any complex infection structures like lobate appressoria and infection cushions. Instead, hyphal anastomosis occurs frequently. Scanning electron microscopy demonstrated the lack of fungal penetration. Hence, the formation of compound appressoria seems to be essential for infection of wheat. Hyphae on flower leaves produced huge amounts of new conidia, thereby circumventing the infection cycle. This abundant sporulation on wheat epidermis was not observed in wild type. Intriguingly, the Fgac1 deletion mutant was able to infect maize cobs as wild type, indicating that cAMP signaling is not important for maize infection. The ΔFgac1 mutant was unable to produce the mycotoxin deoxynivalenol both in vitro and during wheat infection. In this study, we show that cAMP signaling controls important cellular processes such as development of infection structures, pathogenicity, secondary metabolite production and sexual reproduction. For the

  13. Distinct PKC isoforms mediate the activation of cPLA2 and adenylyl cyclase by phorbol ester in RAW264.7 macrophages

    PubMed Central

    Lin, Wan-W; Chen, Bin C

    1998-01-01

    The modulatory effects of protein kinase C (PKC) on the activation of cytosolic phospholipase A2 (cPLA2) and adenylyl cyclase (AC) have recently been described. Since the signalling cascades associated with these events play critical roles in various functions of macrophages, we set out to investigate the crosstalk between PKC and the cPLA2 and AC pathways in mouse RAW 264.7 macrophages and to determine the involvement of individual PKC isoforms. The cPLA2 and AC pathways were studied by measuring the potentiation by the phorbol ester PMA of ionomycin-induced arachidonic acid (AA) release and prostagladin E1 (PGE1)-stimulated cyclic AMP production, respectively.PMA at 1 μM caused a significant increase in AA release both in the presence (371%) and absence (67%) of ionomycin induction, while exposure of RAW 264.7 cells to PMA increased PGE1 stimulation of cyclic AMP levels by 208%.Treatment of cells with staurosporine and Ro 31-8220 inhibited the PMA-induced potentiation of both AA release and cyclic AMP accumulation, while Go 6976 (an inhibitor of classical PKC isoforms) and LY 379196 (a specific inhibitor of PKCβ) inhibited the AA response but failed to affect the enhancement of the cyclic AMP response by PMA.Long term pretreatment of cells with PMA abolished the subsequent effect of PMA in potentiating AA release, but only inhibited the cyclic AMP response by 42%.Neither PD 98059, an inhibitor of MEK, nor genistein, an inhibitor of tyrosine kinases, had any effect on the ability of PMA to potentiate AA or cyclic AMP production.The potentiation of AA release, but not of cyclic AMP formation, by PMA was sensitive to inhibition by wortmannin. This effect was unrelated to the inhibition of PKC activation as deduced from the translocation of PKC activity to the cell membrane.Western blot analysis revealed the presence of eight PKC isoforms (α, βI, βII, δ, ε, μ λ and ξ) in RAW 264.7 cells and PMA was shown to induce the translocation of the α, βI, βII,

  14. Cyclic nucleotide–gated channels, calmodulin, adenylyl cyclase, and calcium/calmodulin-dependent protein kinase II are required for late, but not early, long-term memory formation in the honeybee

    PubMed Central

    Matsumoto, Yukihisa; Sandoz, Jean-Christophe; Devaud, Jean-Marc; Lormant, Flore; Mizunami, Makoto; Giurfa, Martin

    2014-01-01

    Memory is a dynamic process that allows encoding, storage, and retrieval of information acquired through individual experience. In the honeybee Apis mellifera, olfactory conditioning of the proboscis extension response (PER) has shown that besides short-term memory (STM) and mid-term memory (MTM), two phases of long-term memory (LTM) are formed upon multiple-trial conditioning: an early phase (e-LTM) which depends on translation from already available mRNA, and a late phase (l-LTM) which requires de novo transcription and translation. Here we combined olfactory PER conditioning and neuropharmacological inhibition and studied the involvement of the NO–cGMP pathway, and of specific molecules, such as cyclic nucleotide-gated channels (CNG), calmodulin (CaM), adenylyl cyclase (AC), and Ca2+/calmodulin-dependent protein kinase (CaMKII), in the formation of olfactory LTM in bees. We show that in addition to NO–cGMP and cAMP–PKA, CNG channels, CaM, AC, and CaMKII also participate in the formation of a l-LTM (72-h post-conditioning) that is specific for the learned odor. Importantly, the same molecules are dispensable for olfactory learning and for the formation of both MTM (in the minute and hour range) and e-LTM (24-h post-conditioning), thus suggesting that the signaling pathways leading to l-LTM or e-LTM involve different molecular actors. PMID:24741108

  15. Identification of a CAP (adenylyl-cyclase-associated protein) homologous gene in Lentinus edodes and its functional complementation of yeast CAP mutants.

    PubMed

    Zhou, G L; Miyazaki, Y; Nakagawa, T; Tanaka, K; Shishido, K; Matsuda, H; Kawamukai, M

    1998-04-01

    The adenylyl-cyclase-associated protein, CAP, was originally identified in yeasts as a protein that functions in both signal transduction and cytoskeletal organization. This paper reports the identification of a cDNA and genomic DNA that encodes a CAP homologue from the mushroom Lentinus edodes. The L. edodes cap gene contains eight introns and an ORF encoding a 518 amino acid protein. The L. edodes CAP is 35.5% and 40.9% identical at the amino acid level with Saccharomyces cerevisiae CAP and Schizosaccharomyces pombe CAP, respectively. The C-terminal domain shows greater homology (39-46% identity) with yeast CAPs than does the N-terminal domain (27-35% identity). Southern blotting and Northern blotting results suggest that L. edodes cap is a single-copy gene and uniformly expressed. Expression of the L. edodes CAP in both Schiz. pombe and Sacch. cerevisiae complemented defects associated with the loss of the C-terminal domain function of the endogenous CAP. By using a yeast two-hybrid assay, an interaction was demonstrated between the L. edodes CAP and Schiz. pombe actin. This result and the functional complementation test indicate that CAP from L. edodes has a conserved C-terminal domain function. PMID:9579081

  16. Adenylyl cyclase-associated protein 1 in metastasis of squamous cell carcinoma of the head and neck and non-small cell lung cancer

    NASA Astrophysics Data System (ADS)

    Kakurina, G. V.; Kolegova, E. S.; Cheremisina, O. V.; Zavyalov, A. A.; Shishkin, D. A.; Kondakova, I. V.; Choinzonov, E. L.

    2016-08-01

    Progression of tumors and metastasis in particular is one of the main reasons of the high mortality rate among cancer patients. The primary role in developing metastases plays cell locomotion which requires remodeling of the actin cytoskeleton. Form, dynamics, localization and mechanical properties of the actin cytoskeleton are regulated by a variety of actin-binding proteins, which include the adenylyl cyclase-associated protein 1 (CAP1). The study is devoted to the investigation of CAP1 level depending on the presence or absence of metastases in patients with squamous cell carcinoma of the head and neck (SCCHN) and non-small cell lung cancer (NSCLC). The results show the contribution of CAP1 to SCCHN and NSCLC progression. We detected the connection between the tissue protein CAP1 level and the stage of NSCLC and SCCHN disease. Also the levels of the CAP1 protein in tissues of primary tumors and metastases in lung cancer were different. Our data showed that CAP is important in the development of metastases, which suggests further perspectives in the study of this protein for projecting metastasis of NSCLC and SCCHN.

  17. A sperm-specific Na+/H+ exchanger (sNHE) is critical for expression and in vivo bicarbonate regulation of the soluble adenylyl cyclase (sAC)

    PubMed Central

    Wang, Dan; Hu, Jie; Bobulescu, I. Alexandru; Quill, Timothy A.; McLeroy, Paul; Moe, Orson W.; Garbers, David L.

    2007-01-01

    We previously identified a sperm-specific Na+/H+ exchanger (sNHE) principally localized to the flagellum. Disruption of the sNHE gene in mice resulted in absolute male infertility associated with a complete loss of sperm motility. Here, we show that the sNHE-null spermatozoa fail to develop the cAMP-dependent protein tyrosine phosphorylation that coincides with the functional maturation occurring upon incubation in capacitating conditions in vitro. Both the sperm motility defect and the lack of induced protein tyrosine phosphorylation are rescued by the addition of cell-permeable cAMP analogs, suggesting that cAMP metabolism is impaired in spermatozoa lacking sNHE. Our analyses of the bicarbonate-dependent soluble adenylyl cyclase (sAC) signaling pathway in sNHE-null sperm cells reveal that sNHE is required for the expression of full-length sAC, and that it is important for the bicarbonate stimulation of sAC activity in spermatozoa. Furthermore, both codependent expression and coimmunoprecipitation experiments indicate that sNHE and sAC associate with each other. Thus, these two proteins appear to be components of a signaling complex at the sperm flagellar plasma membrane. We propose that the formation of this complex efficiently modulates intracellular pH and bicarbonate levels through the rapid and effective control of sAC and sNHE activities to facilitate sperm motility regulation. PMID:17517652

  18. Regulator of G-protein signaling 6 (RGS6) promotes anxiety and depression by attenuating serotonin-mediated activation of the 5-HT(1A) receptor-adenylyl cyclase axis.

    PubMed

    Stewart, Adele; Maity, Biswanath; Wunsch, Amanda M; Meng, Fantao; Wu, Qi; Wemmie, John A; Fisher, Rory A

    2014-04-01

    Targeting serotonin (5-HT) bioavailability with selective 5-HT reuptake inhibitors (SSRIs) remains the most widely used treatment for mood disorders. However, their limited efficacy, delayed onset of action, and side effects restrict their clinical utility. Endogenous regulator of G-protein signaling (RGS) proteins have been implicated as key inhibitors of 5-HT(1A)Rs, whose activation is believed to underlie the beneficial effects of SSRIs, but the identity of the specific RGS proteins involved remains unknown. We identify RGS6 as the critical negative regulator of 5-HT(1A)R-dependent antidepressant actions. RGS6 is enriched in hippocampal and cortical neurons, 5-HT(1A)R-expressing cells implicated in mood disorders. RGS6(-/-) mice exhibit spontaneous anxiolytic and antidepressant behavior rapidly and completely reversibly by 5-HT(1A)R blockade. Effects of the SSRI fluvoxamine and 5-HT(1A)R agonist 8-OH-DPAT were also potentiated in RGS6(+/-) mice. The phenotype of RGS6(-/-) mice was associated with decreased CREB phosphorylation in the hippocampus and cortex, implicating enhanced Gα(i)-dependent adenylyl cyclase inhibition as a possible causative factor in the behavior observed in RGS6(-/-) animals. Our results demonstrate that by inhibiting serotonergic innervation of the cortical-limbic neuronal circuit, RGS6 exerts powerful anxiogenic and prodepressant actions. These findings indicate that RGS6 inhibition may represent a viable means to treat mood disorders or enhance the efficacy of serotonergic agents. PMID:24421401

  19. Anti-Cdc25 antibodies inhibit guanyl nucleotide-dependent adenylyl cyclase of Saccharomyces cerevisiae and cross-react with a 150-kilodalton mammalian protein.

    PubMed Central

    Gross, E; Marbach, I; Engelberg, D; Segal, M; Simchen, G; Levitzki, A

    1992-01-01

    The CDC25 gene product of the yeast Saccharomyces cerevisiae has been shown to be a positive regulator of the Ras protein. The high degree of homology between yeast RAS and the mammalian proto-oncogene ras suggests a possible resemblance between the mammalian regulator of Ras and the regulator of the yeast Ras (Cdc25). On the basis of this assumption, we have raised antibodies against the conserved C-terminal domain of the Cdc25 protein in order to identify its mammalian homologs. Anti-Cdc25 antibodies raised against a beta-galactosidase-Cdc25 fusion protein were purified by immunoaffinity chromatography and were shown by immunoblotting to specifically recognize the Cdc25 portion of the antigen and a truncated Cdc25 protein, also expressed in bacteria. These antibodies were shown both by immunoblotting and by immunoprecipitation to recognize the CDC25 gene product in wild-type strains and in strains overexpressing Cdc25. The anti-Cdc25 antibodies potently inhibited the guanyl nucleotide-dependent and, approximately 3-fold less potently, the Mn(2+)-dependent adenylyl cyclase activity in S. cerevisiae. The anti-Cdc25 antibodies do not inhibit cyclase activity in a strain harboring RAS2Val-19 and lacking the CDC25 gene product. These results support the view that Cdc25, Ras2, and Cdc35/Cyr1 proteins are associated in a complex. Using these antibodies, we were able to define the conditions to completely solubilize the Cdc25 protein. The results suggest that the Cdc25 protein is tightly associated with the membrane but is not an intrinsic membrane protein, since only EDTA at pH 12 can solubilize the protein. The anti-Cdc25 antibodies strongly cross-reacted with the C-terminal domain of the Cdc25 yeast homolog, Sdc25. Most interestingly, these antibodies also cross-reacted with mammalian proteins of approximately 150 kDa from various tissues of several species of animals. These interactions were specifically blocked by the beta-galactosidase-Cdc25 fusion protein. Images

  20. Cecal ligation and puncture sepsis is associated with attenuated expression of adenylyl cyclase 9 and increased miR142-3p.

