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Sample records for adenylate cyclase

  1. Plant adenylate cyclases.

    Science.gov (United States)

    Lomovatskaya, Lidiya A; Romanenko, Anatoliy S; Filinova, Nadejda V

    2008-01-01

    Adenylate cyclase (AC) (ATP diphosphate-lyase cyclizing; EC 4.6.1.1) is a key component of the adenylate cyclase signaling system and catalyzes the generation of cyclic adenosine monophosphate (cAMP) from ATP. This review summarizes data from the literature and the authors' laboratory on the investigation of plant transmembrane (tmAC) and soluble (sAC) adenylate cyclases, in comparison with some key characteristics of adenylate cyclases of animal cells. Plant sAC has been demonstrated to exhibit similarities with animal sAC with respect to certain characteristics. External factors, such as far-red and red light, temperature, exogenous phytohormones, as well as specific triggering compounds of fungal and bacterial origin exert a significant influence on the activity of plant tmAC and sAC.

  2. Pituitary adenylate cyclase activating polypeptide and migraine

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    Zagami, Alessandro S; Edvinsson, Lars; Goadsby, Peter J

    2014-01-01

    Pituitary adenylate cyclase activating peptide (PACAP) is found in human trigeminocervical complex and can trigger migraine. PACAP levels were measured using a sensitive radioimmunoassay. Stimulation of the superior sagittal sinus (SSS) in cat elevated PACAP levels in cranial blood. Patients...

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

    NARCIS (Netherlands)

    Roelofs, J; Snippe, H; Kleineidam, RG; Van Haastert, PJM

    2001-01-01

    The core of adenylate and guanylate cyclases is formed by an intramolecular ol 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 he

  4. Adenylate cyclases involvement in pathogenicity, a minireview.

    Science.gov (United States)

    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.

  5. Adenyl cyclase in the human placenta.

    Science.gov (United States)

    Sato, K; Ryan, K J

    1971-09-21

    This study demonstrated that the human placenta possesses an adenyl cyclase system responsive to catecholamines and sodium flouride (NaF). 2.5 gm human term placentas were homogenized, centrifuged, washed, resuspended, and used as the enzyme system when placed with various agents. Incubations and the determination of adenosine 3', 5' monophosphate (cyclic AMP) formed were performed. Samples stimulated by .0001 M catecholamines (L-epinephrine or L-norepinephrine) or .01 M NaF had higher levels of cyclic AMP than the controls (p. 005 for catecholamine-treated samples and p. 001 for NaF-treated samples). A concentration of .0001 M L-epinephrine or L-norepinephrine appeared to be a maximum effective dose and .0000001 M a minimum. L=epinephrine was 10 times as effective in the stimulation as L-norepinephrine. With .0001 M, 499 and 439 pmoles/10 minutes per 25 mg of tissue was formed, whereas in the control (no added hormones) 256 pmoles/10 minutes were formed. 3.2% ethanol activated the system by a small amount (p.02). Propranolol alone did not appear to have any effect; however, the effect of .0001 M L-epinephrine was reduced by 95% in the presence of .00001 M propranolol. Propranolol had no effect on NaF-stimulated activity.

  6. Thyrotropin receptor-adenylate cyclase function in human thyroid neoplasms.

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    Saltiel, A R; Powel-Jones, C H; Thomas, C G; Nayfeh, S N

    1981-06-01

    The action of thyrotropin (TSH) on plasma membranes was studied to elucidate the mechanism of hormonal regulation of malignant versus normal human thyroid tissue. Thyroid plasma membranes of six specimens of papillary or follicular carcinoma and six of adenoma, as well as adjacent normal tissue obtained from these patients, were evaluated with respect to binding of 125I-labeled TSH and stimulation of adenylate cyclase. Scatchard analysis of TSH binding revealed the presence of two species of binding sites in normal thyroid of different affinities and capacities. In 11 of 12 tumors studied, the high-affinity binding site remained intact; however, the total number of low-affinity sites was markedly lower than normal tissue. Other parameters of binding were not altered in neoplastic thyroid. In each of these tissues, the hormone responsiveness and kinetics of adenylate cyclase activation were essentially identical to those observed in normal tissue, although basal activity was typically greater in the neoplasm. One carcinoma was totally deficient in both 125I-labeled TSH binding and TSH-stimulatable adenylate cyclase, although basal activity was detected. Furthermore, adenylate cyclase of this specimen was not activated by prostaglandin, in contrast to normal thyroid and other thyroid tumors. These results suggest that: (a) clinical behavior of thyroid carcinomas may not be reflected by TSH receptor-adenylate cyclase function; (b) lack of clinical response as manifest by tumor regression cannot be ascribed to the absence of functional TSH receptors or adenylate cyclase; and (c) decreased low-affinity binding present in tumors is not correlated with altered hormone responsiveness of adenylate cyclase but may reflect more general cancer-induced changes in membrane structure or composition.