    PubMed

    Risøe, Petter K; Ryg, Una; Wang, Yun Yong; Rutkovskiy, Arkady; Smedsrød, Bård; Valen, Guro; Dahle, Maria K

    2011-10-01

    The host inflammatory response in sepsis may be resolved by endogenous anti-inflammatory immune cell responses, avoiding fatal pathogenesis, organ injury, and death. The intracellular signaling mediator cyclic 3'5'-adenosine monophosphate is a potent modulator of inflammatory responses and initiates the polarization of immune cells in a direction that suppresses inflammatory activation. Cyclic 3'5'-adenosine monophosphate is enzymatically produced by adenylyl cyclases (ACs). The expression of ACs is previously shown to be reduced in rat organs after in vivo endotoxemia, concurrent with the progressing systemic inflammation. In the present study, tissue AC gene expression and regulation are explored in a rat model of cecal ligation and puncture (CLP) sepsis. Eighteen hours after CLP operation, expression of several AC isoforms in the liver, spleen, and kidney was reduced, significantly so for AC9 in all tissues. AC9 expression is regulated by the microRNA miR142-3p in T cells. When microRNA was extracted and amplified for miR142-3p expression, it was increasingly expressed 18 h after CLP. A correlation between increased miR142-3p and decreased AC9 expression was found in the liver, kidney, and spleen, and when hepatocytes, Kupffer cells (KCs), and liver sinusoidal endothelial cells were isolated after CLP, reduced AC expression and increased miR142-3p expression were found in KCs and liver sinusoidal endothelial cells. Transfecting a miR142-3p inhibitor probe in rat KCs abolished LPS-mediated AC9 inhibition in vitro. These results indicate that CLP leads to miR142-3p-mediated AC9 reduction in liver macrophages, which may further limit cyclic 3'5'-adenosine monophosphate signaling and the ability of macrophages to resolve the proinflammatory response. PMID:21701418

  1. Adenylyl cyclase 6 mediates the action of cyclic AMP-dependent secretagogues in mouse pancreatic exocrine cells via protein kinase A pathway activation

    PubMed Central

    Sabbatini, Maria E; D’Alecy, Louis; Lentz, Stephen I; Tang, Tong; Williams, John A

    2013-01-01

    Both secretin and vasoactive intestinal polypeptide (VIP) receptors are responsible for the activation of adenylyl cyclases (ACs), which increase intracellular cyclic AMP (cAMP) levels in the exocrine pancreas. There are nine membrane-associated isoforms, each with its own pattern of expression and regulation. In this study we sought to establish which AC isoforms play a regulatory role in pancreatic exocrine cells. Using RT-PCR, AC3, AC4, AC6, AC7 and AC9 were found to be expressed in the pancreas. AC3, AC4, AC6 and AC9 were expressed in both pancreatic acini and ducts, whereas AC7 was expressed only in pancreatic ducts. Based on known regulation by intracellular signals, selective inhibitors and stimulators were used to suggest which isoforms play an important role in the induction of cAMP formation. AC6 appeared to be an important isoform because protein kinase A (PKA), PKC and calcium all inhibited VIP-induced cAMP formation, whereas calcineurin or calmodulin did not modify the response to VIP. Mice with genetically deleted AC6 were studied and showed reduced cAMP formation and PKA activation in both isolated pancreatic acini and duct fragments. The absence of AC6 reduced cAMP-dependent secretagogue-stimulated amylase secretion, and abolished fluid secretion in both in vivo and isolated duct fragments. In conclusion, several AC isoforms are expressed in pancreatic acini and ducts. AC6 mediates a significant part of pancreatic amylase and fluid secretion in response to secretin, VIP and forskolin through cAMP/PKA pathway activation. PMID:23753526

  2. Adenylyl cyclase subtype 1 is essential for late-phase long term potentiation and spatial propagation of synaptic responses in the anterior cingulate cortex of adult mice.

    PubMed

    Chen, Tao; O'Den, Gerile; Song, Qian; Koga, Kohei; Zhang, Ming-Ming; Zhuo, Min

    2014-01-01

    Long-term potentiation (LTP) is a key cellular mechanism for pathological pain in the central nervous system. LTP contains at least two different phases: early-phase LTP (E-LTP) and late-phase LTP (L-LTP). Among several major cortical areas, the anterior cingulate cortex (ACC) is a critical brain region for pain perception and its related emotional changes. Periphery tissue or nerve injuries cause LTP of excitatory synaptic transmission in the ACC. Our previous studies have demonstrated that genetic deletion of calcium-stimulated adenylyl cyclase 1 (AC1) or pharmacological application of a selective AC1 inhibitor NB001 blocked E-LTP in the ACC. However, the effect of AC1 on L-LTP, which requires new protein synthesis and is important for the process of chronic pain, has not been investigated. Here we tested the effects of NB001 on the ACC L-LTP and found that bath application of NB001 (0.1 μM) totally blocked the induction of L-LTP and recruitment of cortical circuitry without affecting basal excitatory transmission. In contrast, gabapentin, a widely used analgesic drug for neuropathic pain, did not block the induction of L-LTP and circuitry recruitment even at a high concentration (100 μM). Gabapentin non-selectively decreased basal synaptic transmission. Our results provide strong evidence that the selective AC1 inhibitor NB001 can be used to inhibit pain-related cortical L-LTP without affecting basal synaptic transmission. It also provides basic mechanisms for possible side effects of gabapentin in the central nervous system and its ineffectiveness in some patients with neuropathic pain. PMID:25304256

  3. 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.

  4. Reconstitution of a fungal meroterpenoid biosynthesis reveals the involvement of a novel family of terpene cyclases.

    PubMed

    Itoh, Takayuki; Tokunaga, Kinya; Matsuda, Yudai; Fujii, Isao; Abe, Ikuro; Ebizuka, Yutaka; Kushiro, Tetsuo

    2010-10-01

    Meroterpenoids are hybrid natural products of both terpenoid and polyketide origin. We identified a biosynthetic gene cluster that is responsible for the production of the meroterpenoid pyripyropene in the fungus Aspergillus fumigatus through reconstituted biosynthesis of up to five steps in a heterologous fungal expression system. The cluster revealed a previously unknown terpene cyclase with an unusual sequence and protein primary structure. The wide occurrence of this sequence in other meroterpenoid and indole-diterpene biosynthetic gene clusters indicates the involvement of these enzymes in the biosynthesis of various terpenoid-bearing metabolites produced by fungi and bacteria. In addition, a novel polyketide synthase that incorporated nicotinyl-CoA as the starter unit and a prenyltransferase, similar to that in ubiquinone biosynthesis, was found to be involved in the pyripyropene biosynthesis. The successful production of a pyripyropene analogue illustrates the catalytic versatility of these enzymes for the production of novel analogues with useful biological activities. PMID:20861902

  5. Reconstitution of a fungal meroterpenoid biosynthesis reveals the involvement of a novel family of terpene cyclases

    NASA Astrophysics Data System (ADS)

    Itoh, Takayuki; Tokunaga, Kinya; Matsuda, Yudai; Fujii, Isao; Abe, Ikuro; Ebizuka, Yutaka; Kushiro, Tetsuo

    2010-10-01

    Meroterpenoids are hybrid natural products of both terpenoid and polyketide origin. We identified a biosynthetic gene cluster that is responsible for the production of the meroterpenoid pyripyropene in the fungus Aspergillus fumigatus through reconstituted biosynthesis of up to five steps in a heterologous fungal expression system. The cluster revealed a previously unknown terpene cyclase with an unusual sequence and protein primary structure. The wide occurrence of this sequence in other meroterpenoid and indole-diterpene biosynthetic gene clusters indicates the involvement of these enzymes in the biosynthesis of various terpenoid-bearing metabolites produced by fungi and bacteria. In addition, a novel polyketide synthase that incorporated nicotinyl-CoA as the starter unit and a prenyltransferase, similar to that in ubiquinone biosynthesis, was found to be involved in the pyripyropene biosynthesis. The successful production of a pyripyropene analogue illustrates the catalytic versatility of these enzymes for the production of novel analogues with useful biological activities.

  6. 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

  7. G-Protein-Coupled Receptor MrgD Is a Receptor for Angiotensin-(1-7) Involving Adenylyl Cyclase, cAMP, and Phosphokinase A.

    PubMed

    Tetzner, Anja; Gebolys, Kinga; Meinert, Christian; Klein, Sabine; Uhlich, Anja; Trebicka, Jonel; Villacañas, Óscar; Walther, Thomas

    2016-07-01

    Angiotensin (Ang)-(1-7) has cardiovascular protective effects and is the opponent of the often detrimental Ang II within the renin-angiotensin system. Although it is well accepted that the G-protein-coupled receptor Mas is a receptor for the heptapeptide, the lack in knowing initial signaling molecules stimulated by Ang-(1-7) prevented definitive characterization of ligand/receptor pharmacology as well as identification of further hypothesized receptors for the heptapeptide. The study aimed to identify a second messenger stimulated by Ang-(1-7) allowing confirmation as well as discovery of the heptapeptide's receptors. Ang-(1-7) elevates cAMP concentration in primary cells, such as endothelial or mesangial cells. Using cAMP as readout in receptor-transfected human embryonic kidney (HEK293) cells, we provided pharmacological proof that Mas is a functional receptor for Ang-(1-7). Moreover, we identified the G-protein-coupled receptor MrgD as a second receptor for Ang-(1-7). Consequently, the heptapeptide failed to increase cAMP concentration in primary mesangial cells with genetic deficiency in both Mas and MrgD Mice deficient in MrgD showed an impaired hemodynamic response after Ang-(1-7) administration. Furthermore, we excluded the Ang II type 2 receptor as a receptor for the heptapeptide but discovered that the Ang II type 2 blocker PD123319 can also block Mas and MrgD receptors. Our results lead to an expansion and partial revision of the renin-angiotensin system, by identifying a second receptor for Ang-(1-7), by excluding Ang II type 2 as a receptor for the heptapeptide, and by enforcing the revisit of such publications which concluded Ang II type 2 function by only using PD123319. PMID:27217404

  8. 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.

  9. 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.

  10. A comparative analysis of the sugar phosphate cyclase superfamily involved in primary and secondary metabolism.

    PubMed

    Wu, Xiumei; Flatt, Patricia M; Schlörke, Oliver; Zeeck, Axel; Dairi, Tohru; Mahmud, Taifo

    2007-01-22

    Sugar phosphate cyclases (SPCs) catalyze the cyclization of sugar phosphates to produce a variety of cyclitol intermediates that serve as the building blocks of many primary metabolites, for example, aromatic amino acids, and clinically relevant secondary metabolites, for example, aminocyclitol/aminoglycoside and ansamycin antibiotics. Feeding experiments with isotopically labeled cyclitols revealed that cetoniacytone A, a unique C(7)N-aminocyclitol antibiotic isolated from an insect endophytic Actinomyces sp., is derived from 2-epi-5-epi-valiolone, a product of SPC. By using heterologous probes from the 2-epi-5-epi-valiolone synthase class of SPCs, an SPC homologue gene, cetA, was isolated from the cetoniacytone producer. cetA is closely related to BE-orf9 found in the BE-40644 biosynthetic gene cluster from Actinoplanes sp. strain A40644. Recombinant expression of cetA and BE-orf9 and biochemical characterization of the gene products confirmed their function as 2-epi-5-epi-valiolone synthases. Further phylogenetic analysis of SPC sequences revealed a new clade of SPCs that might regulate the biosynthesis of a novel set of secondary metabolites. PMID:17195255

  11. Cloning and characterization of peanut allene oxide cyclase gene involved in salt-stressed responses.

    PubMed

    Liu, H H; Wang, Y G; Wang, S P; Li, H J

    2015-01-01

    In this study, the full-length cDNA encoding allene oxide cyclase (AhAOC) was isolated from peanut (Arachis hypogaea L.). The deduced amino acid sequence of AhAOC showed high homology with other plant AOCs. The transcript of AhAOC was found to be abundantly expressed in roots. Expression analysis demonstrated that AhAOC was induced by abscisic acid, methyl-jasmonic acid, salicylic acid, salinity, polyethylene glycol, and cold stresses, particularly by high salinity. Overexpression of AhAOC in rice increased root elongation and plant height compared with expression in control plants and conferred tolerance against salinity. Thus, the AhAOC gene may play an important role in increasing the expression of transcription factors (MYB2 and OsONAC045) and functional genes (DREB1F and LEA3) in transgenic rice under salt stress as well as improve stress tolerance through the accumulation of compatible solutes (proline and soluble sugar). The AhAOC gene is a potential resource for enhancing salt tolerance in crop species. PMID:25867379

  12. Activation of soluble guanylyl cyclase by the nitrovasodilator 3-morpholinosydnonimine involves formation of S-nitrosoglutathione.

    PubMed

    Schrammel, A; Pfeiffer, S; Schmidt, K; Koesling, D; Mayer, B

    1998-07-01

    Soluble guanylyl cyclase (sGC) is the major physiological target of sydnonimine-based vasodilators such as molsidomine. Decomposition of sydnonimines results in the stoichiometric formation of nitric oxide (NO) and superoxide (O2-), which rapidly react to form peroxynitrite. Inasmuch as sGC is activated by NO but not by peroxynitrite, we investigated the mechanisms underlying sGC activation by 3-morpholinosydnonimine (SIN-1). Stimulation of purified bovine lung sGC by SIN-1 was found to be strongly dependent on glutathione (GSH). By contrast, GSH did not affect sGC activation by NO released from 2,2-diethyl-1-nitroso-oxyhydrazine, indicating that NO/O2- released from SIN-1 converted GSH to an activator of sGC. High performance liquid chromatography identified this product as the thionitrite S-nitrosoglutathione. Further, the reaction product decomposed to release NO upon addition of Cu(NO3)2 in the presence of GSH. Activation of sGC was antagonized by the Cu(I)-specific chelator neocuproine, whereas the Cu(II)-selective drug cuprizone was less potent. Carbon dioxide (delivered as NaHCO3) antagonized S-nitrosation by peroxynitrite but not by SIN-1. Thus, NO/O2- released from SIN-1 mediates a CO2-insensitive conversion of GSH to S-nitrosoglutathione, a thionitrite that activates sGC via trace metal-catalyzed release of NO. These results may provide novel insights into the molecular mechanism underlying the nitrovasodilator action of SIN-1. PMID:9658207

  13. The Fumagillin Biosynthetic Gene Cluster in Aspergillus fumigatus Encodes a Cryptic Terpene Cyclase Involved in the Formation of β-trans-Bergamotene

    PubMed Central

    Lin, Hsiao-Ching; Chooi, Yit-Heng; Dhingra, Sourabh; Xu, Wei; Calvo, Ana M.; Tang, Yi

    2013-01-01

    Fumagillin 1 is a meroterpenoid from Aspergillus fumigatus that is known for its anti-angiogenic activity by binding to human methionine aminopeptidase 2. The genetic and molecular basis for biosynthesis of 1 had been an enigma despite the availability of the A. fumigatus genome sequence. Here, we reported the identification and verification of the fma gene cluster, followed by characterization of the polyketide synthase and acyltransferase involved in biosynthesis of the dioic acid portion of 1. More significantly, we uncovered the elusive β-trans-bergamotene synthase in A. fumigatus as a membrane-bound terpene cyclase. PMID:23488861

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. Abietadiene synthase from grand fir (Abies grandis). cDNA isolation, characterization, and bacterial expression of a bifunctional diterpene cyclase involved in resin acid biosynthesis.

    PubMed

    Vogel, B S; Wildung, M R; Vogel, G; Croteau, R

    1996-09-20

    (-)-Abietic acid, the principal diterpenoid resin acid of the wound-induced oleoresin secreted by grand fir (Abies grandis), is synthesized by the cyclization of geranylgeranyl diphosphate to (-)-abieta-7(8),13(14)-diene, followed by sequential three-step oxidation of the C-18 methyl group of the olefin to a carboxyl function. The enzyme catalyzing the cyclization reaction, abietadiene synthase, was purified from stems of wounded grand fir saplings and was digested with trypsin. Amino acid sequence information from the resulting peptides allowed construction of degenerate oligonucleotide primers, which amplified a 551-base pair fragment from a wound-induced stem cDNA library. This hybridization probe was then utilized to screen the wound-induced stem cDNA library, from which three cDNA clones were isolated that were functionally expressed in Escherichia coli, thereby confirming that a single protein catalyzes the complex, multistep cyclization of geranylgeranyl diphosphate to abietadiene. cDNA isolate Ac22.1, which yielded the highest expressed level of cyclase activity, was 2861 base pairs in length and encoded an 868-amino acid open reading frame that included a putative plastidial transit peptide. Deduced amino acid sequence comparison to other terpene cyclases revealed an amino-terminal region of the abietadiene synthase, which resembles those of enzymes that employ substrate double bond protonation to initiate the carbocationic reaction cascade, and a carboxyl-terminal region of the synthase, which resembles those of enzymes that employ ionization of the substrate allylic diphosphate ester function to initiate the cyclization reaction. This apparent fusion of segments of the two distinct terpenoid cyclase types is consistent with the novel mechanism of the bifunctional abietadiene synthase in catalyzing both protonation-initiated and ionization-initiated cyclization steps. PMID:8798524

  20. 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

  1. Absence of the cbb3 Terminal Oxidase Reveals an Active Oxygen-Dependent Cyclase Involved in Bacteriochlorophyll Biosynthesis in Rhodobacter sphaeroides

    PubMed Central

    Chen, Guangyu E.; Martin, Elizabeth C.; Hunter, C. Neil

    2016-01-01

    ABSTRACT The characteristic green color associated with chlorophyll pigments results from the formation of an isocyclic fifth ring on the tetrapyrrole macrocycle during the biosynthesis of these important molecules. This reaction is catalyzed by two unrelated cyclase enzymes employing different chemistries. Oxygenic phototrophs such as plants and cyanobacteria utilize an oxygen-dependent enzyme, the major component of which is a diiron protein named AcsF, while BchE, an oxygen-sensitive [4Fe-4S] cluster protein, dominates in phototrophs inhabiting anoxic environments, such as the purple phototrophic bacterium Rhodobacter sphaeroides. We identify a potential acsF in this organism and assay for activity of the encoded protein in a strain lacking bchE under various aeration regimes. Initially, cells lacking bchE did not demonstrate AcsF activity under any condition tested. However, on removal of a gene encoding a subunit of the cbb3-type respiratory terminal oxidase, cells cultured under regimes ranging from oxic to micro-oxic exhibited cyclase activity, confirming the activity of the oxygen-dependent enzyme in this model organism. Potential reasons for the utilization of an oxygen-dependent enzyme in anoxygenic phototrophs are discussed. IMPORTANCE The formation of the E ring of bacteriochlorophyll pigments is the least well characterized step in their biosynthesis, remaining enigmatic for over 60 years. Two unrelated enzymes catalyze this cyclization step; O2-dependent and O2-independent forms dominate in oxygenic and anoxygenic phototrophs, respectively. We uncover the activity of an O2-dependent enzyme in the anoxygenic purple phototrophic bacterium Rhodobacter sphaeroides, initially by inactivation of the high-affinity terminal respiratory oxidase, cytochrome cbb3. We propose that the O2-dependent form allows for the biosynthesis of a low level of bacteriochlorophyll under oxic conditions, so that a rapid initiation of photosynthetic processes is possible for

  2. HpDTC1, a Stress-Inducible Bifunctional Diterpene Cyclase Involved in Momilactone Biosynthesis, Functions in Chemical Defence in the Moss Hypnum plumaeforme

    PubMed Central

    Okada, Kazunori; Kawaide, Hiroshi; Miyamoto, Koji; Miyazaki, Sho; Kainuma, Ryosuke; Kimura, Honoka; Fujiwara, Kaoru; Natsume, Masahiro; Nojiri, Hideaki; Nakajima, Masatoshi; Yamane, Hisakazu; Hatano, Yuki; Nozaki, Hiroshi; Hayashi, Ken-ichiro

    2016-01-01

    Momilactones, which are diterpenoid phytoalexins with antimicrobial and allelopathic functions, have been found only in rice and the moss Hypnum plumaeforme. Although these two evolutionarily distinct plant species are thought to produce momilactones as a chemical defence, the momilactone biosynthetic pathway in H. plumaeforme has been unclear. Here, we identified a gene encoding syn-pimara-7,15-diene synthase (HpDTC1) responsible for the first step of momilactone biosynthesis in the moss. HpDTC1 is a bifunctional diterpene cyclase that catalyses a two-step cyclization reaction of geranylgeranyl diphosphate to syn-pimara-7,15-diene. HpDTC1 transcription was up-regulated in response to abiotic and biotic stress treatments. HpDTC1 promoter-GUS analysis in transgenic Physcomitrella patens showed similar transcriptional responses as H. plumaeforme to the stresses, suggesting that a common response system to stress exists in mosses. Jasmonic acid (JA), a potent signalling molecule for inducing plant defences, could not activate HpDTC1 expression. In contrast, 12-oxo-phytodienoic acid, an oxylipin precursor of JA in vascular plants, enhanced HpDTC1 expression and momilactone accumulation, implying that as-yet-unknown oxylipins could regulate momilactone biosynthesis in H. plumaeforme. These results demonstrate the existence of an evolutionarily conserved chemical defence system utilizing momilactones and suggest the molecular basis of the regulation for inductive production of momilactones in H. plumaeforme. PMID:27137939

  3. HpDTC1, a Stress-Inducible Bifunctional Diterpene Cyclase Involved in Momilactone Biosynthesis, Functions in Chemical Defence in the Moss Hypnum plumaeforme.

    PubMed

    Okada, Kazunori; Kawaide, Hiroshi; Miyamoto, Koji; Miyazaki, Sho; Kainuma, Ryosuke; Kimura, Honoka; Fujiwara, Kaoru; Natsume, Masahiro; Nojiri, Hideaki; Nakajima, Masatoshi; Yamane, Hisakazu; Hatano, Yuki; Nozaki, Hiroshi; Hayashi, Ken-Ichiro

    2016-01-01

    Momilactones, which are diterpenoid phytoalexins with antimicrobial and allelopathic functions, have been found only in rice and the moss Hypnum plumaeforme. Although these two evolutionarily distinct plant species are thought to produce momilactones as a chemical defence, the momilactone biosynthetic pathway in H. plumaeforme has been unclear. Here, we identified a gene encoding syn-pimara-7,15-diene synthase (HpDTC1) responsible for the first step of momilactone biosynthesis in the moss. HpDTC1 is a bifunctional diterpene cyclase that catalyses a two-step cyclization reaction of geranylgeranyl diphosphate to syn-pimara-7,15-diene. HpDTC1 transcription was up-regulated in response to abiotic and biotic stress treatments. HpDTC1 promoter-GUS analysis in transgenic Physcomitrella patens showed similar transcriptional responses as H. plumaeforme to the stresses, suggesting that a common response system to stress exists in mosses. Jasmonic acid (JA), a potent signalling molecule for inducing plant defences, could not activate HpDTC1 expression. In contrast, 12-oxo-phytodienoic acid, an oxylipin precursor of JA in vascular plants, enhanced HpDTC1 expression and momilactone accumulation, implying that as-yet-unknown oxylipins could regulate momilactone biosynthesis in H. plumaeforme. These results demonstrate the existence of an evolutionarily conserved chemical defence system utilizing momilactones and suggest the molecular basis of the regulation for inductive production of momilactones in H. plumaeforme. PMID:27137939

  4. Stimulation of Hippocampal Adenylyl Cyclase Activity Dissociates Memory Consolidation Processes for Response and Place Learning

    ERIC Educational Resources Information Center

    Martel, Guillaume; Millard, Annabelle; Jaffard, Robert; Guillou, Jean-Louis

    2006-01-01

    Procedural and declarative memory systems are postulated to interact in either a synergistic or a competitive manner, and memory consolidation appears to be a highly critical stage for this process. However, the precise cellular mechanisms subserving these interactions remain unknown. To investigate this issue, 24-h retention performances were…

  5. Regulation of NANC neural bronchoconstriction in vivo in the guinea-pig: involvement of nitric oxide, vasoactive intestinal peptide and soluble guanylyl cyclase.

    PubMed Central

    Lei, Y. H.; Barnes, P. J.; Rogers, D. F.

    1993-01-01

    1. We investigated the effect of the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) and the peptidase alpha-chymotrypsin on non-adrenergic, non-cholinergic (NANC neural) bronchoconstriction induced by electrical stimulation of the vagus nerves and by capsaicin in anaesthetized guinea-pigs in vivo using pulmonary insufflation pressure (PIP) as an index of bronchial tone. We also investigated the contribution of soluble guanylyl cyclase (SGC) to NANC neural relaxant mechanisms. 2. In the presence of atropine and propranolol, electrical stimulation of the vagus nerves induced a frequency-dependent increase in PIP above baseline of 67% at 2.5 Hz, of 128% at 5 Hz and of 230% at 10 Hz. L-NAME (1-50 mg kg-1, i.v.), at doses inducing increases in systemic blood pressure, dose-relatedly potentiated NANC bronchoconstriction. At 10 mg kg-1 i.v., L-NAME significantly (P < 0.05) potentiated NANC bronchoconstriction by a further 106% at 2.5 Hz and a further 147% at 5 Hz but did not potentiate the increase in PIP at 10 Hz. L-NAME did not induce bronchoconstriction in sham-stimulated control animals. D-NAME did not potentiate NANC bronchoconstriction. Raising systemic blood pressure with phenylephrine did not potentiate vagally-induced bronchoconstriction (2.5 Hz). 3. The NO precursor L-arginine, but not D-arginine, (100 mg kg-1, i.v.) significantly reversed the potentiation by L-NAME of NANC bronchoconstriction. L-Arginine alone significantly inhibited neurogenic bronchoconstriction at 10 Hz (by 74%); the inhibition of 25% at 2.5 Hz was not significant.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7679032

  6. 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

  7. 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

  8. Presynaptic kainate receptor facilitation of glutamate release involves protein kinase A in the rat hippocampus

    PubMed Central

    Rodríguez-Moreno, Antonio; Sihra, Talvinder S

    2004-01-01

    We have explored the mechanisms involved in the facilitation of glutamate release mediated by the activation of kainate receptors in the rat hippocampus using isolated nerve terminal (synaptosome) and slice preparations. In hippocampal nerve terminals, kainate (KA) produced an increase of glutamate release at concentrations of agonist ranging from 10 to 1000 μm. In hippocampal slices, KA at low nanomolar concentrations (20–50 nm) also produced an increase of evoked excitatory postsynaptic currents (eEPSCs) at mossy fibre–CA3 synapses. In both, synaptosomes and slices, the effect of KA was antagonized by CNQX, and persisted after pretreatment with a cocktail of antagonists for other receptors whose activation could potentially have produced facilitation of release. These data indicate that the facilitation of glutamate release observed is mediated by the activation of presynaptic glutamate receptors of the kainate type. Mechanistically, the observed effects of KA appear to be the same in synaptosomal and slice preparations. Thus, the effect of KA on glutamate release and mossy fibre–CA3 synaptic transmission was occluded by the stimulation of adenylyl cyclase by forskolin and suppressed by the inhibition of protein kinase A by H-89 or Rp-Br-cAMP. We conclude that kainate receptors present at presynaptic terminals in the rat hippocampus mediate the facilitation of glutamate release through a mechanism involving the activation of an adenylyl cyclase–second messenger cAMP–protein kinase A signalling cascade. PMID:15107475

  9. 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.

  10. 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

  11. 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

  12. 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

  13. Apparent dopamine D1 and D2 receptors in the weaver mutant mouse: receptor binding and coupling to adenylyl cyclase.

    PubMed

    Dewar, K M; Paquet, M; Sequeira, A

    1999-01-01

    Weaver mutant mice have a selective degeneration of the nigrostriatal dopamine pathway arising between 7-21 days after birth. The goal of this study was to investigate the effects of this mutation on different parameters of the nigrostriatal and mesolimbic dopamine system: apparent D1 and D2 receptor binding sites as well as their signal transduction pathway. Using quantitative autoradiography of ligands for dopamine D1, D2 receptors and the dopamine uptake site, we found a significant loss in apparent D1 receptor binding sites throughout the neostriatum, significant increase of apparent D2 receptor binding in the dorsal aspect of the neostriatum, and almost complete loss of DA uptake sites in these regions of the weaver mouse. In contrast to the neostriatum, the density of dopamine receptors and uptake sites in the nucleus accumbens of the weaver mouse did not differ from controls. Despite alterations in the binding of apparent D1 and D2 receptors, there was no significant difference in either basal, DA stimulated or GTPgammaS stimulated cAMP production. These findings suggest the down-regulation of apparent D1 receptor binding sites reported in this model, probably does not reflect an important physiological mechanism through which these animals compensate for loss of dopamine innervation. PMID:10443552

  14. 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

  15. 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

  16. 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

  17. 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

  18. [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

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

    PubMed

    Bennett, V; Cuatrecasas, P

    1975-06-01

    cyclase activity of cultured melanoma cells indicates a half-time of about 36 hr. The rate of exponentially increasing activity and extent of enzyme activation are related to the number of bound toxin molecules according to a Langmuir adsorption isotherm and are half-maximal when about 2000 molecules of toxin are bound per cell. It is proposed that initially cholera toxin binds ineffectively, but that it is converted to an active form during the lag phase. This process may involve lateral motion of toxin-GM1 ganglioside complex within the plane of the membrane. The kinetics of adenylate cyclase activation are consistent with the possibility that during the exponential phase a bimolecular association is proceeding between the active form of the cholera toxin and some other membrane component. The possibility is considered that the cholera toxin molecule may bind directly to adenylate cyclase. These considerations may prove useful in understanding the possible interactions of active hormone-receptor complexes with adenylate cyclase in cell membranes. PMID:805248

  20. Adenylylation of mycobacterial Glnk (PII) protein is induced by nitrogen limitation

    PubMed Central

    Williams, Kerstin J.; Bennett, Mark H.; Barton, Geraint R.; Jenkins, Victoria A.; Robertson, Brian D.

    2013-01-01

    Summary PII proteins are pivotal regulators of nitrogen metabolism in most prokaryotes, controlling the activities of many targets, including nitrogen assimilation enzymes, two component regulatory systems and ammonium transport proteins. Escherichia coli contains two PII-like proteins, PII (product of glnB) and GlnK, both of which are uridylylated under nitrogen limitation at a conserved Tyrosine-51 residue by GlnD (a uridylyl transferase). PII-uridylylation in E. coli controls glutamine synthetase (GS) adenylylation by GlnE and mediates the NtrB/C transcriptomic response. Mycobacteria contain only one PII protein (GlnK) which in environmental Actinomycetales is adenylylated by GlnD under nitrogen limitation. However in mycobacteria, neither the type of GlnK (PII) covalent modification nor its precise role under nitrogen limitation is known. In this study, we used LC-Tandem MS to analyse the modification state of mycobacterial GlnK (PII), and demonstrate that during nitrogen limitation GlnK from both non-pathogenic Mycobacterium smegmatis and pathogenic Mycobacterium tuberculosis is adenylylated at the Tyrosine-51 residue; we also show that GlnD is the adenylyl transferase enzyme responsible. Further analysis shows that in contrast to E. coli, GlnK (PII) adenylylation in M. tuberculosis does not regulate GS adenylylation, nor does it mediate the transcriptomic response to nitrogen limitation. PMID:23352854

  1. 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

  2. Glutamine Synthetase Regulation, Adenylylation State, and Strain Specificity Analyzed by Polyacrylamide Gel Electrophoresis

    PubMed Central

    Bender, Robert A.; Streicher, Stanley L.

    1979-01-01

    We used polyacrylamide gel electrophoresis to examine the regulation and adenylylation states of glutamine synthetases (GSs) from Escherichia coli (GSE) and Klebsiella aerogenes (GSK). In gels containing sodium dodecyl sulfate (SDS), we found that GSK had a mobility which differed significantly from that of GSE. In addition, for both GSK and GSE, adenylylated subunits (GSK-adenosine 5′-monophosphate [AMP] and GSE-AMP) had lesser mobilities in SDS gels than did the corresponding non-adenylylated subunits. The order of mobilities was GSK-AMP < GSK < GSE-AMP < GSE. We were able to detect these mobility differences with purified and partially purified preparations of GS, crude cell extracts, and whole cell lysates. SDS gel electrophoresis thus provided a means of estimating the adenylylation state and the quantity of GS present independent of enzymatic activity measurements and of determining the strain origin. Using SDS gels, we showed that: (i) the constitutively produced GS in strains carrying the glnA4 allele was mostly adenylylated, (ii) the GS-like polypeptide produced by strains carrying the glnA51 allele was indistinguishable from wild-type GSK, and (iii) strains carrying the glnA10 allele contained no polypeptide having the mobility of GSK or GSK-AMP. Using native polyacrylamide gels, we detected the increased amount of dodecameric GS present in cells grown under nitrogen limitation compared with cells grown under conditions of nitrogen excess. In native gels there was neither a significant difference in the mobilities of adenylylated and non-adenylylated GSs nor a GS-like protein in cells carrying the glnA10 allele. Images PMID:33958

  3. 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

  4. 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

  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. 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

  7. 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

  8. 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

  9. Cobinamides Are Novel Coactivators of Nitric Oxide Receptor That Target Soluble Guanylyl Cyclase Catalytic Domain

    PubMed Central

    Sharina, Iraida; Sobolevsky, Michael; Doursout, Marie-Francoise; Gryko, Dorota

    2012-01-01

    Soluble guanylyl cyclase (sGC), a ubiquitously expressed heme-containing receptor for nitric oxide (NO), is a key mediator of NO-dependent processes. In addition to NO, a number of synthetic compounds that target the heme-binding region of sGC and activate it in a NO-independent fashion have been described. We report here that dicyanocobinamide (CN2-Cbi), a naturally occurring intermediate of vitamin B12 synthesis, acts as a sGC coactivator both in vitro and in intact cells. Heme depletion or heme oxidation does not affect CN2-Cbi-dependent activation. Deletion mutagenesis demonstrates that CN2-Cbi targets a new regulatory site and functions though a novel mechanism of sGC activation. Unlike all known sGC regulators that target the N-terminal regulatory regions, CN2-Cbi directly targets the catalytic domain of sGC, resembling the effect of forskolin on adenylyl cyclases. CN2-Cbi synergistically enhances sGC activation by NO-independent regulators 3-(4-amino-5-cyclopropylpyrimidine-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine (BAY41-2272), 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino) methyl [benzoic]-acid (cinaciguat or BAY58-2667), and 5-chloro-2-(5-chloro-thiophene-2-sulfonylamino-N-(4-(morpholine-4-sulfonyl)-phenyl)-benzamide sodium salt (ataciguat or HMR-1766). BAY41-2272 and CN2-Cbi act reciprocally by decreasing the EC50 values. CN2-Cbi increases intracellular cGMP levels and displays vasorelaxing activity in phenylephrine-constricted rat aortic rings in an endothelium-independent manner. Both effects are synergistically potentiated by BAY41-2272. These studies uncover a new mode of sGC regulation and provide a new tool for understanding the mechanism of sGC activation and function. CN2-Cbi also offers new possibilities for its therapeutic applications in augmenting the effect of other sGC-targeting drugs. PMID:22171090

  10. The human D2 dopamine receptor synergizes with the A2A adenosine receptor to stimulate adenylyl cyclase in PC12 cells.

    PubMed

    Kudlacek, Oliver; Just, Herwig; Korkhov, Vladimir M; Vartian, Nina; Klinger, Markus; Pankevych, Halyna; Yang, Qiong; Nanoff, Christian; Freissmuth, Michael; Boehm, Stefan

    2003-07-01

    The adenosine A(2A) receptor and the dopamine D(2) receptor are prototypically coupled to G(s) and G(i)/G(o), respectively. In striatal intermediate spiny neurons, these receptors are colocalized in dendritic spines and act as mutual antagonists. This antagonism has been proposed to occur at the level of the receptors or of receptor-G protein coupling. We tested this model in PC12 cells which endogenously express A(2A) receptors. The human D(2) receptor was introduced into PC12 cells by stable transfection. A(2A)-agonist-mediated inhibition of D(2) agonist binding was absent in PC12 cell membranes but present in HEK293 cells transfected as a control. However, in the resulting PC12 cell lines, the action of the D(2) agonist quinpirole depended on the expression level of the D(2) receptor: at low and high receptor levels, the A(2A)-agonist-induced elevation of cAMP was enhanced and inhibited, respectively. Forskolin-stimulated cAMP formation was invariably inhibited by quinpirole. The effects of quinpirole were abolished by pretreatment with pertussis toxin. A(2A)-receptor-mediated cAMP formation was inhibited by other G(i)/G(o)-coupled receptors that were either endogenously present (P(2y12)-like receptor for ADP) or stably expressed after transfection (A(1) adenosine, metabotropic glutamate receptor-7A). Similarly, voltage activated Ca(2+) channels were inhibited by the endogenous P(2Y) receptor and by the heterologously expressed A(1) receptor but not by the D(2) receptor. These data indicate functional segregation of signaling components. Our observations are thus compatible with the proposed model that D(2) and A(2A) receptors are closely associated, but they highlight the fact that this interaction can also support synergism. PMID:12784121

  11. 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

  12. A Novel Link between Fic (Filamentation Induced by cAMP)-mediated Adenylylation/AMPylation and the Unfolded Protein Response*

    PubMed Central

    Sanyal, Anwesha; Chen, Andy J.; Nakayasu, Ernesto S.; Lazar, Cheri S.; Zbornik, Erica A.; Worby, Carolyn A.; Koller, Antonius; Mattoo, Seema

    2015-01-01

    The maintenance of endoplasmic reticulum (ER) homeostasis is a critical aspect of determining cell fate and requires a properly functioning unfolded protein response (UPR). We have discovered a previously unknown role of a post-translational modification termed adenylylation/AMPylation in regulating signal transduction events during UPR induction. A family of enzymes, defined by the presence of a Fic (filamentation induced by cAMP) domain, catalyzes this adenylylation reaction. The human genome encodes a single Fic protein, called HYPE (Huntingtin yeast interacting protein E), with adenylyltransferase activity but unknown physiological target(s). Here, we demonstrate that HYPE localizes to the lumen of the endoplasmic reticulum via its hydrophobic N terminus and adenylylates the ER molecular chaperone, BiP, at Ser-365 and Thr-366. BiP functions as a sentinel for protein misfolding and maintains ER homeostasis. We found that adenylylation enhances BiP's ATPase activity, which is required for refolding misfolded proteins while coping with ER stress. Accordingly, HYPE expression levels increase upon stress. Furthermore, siRNA-mediated knockdown of HYPE prevents the induction of an unfolded protein response. Thus, we identify HYPE as a new UPR regulator and provide the first functional data for Fic-mediated adenylylation in mammalian signaling. PMID:25601083

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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

  20. Nicotinamide Mononucleotide Adenylyl Transferase 2: A Promising Diagnostic and Therapeutic Target for Colorectal Cancer

    PubMed Central

    Cui, Chunhui; Qi, Jia; Deng, Quanwen; Chen, Rihong; Zhai, Duanyang; Yu, Jinlong

    2016-01-01

    Colorectal cancer (CRC) is one of the most common cancers all over the world. It is essential to search for more effective diagnostic and therapeutic methods for CRC. Abnormal nicotinamide adenine dinucleotide (NAD) metabolism has been considered as a characteristic of cancer cells. In this study, nicotinamide mononucleotide adenylyl transferases (NMNATs) as well as p53-mediated cancer signaling pathways were investigated in patients with colorectal cancer. The CRC tissues and adjacent normal tissues were obtained from 95 untreated colorectal cancer patients and were stained for expression of nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) and p53. The survival rate was analyzed by the Kaplan-Meier method and the log-rank test. The multivariate Cox proportional hazard regression analysis was conducted as well. Our data demonstrated that expression of NMNAT2 and p53 was significantly higher in CRC tissues, while NMNAT2 expression is in correlation with the invasive depth of tumors and TNM stage. Significant positive correlation was found between the expression of NMNAT2 and the expression of p53. However, NMNAT2 expression was not a statistically significant prognostic factor for overall survival. In conclusion, our results indicated that NMNAT2 might participate in tumorigenesis of CRC in a p53-dependent manner and NMNAT2 expression might be a potential therapeutic target for CRC. PMID:27218101

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. Adenylyl cylases 1 and 8 mediate select striatal-dependent behaviors and sensitivity to ethanol stimulation in the adolescent period following acute neonatal ethanol exposure.

    PubMed

    Susick, Laura L; Lowing, Jennifer L; Bosse, Kelly E; Hildebrandt, Clara C; Chrumka, Alexandria C; Conti, Alana C

    2014-08-01

    Neonatal alcohol exposure in rodents causes dramatic neurodegenerative effects throughout the developing nervous system, particularly in the striatum, acutely after exposure. These acute neurodegenerative effects are augmented in mice lacking adenylyl cyclases 1 and 8 (AC1/8) as neonatal mice with a genetic deletion of both AC isoforms (DKO) have increased vulnerability to ethanol-induced striatal neurotoxicity compared to wild type (WT) controls. While neonatal ethanol exposure is known to negatively impact cognitive behaviors, such as executive functioning and working memory in adolescent and adult animals, the threshold of ethanol exposure required to impinge upon developmental behaviors in mice has not been extensively examined. Therefore, the purpose of this study was to determine the behavioral effects of neonatal ethanol exposure using various striatal-dependent developmental benchmarks and to assess the impact of AC1/8 deletion on this developmental progression. WT and DKO mice were treated with 2.5 g/kg ethanol or saline on postnatal day (P)6 and later subjected to the wire suspension, negative geotaxis, postural reflex, grid hang, tail suspension and accelerating rotarod tests at various time points. At P30, mice were evaluated for their hypnotic responses to 4.0 g/kg ethanol by using the loss of righting reflex assay and ethanol-induced stimulation of locomotor activity after 2.0 g/kg ethanol. Ethanol exposure significantly impaired DKO performance in the negative geotaxis test while genetic deletion of AC1/8 alone increased grid hang time and decreased immobility time in the tail suspension test with a concomitant increase in hindlimb clasping behavior. Locomotor stimulation was significantly increased in animals that received ethanol as neonates, peaking significantly in ethanol-treated DKO mice compared to ethanol-treated WT controls, while sedation duration following high-dose ethanol challenge was unaffected. These data indicate that the

  8. 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

  9. 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

  10. 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

  11. A Rhodopsin-Guanylyl Cyclase Gene Fusion Functions in Visual Perception in a Fungus

    PubMed Central

    Avelar, Gabriela M.; Schumacher, Robert I.; Zaini, Paulo A.; Leonard, Guy; Richards, Thomas A.; Gomes, Suely L.

    2014-01-01

    Summary Sensing light is the fundamental property of visual systems, with vision in animals being based almost exclusively on opsin photopigments [1]. Rhodopsin also acts as a photoreceptor linked to phototaxis in green algae [2, 3] and has been implicated by chemical means as a light sensor in the flagellated swimming zoospores of the fungus Allomyces reticulatus [4]; however, the signaling mechanism in these fungi remains unknown. Here we use a combination of genome sequencing and molecular inhibition experiments with light-sensing phenotype studies to examine the signaling pathway involved in visual perception in the closely related fungus Blastocladiella emersonii. Our data show that in these fungi, light perception is accomplished by the function of a novel gene fusion (BeGC1) of a type I (microbial) rhodopsin domain and guanylyl cyclase catalytic domain. Photobleaching of rhodopsin function prevents accumulation of cGMP levels and phototaxis of fungal zoospores exposed to green light, whereas inhibition of guanylyl cyclase activity negatively affects fungal phototaxis. Immunofluorescence microscopy localizes the BeGC1 protein to the external surface of the zoospore eyespot positioned close to the base of the swimming flagellum [4, 5], demonstrating this is a photoreceptive organelle composed of lipid droplets. Taken together, these data indicate that Blastocladiomycota fungi have a cGMP signaling pathway involved in phototaxis similar to the vertebrate vision-signaling cascade but composed of protein domain components arranged as a novel gene fusion architecture and of distant evolutionary ancestry to type II rhodopsins of animals. PMID:24835457

  12. A rhodopsin-guanylyl cyclase gene fusion functions in visual perception in a fungus.

    PubMed

    Avelar, Gabriela M; Schumacher, Robert I; Zaini, Paulo A; Leonard, Guy; Richards, Thomas A; Gomes, Suely L

    2014-06-01

    Sensing light is the fundamental property of visual systems, with vision in animals being based almost exclusively on opsin photopigments [1]. Rhodopsin also acts as a photoreceptor linked to phototaxis in green algae [2, 3] and has been implicated by chemical means as a light sensor in the flagellated swimming zoospores of the fungus Allomyces reticulatus [4]; however, the signaling mechanism in these fungi remains unknown. Here we use a combination of genome sequencing and molecular inhibition experiments with light-sensing phenotype studies to examine the signaling pathway involved in visual perception in the closely related fungus Blastocladiella emersonii. Our data show that in these fungi, light perception is accomplished by the function of a novel gene fusion (BeGC1) of a type I (microbial) rhodopsin domain and guanylyl cyclase catalytic domain. Photobleaching of rhodopsin function prevents accumulation of cGMP levels and phototaxis of fungal zoospores exposed to green light, whereas inhibition of guanylyl cyclase activity negatively affects fungal phototaxis. Immunofluorescence microscopy localizes the BeGC1 protein to the external surface of the zoospore eyespot positioned close to the base of the swimming flagellum [4, 5], demonstrating this is a photoreceptive organelle composed of lipid droplets. Taken together, these data indicate that Blastocladiomycota fungi have a cGMP signaling pathway involved in phototaxis similar to the vertebrate vision-signaling cascade but composed of protein domain components arranged as a novel gene fusion architecture and of distant evolutionary ancestry to type II rhodopsins of animals. PMID:24835457

  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. 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.

  15. Adenylylation of Tyr77 stabilizes Rab1b GTPase in an active state: A molecular dynamics simulation analysis

    PubMed Central

    Luitz, Manuel P.; Bomblies, Rainer; Ramcke, Evelyn; Itzen, Aymelt; Zacharias, Martin

    2016-01-01

    The pathogenic pathway of Legionella pneumophila exploits the intercellular vesicle transport system via the posttranslational attachment of adenosine monophosphate (AMP) to the Tyr77 sidechain of human Ras like GTPase Rab1b. The modification, termed adenylylation, is performed by the bacterial enzyme DrrA/SidM, however the effect on conformational properties of the molecular switch mechanism of Rab1b remained unresolved. In this study we find that the adenylylation of Tyr77 stabilizes the active Rab1b state by locking the switch in the active signaling conformation independent of bound GTP or GDP and that electrostatic interactions due to the additional negative charge in the switch region make significant contributions. The stacking interaction between adenine and Phe45 however, seems to have only minor influence on this stabilisation. The results may also have implications for the mechanistic understanding of conformational switching in other signaling proteins. PMID:26818796

  16. Functional analysis of allene oxide cyclase, MpAOC, in the liverwort Marchantia polymorpha.

    PubMed

    Yamamoto, Yusuke; Ohshika, Jun; Takahashi, Tomohiro; Ishizaki, Kimitsune; Kohchi, Takayuki; Matusuura, Hideyuki; Takahashi, Kosaku

    2015-08-01

    12-Oxo-phytodienoic acid (OPDA) is an intermediate in jasmonic acid (JA) biosynthesis. OPDA exerts JA-dependent and JA-independent biological effects; therefore, it is considered a signaling molecule in flowering plants. OPDA is induced by bacterial infection and wounding and inhibits growth in the moss Physcomitrella patens. The functions of OPDA and allene oxide cyclase (AOC) in the liverwort Marchantia polymorpha were explored, which represents the most basal lineage of extant land plants. The analysis of OPDA showed that it is present in M. polymorpha and is increased by wounding. OPDA has been suggested to be involved in the response to environmental stresses. Moreover, OPDA showed growth inhibitory activity in M. polymorpha. Nonetheless JA in M. polymorpha was not found in this study. AOC synthesizes OPDA from an unstable allene oxide. A database search of the M. polymorpha genome identified only a putative gene encoding allene oxide cyclase (MpAOC). Recombinant MpAOC showed AOC activity similar to that in flowering plants. MpAOC was localized to chloroplasts, as in flowering plants. Expression of MpAOC was induced by wounding and OPDA treatment, and positive feedback regulation of OPDA was demonstrated in M. polymorpha. Overexpression of MpAOC increased the endogenous OPDA level and suppressed growth in M. polymorpha. These results indicate the role of OPDA as a signaling molecule regulating growth and the response to wounding in the liverwort M. polymorpha. PMID:25892411

  17. 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 signaling. A structural comparison reveals resemblance of the oligomeric states to assemblies of GAF domains, widely used regulatory domains in signaling molecules conserved from archaea to mammals, suggesting a similar mechanism of regulation. PMID:18366254

  18. Identification of guanylate cyclases and related signaling proteins in sperm tail from sea stars by mass spectrometry.

    PubMed

    Nakachi, Mia; Matsumoto, Midori; Terry, Philip M; Cerny, Ronald L; Moriyama, Hideaki

    2008-01-01

    Marine invertebrates employ external fertilization to take the advantages of sexual reproduction as one of excellent survival strategies. To prevent mismatching, successful fertilization can be made only after going though strictly defined steps in the fertilization. In sea stars, the fertilization process starts with the chemotaxis of sperm followed by hyperactivation of sperm upon arriving onto the egg coat, and then sperm penetrate to the egg coat before achieving the fusion. To investigate whether the initiation of chemotaxis and the following signaling has species specificity, we conducted comparative studies in the protein level among sea stars, Asterias amurensis, A. forbesi, and Asterina pectinifera. Since transcription of messenger ribonucleic acid (mRNA) has been suppressed in gamete, the roles of sperm proteins during the fertilization cannot be investigated by examining the mRNA profile. Therefore, proteomics analysis by mass spectrometry was used in this study. In sea stars, upon receiving asteroidal sperm-activating peptide (asterosap), the receptor membrane-bound guanylate cyclases in the sperm tail trigger sperm chemotaxis. We confirmed the presence of membrane-bound guanylate cyclases in the three sea star species, and they all had the same structural domains including the extracellular domain, kinase-like domain, and guanylate cyclase domain. The majority of peptides recovered were from alpha-helices distributed on the solvent side of the protein. More peptides were recovered from the intracellular domains. The transmembrane domain has not been recovered. The functions of the receptors seemed to be conserved among the species. Furthermore, we identified proteins that may be involved in the guanylate cyclase-triggered signaling pathway. PMID:18461395

  19. Identification of terminal adenylyl transferase activity of the poliovirus polymerase 3Dpol.

    PubMed Central

    Neufeld, K L; Galarza, J M; Richards, O C; Summers, D F; Ehrenfeld, E

    1994-01-01

    A terminal adenylyl transferase (TATase) activity has been identified in preparations of purified poliovirus RNA-dependent RNA polymerase (3Dpol). Highly purified 3Dpol is capable of adding [32P]AMP to the 3' ends of chemically synthesized 12-nucleotide (nt)-long RNAs. The purified 52-kDa polypeptide, isolated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renatured, retained the TATase activity. Two 3Dpol mutants, purified from Escherichia coli expression systems, displayed no detectable polymerase activity and were unable to catalyze TATase activity. Likewise, extracts from the parental E. coli strain that harbored no expression plasmid were unable to catalyze formation of the TATase products. With the RNA oligonucleotide 5'-CCUGCUUUUGCA-3' used as an acceptor, the products formed by wild-type 3Dpol were 9 and 18 nt longer than the 12-nt oligomer. GTP, CTP, and UTP did not serve as substrates for transfer to this RNA, either by themselves or when all deoxynucleoside triphosphates were present in the reaction. Results from kinetic and stoichiometric analyses suggest that the reaction is catalytic and shows substrate and enzyme dependence. The 3'-terminal 13 nt of poliovirus minus-strand RNA also served as an acceptor for TATase activity, raising the possibility that this activity functions in poliovirus RNA replication. The efficiency of utilization and the nature of the products formed during the reaction were dependent on the acceptor RNA. Images PMID:8057462

  20. 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

  1. 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

  2. Regulation of poly(ADP-ribose) polymerase 1 activity by the phosphorylation state of the nuclear NAD biosynthetic enzyme NMN adenylyl transferase 1

    PubMed Central

    Berger, Felicitas; Lau, Corinna; Ziegler, Mathias

    2007-01-01

    Nuclear NAD+ metabolism constitutes a major component of signaling pathways. It includes NAD+-dependent protein deacetylation by members of the Sir2 family and protein modification by poly(ADP-ribose) polymerase 1 (PARP-1). PARP-1 has emerged as an important mediator of processes involving DNA rearrangements. High-affinity binding to breaks in DNA activates PARP-1, which attaches poly(ADP-ribose) (PAR) to target proteins. NMN adenylyl transferases (NMNATs) catalyze the final step of NAD+ biosynthesis. We report here that the nuclear isoform NMNAT-1 stimulates PARP-1 activity and binds to PAR. Its overexpression in HeLa cells promotes the relocation of apoptosis-inducing factor from the mitochondria to the nucleus, a process known to depend on poly(ADP-ribosyl)ation. Moreover, NMNAT-1 is subject to phosphorylation by protein kinase C, resulting in reduced binding to PAR. Mimicking phosphorylation, substitution of the target serine residue by aspartate precludes PAR binding and stimulation of PARP-1. We conclude that, depending on its state of phosphorylation, NMNAT-1 binds to activated, automodifying PARP-1 and thereby amplifies poly(ADP-ribosyl)ation. PMID:17360427

  3. 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

  4. 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.

  5. 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

  6. NO-independent stimulators and activators of soluble guanylate cyclase: discovery and therapeutic potential

    PubMed Central

    Evgenov, Oleg V.; Pacher, Pál; Schmidt, Peter M.; Haskó, György; Schmidt, Harald H. H. W.; Stasch, Johannes-Peter

    2008-01-01

    Soluble guanylate cyclase (sGC) is a key signal-transduction enzyme activated by nitric oxide (NO). Impaired bioavailability and/or responsiveness to endogenous NO has been implicated in the pathogenesis of cardiovascular and other diseases. Current therapies that involve the use of organic nitrates and other NO donors have limitations, including non-specific interactions of NO with various biomolecules, lack of response and the development of tolerance following prolonged administration. Compounds that activate sGC in an NO-independent manner might therefore provide considerable therapeutic advantages. Here we review the discovery, biochemistry, pharmacology and clinical potential of haem-dependent sGC stimulators (including YC-1, BAY 41-2272, BAY 41-8543, CFM-1571 and A-350619) and haem-independent sGC activators (including BAY 58-2667 and HMR-1766). PMID:16955067

  7. 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.

  8. Characterization of a Fungal Thioesterase Having Claisen Cyclase and Deacetylase Activities in Melanin Biosynthesis

    PubMed Central

    Vagstad, Anna L; Hill, Eric A; Labonte, Jason W; Townsend, Craig A

    2012-01-01

    Summary Melanins are a broad class of darkly-pigmented macromolecules formed by oxidative polymerization of phenolic monomers. In fungi, melanins are known virulence factors that contribute to pathogenicity. Their biosynthesis generally involves polymerization of 1,8-dihydroxynaphthalene via a 1,3,6,8- tetrahydroxynaphthalene (THN) precursor assembled by multidomain, nonreducing polyketide synthases. Multiple, convergent routes to THN have evolved in fungi. Parallel heptaketide and hexaketide pathways exist that utilize conventional C-terminal thioesterase/Claisen cyclase domains and separate side-chain deacylases. Here, in vitro characterization of Pks1 from Colletotrichum lagenarium establishes a true THN synthase with a bifunctional thioesterase (TE) catalyzing both cyclization and deacetylation of an enzyme-bound hexaketide substrate. Chimeric TE domains were generated by swapping lid regions of active sites between classes of melanin TEs to gain insight into this unprecedented catalysis of carbon–carbon bond making and breaking by an α/β-hydrolase fold enzyme. PMID:23261597

  9. 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

  10. Identification of Arabidopsis Cyclase-associated Protein 1 as the First Nucleotide Exchange Factor for Plant Actin

    PubMed Central

    Chaudhry, Faisal; Guérin, Christophe; von Witsch, Matthias

    2007-01-01

    The actin cytoskeleton powers organelle movements, orchestrates responses to abiotic stresses, and generates an amazing array of cell shapes. Underpinning these diverse functions of the actin cytoskeleton are several dozen accessory proteins that coordinate actin filament dynamics and construct higher-order assemblies. Many actin-binding proteins from the plant kingdom have been characterized and their function is often surprisingly distinct from mammalian and fungal counterparts. The adenylyl cyclase-associated protein (CAP) has recently been shown to be an important regulator of actin dynamics in vivo and in vitro. The disruption of actin organization in cap mutant plants indicates defects in actin dynamics or the regulated assembly and disassembly of actin subunits into filaments. Current models for actin dynamics maintain that actin-depolymerizing factor (ADF)/cofilin removes ADP–actin subunits from filament ends and that profilin recharges these monomers with ATP by enhancing nucleotide exchange and delivery of subunits onto filament barbed ends. Plant profilins, however, lack the essential ability to stimulate nucleotide exchange on actin, suggesting that there might be a missing link yet to be discovered from plants. Here, we show that Arabidopsis thaliana CAP1 (AtCAP1) is an abundant cytoplasmic protein; it is present at a 1:3 M ratio with total actin in suspension cells. AtCAP1 has equivalent affinities for ADP– and ATP–monomeric actin (Kd ∼ 1.3 μM). Binding of AtCAP1 to ATP–actin monomers inhibits polymerization, consistent with AtCAP1 being an actin sequestering protein. However, we demonstrate that AtCAP1 is the first plant protein to increase the rate of nucleotide exchange on actin. Even in the presence of ADF/cofilin, AtCAP1 can recharge actin monomers and presumably provide a polymerizable pool of subunits to profilin for addition onto filament ends. In turnover assays, plant profilin, ADF, and CAP act cooperatively to promote flux of

  11. 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.

  12. Isolation and characterization of glutaminyl cyclases from Drosophila: evidence for enzyme forms with different subcellular localization.

    PubMed

    Schilling, Stephan; Lindner, Christiane; Koch, Birgit; Wermann, Michael; Rahfeld, Jens-Ulrich; von Bohlen, Alex; Rudolph, Thomas; Reuter, Gunter; Demuth, Hans-Ulrich

    2007-09-25

    Glutaminyl cyclases (QCs) present in plants and vertebrates catalyze the formation of pyroglutamic acid (pGlu) from N-terminal glutamine. Pyroglutamyl hormones also identified in invertebrates imply the involvement of QC activity during their posttranslational maturation. Database mining led to the identification of two genes in Drosophila, which putatively encode QCs, CG32412 (DromeQC) and CG5976 (isoDromeQC). Analysis of their primary structure suggests different subcellular localizations. While DromeQC appeared to be secreted due to an N-terminal signal peptide, isoDromeQC contains either an N-terminal mitochondrial targeting or a secretion signal due to generation of different transcripts from gene CG5976. According to the prediction, homologous expression of the corresponding cDNAs in S2 cells revealed either secreted protein in the medium or intracellular QC activity. Subcellular fractionation and immunochemistry support export of isoDromeQC into the mitochondrion. For enzymatic characterization, DromeQC and isoDromeQC were expressed heterologously in Pichia pastoris and Escherichia coli, respectively. Compared to mammalian QCs, the specificity constants were about 1 order of magnitude lower for most of the analyzed substrates. The pH dependence of the specificity constant was similar for both enzymes, indicating the necessity of an unprotonated substrate amino group and two protonated groups of the enzyme, resulting in an asymmetric bell-shaped characteristic. The determination of the metal content of DromeQC revealed equimolar protein-bound zinc. These results prove conserved enzymatic mechanisms between QCs from invertebrates and mammals. Drosophila is the first organism for which isoenzymes of glutaminyl cyclase have been isolated. The identification of a mitochondrial QC points toward yet undiscovered physiological functions of these enzymes. PMID:17722885

  13. Topological mimicry and epitope duplication in the guanylyl cyclase C receptor.

    PubMed Central

    Nandi, A.; Suguna, K.; Surolia, A.; Visweswariah, S. S.

    1998-01-01

    Guanylyl cyclase C (GCC) is the receptor for the gastrointestinal hormones, guanylin, and uroguanylin, in addition to the bacterial heat-stable enterotoxins, which are one of the major causes of watery diarrhea the world over. GCC is expressed in intestinal cells, colorectal tumor tissue and tumors originating from metastasis of the colorectal carcinoma. We have earlier generated a monoclonal antibody to human GCC, GCC:B10, which was useful for the immunohistochemical localization of the receptor in the rat intestine (Nandi A et al., 1997, J Cell Biochem 66:500-511), and identified its epitope to a 63-amino acid stretch in the intracellular domain of GCC. In view of the potential that this antibody has for the identification of colorectal tumors, we have characterized the epitope for GCC:B10 in this study. Overlapping peptide synthesis indicated that the epitope was contained in the sequence HIPPENIFPLE. This sequence was unique to GCC, and despite a short stretch of homology with serum amyloid protein and pertussis toxin, no cross reactivity was detected. The core epitope was delineated using a random hexameric phage display library, and two categories of sequences were identified, containing either a single, or two adjacent proline residues. No sequence identified by phage display was identical to the epitope present in GCC, indicating that phage sequences represented mimotopes of the native epitope. Alignment of these sequences with HIPPENIFPLE suggested duplication of the recognition motif, which was confirmed by peptide synthesis. These studies allowed us not only to define the requirements of epitope recognition by GCC:B10 monoclonal antibody, but also to describe a novel means of epitope recognition involving topological mimicry and probable duplication of the cognate epitope in the native guanylyl cyclase C receptor sequence. PMID:9792105

  14. 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

  15. 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

  16. Rex (encoded by DVU_0916) in Desulfovibrio vulgaris Hildenborough is a repressor of sulfate adenylyl transferase and is regulated by NADH.

    PubMed

    Christensen, G A; Zane, G M; Kazakov, A E; Li, X; Rodionov, D A; Novichkov, P S; Dubchak, I; Arkin, A P; Wall, J D

    2015-01-01

    Although the enzymes for dissimilatory sulfate reduction by microbes have been studied, the mechanisms for transcriptional regulation of the encoding genes remain unknown. In a number of bacteria the transcriptional regulator Rex has been shown to play a key role as a repressor of genes producing proteins involved in energy conversion. In the model sulfate-reducing microbe Desulfovibrio vulgaris Hildenborough, the gene DVU_0916 was observed to resemble other known Rex proteins. Therefore, the DVU_0916 protein has been predicted to be a transcriptional repressor of genes encoding proteins that function in the process of sulfate reduction in D. vulgaris Hildenborough. Examination of the deduced DVU_0916 protein identified two domains, one a winged helix DNA-binding domain common for transcription factors, and the other a Rossman fold that could potentially interact with pyridine nucleotides. A deletion of the putative rex gene was made in D. vulgaris Hildenborough, and transcript expression studies of sat, encoding sulfate adenylyl transferase, showed increased levels in the D. vulgaris Hildenborough Rex (RexDvH) mutant relative to the parental strain. The RexDvH-binding site upstream of sat was identified, confirming RexDvH to be a repressor of sat. We established in vitro that the presence of elevated NADH disrupted the interaction between RexDvH and DNA. Examination of the 5' transcriptional start site for the sat mRNA revealed two unique start sites, one for respiring cells that correlated with the RexDvH-binding site and a second for fermenting cells. Collectively, these data support the role of RexDvH as a transcription repressor for sat that senses the redox status of the cell. PMID:25313388

  17. 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

  18. A Wheat Allene Oxide Cyclase Gene Enhances Salinity Tolerance via Jasmonate Signaling1[C][W

    PubMed Central

    Zhao, Yang; Dong, Wei; Zhang, Naibo; Ai, Xinghui; Wang, Mengcheng; Huang, Zhigang; Xiao, Langtao; Xia, Guangmin

    2014-01-01

    One of the two branches of the α-linolenic acid metabolism pathway is catalyzed by 12-oxo-phytodienoic acid reductase I, and the other is involved in jasmonic acid (JA) synthesis. The former is known to be active in the response to salinity tolerance in wheat (Triticum aestivum), but the participation of the latter in this response has not been established as yet. Here, the salinity-responsive bread wheat gene TaAOC1, which encodes an allene oxide cyclase involved in the α-linolenic acid metabolism pathway, was constitutively expressed in both bread wheat and Arabidopsis (Arabidopsis thaliana). In both species, transgenic lines exhibited an enhanced level of tolerance to salinity. The transgenic plants accumulated a higher content of JA and developed shorter roots. Both the shortened roots and the salinity tolerance were abolished in a background lacking a functional AtMYC2, a key component of the JA and abscisic acid signaling pathway, but were still expressed in a background deficient with respect to abscisic acid synthesis. We provide the first evidence, to our knowledge, suggesting that JA is also involved in the plant salinity response and that the α-linolenic acid metabolism pathway has a regulatory role over this response. PMID:24326670

  19. 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

  20. Crystal Structure of the Signaling Helix Coiled-coil Domain of the b1 Subunit of the Soluble guanylyl Cyclase

    SciTech Connect

    Ma, X.; Beuve, A; van den Akker, F

    2010-01-01

    The soluble guanylyl cyclase (sGC) is a heterodimeric enzyme that, upon activation by nitric oxide, stimulates the production of the second messenger cGMP. Each sGC subunit harbor four domains three of which are used for heterodimerization: H-NOXA/H-NOBA domain, coiled-coil domain (CC), and catalytic guanylyl cyclase domain. The CC domain has previously been postulated to be part of a larger CC family termed the signaling helix (S-helix) family. Homodimers of sGC have also been observed but are not functionally active yet are likely transient awaiting their intended heterodimeric partner. To investigate the structure of the CC S-helix region, we crystallized and determined the structure of the CC domain of the sGC{beta}1 subunit comprising residues 348-409. The crystal structure was refined to 2.15 {angstrom} resolution. The CC structure of sGC{beta}1 revealed a tetrameric arrangement comprised of a dimer of CC dimers. Each monomer is comprised of a long a-helix, a turn near residue P399, and a short second a-helix. The CC structure also offers insights as to how sGC homodimers are not as stable as (functionally) active heterodimers via a possible role for inter-helix salt-bridge formation. The structure also yielded insights into the residues involved in dimerization. In addition, the CC region is also known to harbor a number of congenital and man-made mutations in both membrane and soluble guanylyl cyclases and those function-affecting mutations have been mapped onto the CC structure. This mutant analysis indicated an importance for not only certain dimerization residue positions, but also an important role for other faces of the CC dimer which might perhaps interact with adjacent domains. Our results also extend beyond guanylyl cyclases as the CC structure is, to our knowledge, the first S-helix structure and serves as a model for all S-helix containing family members.

  1. 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

  2. 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.

  3. 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

  4. Identification of a Diguanylate Cyclase and Its Role in Porphyromonas gingivalis Virulence

    PubMed Central

    Chaudhuri, Swarnava; Pratap, Siddharth; Paromov, Victor; Li, Zhijun; Mantri, Chinmay K.

    2014-01-01

    Porphyromonas gingivalis is a Gram-negative obligate anaerobic bacterium and is considered a keystone pathogen in the initiation of periodontitis, one of the most widespread infectious diseases. Bacterial bis-(3′-5′) cyclic GMP (cyclic di-GMP [c-di-GMP]) serves as a second messenger and is involved in modulating virulence factors in numerous bacteria. However, the role of this second messenger has not been investigated in P. gingivalis, mainly due to a lack of an annotation regarding diguanylate cyclases (DGCs) in this bacterium. Using bioinformatics tools, we found a protein, PGN_1932, containing a GGDEF domain. A deletion mutation in the pgn_1932 gene had a significant effect on the intracellular c-di-GMP level in P. gingivalis. Genetic analysis showed that expression of the fimA and rgpA genes, encoding the major protein subunit of fimbriae and an arginine-specific proteinase, respectively, was downregulated in the pgn_1932 mutant. Correspondingly, FimA protein production and the fimbrial display on the mutant were significantly reduced. Mutations in the pgn_1932 gene also had a significant impact on the adhesive and invasive capabilities of P. gingivalis, which are required for its pathogenicity. These findings provide evidence that the PGN_1932 protein is both responsible for synthesizing c-di-GMP and involved in biofilm formation and host cell invasion by P. gingivalis by controlling the expression and biosynthesis of FimA. PMID:24733094

  5. RNA splicing in regulation of Nitric Oxide receptor Soluble Guanylyl Cyclase

    PubMed Central

    Sharina, Iraida G.; Cote, Gilbert J.; Martin, Emil; Doursout, Marie-Francoise; Murad, Ferid

    2011-01-01

    Soluble guanylyl cyclase (sGC) is a key protein in the nitric oxide (NO)/-cGMP signaling pathway. sGC activity is involved in a number of important physiological processes including smooth muscle relaxation, neurotransmission and platelet aggregation and adhesion. Regulation of sGC expression and activity emerges as a crucial factor in control of sGC function in normal and pathological conditions. Recently accumulated evidence strongly indicates that the regulation of sGC expression is a complex process modulated on several levels including transcription, post-transcriptional regulation, translation and protein stability. Presently our understanding of mechanisms governing regulation of sGC expression remains very limited and awaits systematic investigation. Among other ways, the expression of sGC subunits is modulated at the levels of mRNA abundance and transcript diversity. In this review we summarize available information on different mechanisms (including transcriptional activation, mRNA stability and alternative splicing) involved in the modulation of mRNA levels of sGC subunits in response to various environmental clues. We also summarize and cross-reference the information on human sGC splice forms available in the literature and in genomic databases. This review highlights the fact that the study of the biological role and regulation of sGC splicing will bring new insights to our understanding of NO/cGMP biology. PMID:21867767

  6. 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

  7. Isolation and Characterization of a Lycopene ε-Cyclase Gene of Chlorella (Chromochloris) zofingiensis. Regulation of the Carotenogenic Pathway by Nitrogen and Light

    PubMed Central

    Cordero, Baldo F.; Couso, Inmaculada; Leon, Rosa; Rodriguez, Herminia; Vargas, Maria Angeles

    2012-01-01

    The isolation and characterization of the lycopene ε-cyclase gene from the green microalga Chlorella (Chromochloris) zofingiensis (Czlcy-e) was performed. This gene is involved in the formation of the carotenoids α-carotene and lutein. Czlcy-e gene encoded a polypeptide of 654 amino acids. A single copy of Czlcy-e was found in C. zofingiensis. Functional analysis by heterologous complementation in Escherichia coli showed the ability of this protein to convert lycopene to δ-carotene. In addition, the regulation of the carotenogenic pathway by light and nitrogen was also studied in C. zofingiensis. High irradiance stress did not increase mRNA levels of neither lycopene β-cyclase gene (lcy-b) nor lycopene ε-cyclase gene (lcy-e) as compared with low irradiance conditions, whereas the transcript levels of psy, pds, chyB and bkt genes were enhanced, nevertheless triggering the synthesis of the secondary carotenoids astaxanthin, canthaxanthin and zeaxanthin and decreasing the levels of the primary carotenoids α-carotene, lutein, violaxanthin and β-carotene. Nitrogen starvation per se enhanced mRNA levels of all genes considered, except lcy-e and pds, but did not trigger the synthesis of astaxanthin, canthaxanthin nor zeaxanthin. The combined effect of both high light and nitrogen starvation stresses enhanced significantly the accumulation of these carotenoids as well as the transcript levels of bkt gene, as compared with the effect of only high irradiance stress. PMID:23118722

  8. Prunetin signals via G-protein-coupled receptor, GPR30(GPER1): Stimulation of adenylyl cyclase and cAMP-mediated activation of MAPK signaling induces Runx2 expression in osteoblasts to promote bone regeneration.

    PubMed

    Khan, Kainat; Pal, Subhashis; Yadav, Manisha; Maurya, Rakesh; Trivedi, Arun Kumar; Sanyal, Sabyasachi; Chattopadhyay, Naibedya

    2015-12-01

    Prunetin is found in red clover and fruit of Prunus avium (red cherry). The effect of prunetin on osteoblast function, its mode of action and bone regeneration in vivo were investigated. Cultures of primary osteoblasts, osteoblastic cell line and HEK293T cells were used for various in vitro studies. Adult female rats received drill-hole injury at the femur diaphysis to assess the bone regenerative effect of prunetin. Prunetin at 10nM significantly (a) increased proliferation and differentiation of primary cultures of osteoblasts harvested from rats and (b) promoted formation of mineralized nodules by bone marrow stromal/osteoprogenitor cells. At this concentration, prunetin did not activate any of the two nuclear estrogen receptors (α and β). However, prunetin triggered signaling via a G-protein-coupled receptor, GPR30/GPER1, and enhanced cAMP levels in osteoblasts. G15, a selective GPR30 antagonist, abolished prunetin-induced increases in osteoblast proliferation, differentiation and intracellular cAMP. In osteoblasts, prunetin up-regulated runt-related transcription factor 2 (Runx2) protein through cAMP-dependent Erk/MAP kinase activation that ultimately resulted in the up-regulation of GPR30. Administration of prunetin at 0.25mg/kg given to rats stimulated bone regeneration at the site of drill hole and up-regulated Runx2 expression in the fractured callus and the effect was comparable to human parathyroid hormone, the only clinically used osteogenic therapy. We conclude that prunetin promotes osteoinduction in vivo and the mechanism is defined by signaling through GPR30 resulting in the up-regulation of the key osteogenic gene Runx2 that in turn up-regulates GPR30. PMID:26345541

  9. Cyclic Nucleotide-Gated Channels, Calmodulin, Adenylyl Cyclase, and Calcium/Calmodulin-Dependent Protein Kinase II Are Required for Late, but Not Early, Long-Term Memory Formation in the Honeybee

    ERIC Educational Resources Information Center

    Matsumoto, Yukihisa; Sandoz, Jean-Christophe; Devaud, Jean-Marc; Lormant, Flore; Mizunami, Makoto; Giurfa, Martin

    2014-01-01

    Memory is a dynamic process that allows encoding, storage, and retrieval of information acquired through individual experience. In the honeybee "Apis mellifera," olfactory conditioning of the proboscis extension response (PER) has shown that besides short-term memory (STM) and mid-term memory (MTM), two phases of long-term memory (LTM)…

  10. 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

  11. 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.

  12. 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

  13. Ectopic expression of guanylyl cyclase C in adenocarcinomas of the esophagus and stomach.

    PubMed

    Park, Jason; Schulz, Stephanie; Haaf, Janis; Kairys, John C; Waldman, Scott A

    2002-08-01

    Guanylyl cyclase C (GC-C), a receptor specifically expressed in cells originating from differentiated intestinal epithelium, is a marker and therapeutic target for colorectal cancer metastases. Intestinal metaplasia, in which epithelial cells assume histological and molecular characteristics of differentiated intestinal enterocytes, is a common precursor to adenocarcinomas of the esophagus and stomach. Thus, those tumors, tissues adjacent to them, and their associated regional lymph nodes were assessed for GC-C expression by reverse transcription coupled with the PCR. GC-C mRNA was detected in five of five and eight of nine esophageal and gastric adenocarcinomas, respectively. Also, GC-C mRNA was detected in three of five and six of seven tissues adjacent to, but not histologically involved in, esophageal and gastric adenocarcinomas, respectively, reflecting molecular changes associated with neoplastic transformation preceding histopathological changes. In contrast, three normal gastric specimens did not express GC-C. Furthermore, GC-C mRNA was detected in 1 of 1 lymph node containing tumor cells by histopathology from a patient with gastric adenocarcinoma and in 3 of 11 lymph nodes, all of which were free of tumor cells by histopathology, from a patient with a gastroesophageal junction tumor. This is the first demonstration that GC-C is ectopically expressed by primary and metastatic adenocarcinomas of the esophagus and stomach and suggests that GC-C may be a sensitive and specific clinical marker and target for adenocarcinomas of the upper gastrointestinal tract. PMID:12163327

  14. Multiple diguanylate cyclase-coordinated regulation of pyoverdine synthesis in Pseudomonas aeruginosa.

    PubMed

    Chen, Yicai; Yuan, Mingjun; Mohanty, Anee; Yam, Joey Kuok Hoong; Liu, Yang; Chua, Song Lin; Nielsen, Thomas E; Tolker-Nielsen, Tim; Givskov, Michael; Cao, Bin; Yang, Liang

    2015-06-01

    The nucleotide signalling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) plays an essential role in regulating microbial virulence and biofilm formation. C-di-GMP is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes. One intrinsic feature of c-di-GMP signalling is the abundance of DGCs and PDEs encoded by many bacterial species. It is unclear whether the different DGCs or PDEs coordinately establish the c-di-GMP regulation or function independently of each other. Here, we provide evidence that multiple DGCs are involved in regulation of c-di-GMP on synthesis of the major iron siderophore pyoverdine in Pseudomonas aeruginosa. Constitutive expression of the WspG or YedQ DGC in P. aeruginosa is able to induce its pyoverdine synthesis. Induction of pyoverdine synthesis by high intracellular c-di-GMP depends on the synthesis of exopolysaccharides and another two DGCs, SiaD and SadC. SiaD was found to boost the c-di-GMP synthesis together with constitutively expressing YedQ. The exopolysaccharides and the SiaD DGC were found to modulate the expression of the RsmY/RsmZ ncRNAs. Induction of the RsmY/RsmZ ncRNAs might enhance the pyoverdine synthesis through SadC. Our study sheds light on a novel multiple DGC-coordinated c-di-GMP regulatory mechanism of bacteria. PMID:25683454

  15. Levels of Lycopene β-Cyclase 1 Modulate Carotenoid Gene Expression and Accumulation in Daucus carota

    PubMed Central

    Moreno, Juan Camilo; Pizarro, Lorena; Fuentes, Paulina; Handford, Michael; Cifuentes, Victor; Stange, Claudia

    2013-01-01

    Plant carotenoids are synthesized and accumulated in plastids through a highly regulated pathway. Lycopene β-cyclase (LCYB) is a key enzyme involved directly in the synthesis of α-carotene and β-carotene through the cyclization of lycopene. Carotenoids are produced in both carrot (Daucus carota) leaves and reserve roots, and high amounts of α-carotene and β-carotene accumulate in the latter. In some plant models, the presence of different isoforms of carotenogenic genes is associated with an organ-specific function. D. carota harbors two Lcyb genes, of which DcLcyb1 is expressed in leaves and storage roots during carrot development, correlating with an increase in carotenoid levels. In this work, we show that DcLCYB1 is localized in the plastid and that it is a functional enzyme, as demonstrated by heterologous complementation in Escherichia coli and over expression and post transcriptional gene silencing in carrot. Transgenic plants with higher or reduced levels of DcLcyb1 had incremented or reduced levels of chlorophyll, total carotenoids and β-carotene in leaves and in the storage roots, respectively. In addition, changes in the expression of DcLcyb1 are accompanied by a modulation in the expression of key endogenous carotenogenic genes. Our results indicate that DcLcyb1 does not possess an organ specific function and modulate carotenoid gene expression and accumulation in carrot leaves and storage roots. PMID:23555569

  16. 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

  17. Guanylyl Cyclase/Atrial Natriuretic Peptide Receptor-A: Role in the Pathophysiology of Cardiovascular Regulation

    PubMed Central

    Pandey, Kailash N.

    2012-01-01

    Atrial natriuretic factor (ANF), also known as atrial natriuretic peptide (ANP), is an endogenous and potent hypotensive hormone that elicits natriuretic, diuretic, vasorelaxant, and anti-proliferative effects, which are important in the control of blood pressure and cardiovascular events. One principal locus involved in the regulatory action of ANP and brain natriuretic peptide (BNP) is guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA). Studies on ANP, BNP, and their receptor, GC-A/NPRA, have greatly increased our knowledge of the control of hypertension and cardiovascular disorders. Cellular, biochemical, and molecular studies have helped to delineate the receptor function and signaling mechanisms of NPRA. Gene-targeted and transgenic mouse models have advanced our understanding of the importance of ANP, BNP, and GC-A/NPRA in disease states at the molecular level. Importantly, ANP and BNP are used as critical markers of cardiac events; however, their therapeutic potentials for the diagnosis and treatment of hypertension, heart failure, and stroke have just begun to be realized. We are now just at the initial stage of molecular therapeutics and pharmacogenomic advancement of the natriuretic peptides. More investigations should be undertaken and ongoing ones be extended in this important field. PMID:21815745

  18. Guanylyl cyclase / atrial natriuretic peptide receptor-A: role in the pathophysiology of cardiovascular regulation.

    PubMed

    Pandey, Kailash N

    2011-08-01

    Atrial natriuretic factor (ANF), also known as atrial natriuretic peptide (ANP), is an endogenous and potent hypotensive hormone that elicits natriuretic, diuretic, vasorelaxant, and anti-proliferative effects, which are important in the control of blood pressure and cardiovascular events. One principal locus involved in the regulatory action of ANP and brain natriuretic peptide (BNP) is guanylyl cyclase / natriuretic peptide receptor-A (GC-A/NPRA). Studies on ANP, BNP, and their receptor, GC-A/NPRA, have greatly increased our knowledge of the control of hypertension and cardiovascular disorders. Cellular, biochemical, and molecular studies have helped to delineate the receptor function and signaling mechanisms of NPRA. Gene-targeted and transgenic mouse models have advanced our understanding of the importance of ANP, BNP, and GC-A/NPRA in disease states at the molecular level. Importantly, ANP and BNP are used as critical markers of cardiac events; however, their therapeutic potentials for the diagnosis and treatment of hypertension, heart failure, and stroke have just begun to be realized. We are now just at the initial stage of molecular therapeutics and pharmacogenomic advancement of the natriuretic peptides. More investigations should be undertaken and ongoing ones be extended in this important field. PMID:21815745

  19. 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

  20. Mapping Soluble Guanylyl Cyclase and Protein Disulfide Isomerase Regions of Interaction

    PubMed Central

    Jain, Mohit; Liu, Tong; Li, Hong; Beuve, Annie

    2015-01-01

    Soluble guanylyl cyclase (sGC) is a heterodimeric nitric oxide (NO) receptor that produces cyclic GMP. This signaling mechanism is a key component in the cardiovascular system. NO binds to heme in the β subunit and stimulates the catalytic conversion of GTP to cGMP several hundred fold. Several endogenous factors have been identified that modulate sGC function in vitro and in vivo. In previous work, we determined that protein disulfide isomerase (PDI) interacts with sGC in a redox-dependent manner in vitro and that PDI inhibited NO-stimulated activity in cells. To our knowledge, this was the first report of a physical interaction between sGC and a thiol-redox protein. To characterize this interaction between sGC and PDI, we first identified peptide linkages between sGC and PDI, using a lysine cross-linking reagent and recently developed mass spectrometry analysis. Together with Flag-immunoprecipitation using sGC domain deletions, wild-type (WT) and mutated PDI, regions of sGC involved in this interaction were identified. The observed data were further explored with computational modeling to gain insight into the interaction mechanism between sGC and oxidized PDI. Our results indicate that PDI interacts preferentially with the catalytic domain of sGC, thus providing a mechanism for PDI inhibition of sGC. A model in which PDI interacts with either the α or the β catalytic domain is proposed. PMID:26618351

  1. 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

  2. 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

  3. 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

  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. 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.

  6. The Diguanylate Cyclase SadC Is a Central Player in Gac/Rsm-Mediated Biofilm Formation in Pseudomonas aeruginosa

    PubMed Central

    Moscoso, Joana A.; Jaeger, Tina; Valentini, Martina; Hui, Kailyn; Jenal, Urs

    2014-01-01

    Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen and a threat for immunocompromised and cystic fibrosis patients. It is responsible for acute and chronic infections and can switch between these lifestyles upon taking an informed decision involving complex regulatory networks. The RetS/LadS/Gac/Rsm network and the cyclic-di-GMP (c-di-GMP) signaling pathways are both central to this phenomenon redirecting the P. aeruginosa population toward a biofilm mode of growth, which is associated with chronic infections. While these two pathways were traditionally studied independently from each other, we recently showed that cellular levels of c-di-GMP are increased in the hyperbiofilm retS mutant. Here, we have formally established the link between the two networks by showing that the SadC diguanylate cyclase is central to the Gac/Rsm-associated phenotypes, notably, biofilm formation. Importantly, SadC is involved in the signaling that converges onto the RsmA translational repressor either via RetS/LadS or via HptB/HsbR. Although the level of expression of the sadC gene does not seem to be impacted by the regulatory cascade, the production of the SadC protein is tightly repressed by RsmA. This adds to the growing complexity of the signaling network associated with c-di-GMP in P. aeruginosa. While this organism possesses more than 40 c-di-GMP-related enzymes, it remains unclear how signaling specificity is maintained within the c-di-GMP network. The finding that SadC but no other diguanylate cyclase is related to the formation of biofilm governed by the Gac/Rsm pathway further contributes to understanding of this insulation mechanism. PMID:25225264

  7. 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

  8. 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

  9. 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

  10. 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