  7. Monospecific antibody against Bordetella pertussis Adenylate Cyclase protects from Pertussis

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    Yasmeen Faiz Kazi

    2012-06-01

    Full Text Available Objectives: Acellular pertussis vaccines has been largely accepted world-wide however, there are reports about limitedantibody response against these vaccines suggesting that multiple antigens should be included in acellular vaccinesto attain full protection. The aim of present study was to evaluate the role of Bordetella pertussis adenylate cyclase as aprotective antigen.Materials and methods: Highly mono-specific antibody against adenylate cyclase (AC was raised in rabbits usingnitrocellulose bound adenylate cyclase and the specificity was assessed by immuoblotting. B.pertussis 18-323, wasincubated with the mono-specific serum and without serum as a control. Mice were challenged intra-nasally and pathophysiolgicalresponses were recorded.Results: The production of B.pertussis adenylate cyclase monospecific antibody that successfully recognized on immunoblotand gave protection against fatality (p< 0.01 and lung consolidation (p <0.01. Mouse weight gain showedsignificant difference (p< 0.05.Conclusion: These preliminary results highlight the role of the B.pertussis adenylate cyclase as a potential pertussisvaccine candidate. B.pertussis AC exhibited significant protection against pertussis in murine model. J Microbiol InfectDis 2012; 2(2: 36-43Key words: Pertussis; monospecific; antibody; passive-protection

  8. Pertussis toxin inhibits cAMP-induced desensitization of adenylate cyclase in Dictyostelium discoideum

    NARCIS (Netherlands)

    Snaar-Jagalska, B. Ewa; Haastert, Peter J.M. van

    1990-01-01

    cAMP binds to surface receptors of Dictyostelium discoideum cells, transducing the signal to adenylate cyclase, guanylate cyclase and to chemotaxis. The activation of adenylate cyclase is maximal after 1 min and then declines to basal levels due to desensitization, which is composed of two component

  9. Restoration of adenylate cyclase responsiveness in murine myeloid leukemia permits inhibition of proliferation by hormone. Butyrate augments catalytic activity of adenylate cyclase.

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    Inhorn, L; Fleming, J W; Klingberg, D; Gabig, T G; Boswell, H S

    1988-04-01

    Mechanisms of leukemic cell clonal dominance may include aberrations of transmembrane signaling. In particular, neoplastic transformation has been associated with reduced capacity for hormone-stimulated adenylate cyclase activity. In the present study, prostaglandin E, a hormonal activator of adenylate cyclase that has antiproliferative activity in myeloid cells, and cholera toxin, an adenylate cyclase agonist that functions at a postreceptor site by activating the adenylate cyclase stimulatory GTP-binding protein (Gs), were studied for antiproliferative activity in two murine myeloid cell lines. FDC-P1, an interleukin 3 (IL 3)-dependent myeloid cell line and a tumorigenic IL 3-independent subline, FI, were resistant to these antiproliferative agents. The in vitro ability of the "differentiation" agent, sodium butyrate, to reverse their resistance to adenylate cyclase agonists was studied. The antiproliferative action of butyrate involved augmentation of transmembrane adenylate cyclase activity. Increased adenylate cyclase catalyst activity was the primary alteration of this transmembrane signaling group leading to the functional inhibitory effects on leukemia cells, although alterations in regulatory G-proteins appear to play a secondary role.

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

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    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.

  11. Evidence for adenylate cyclase as a scaffold protein for Ras2-Ira interaction in Saccharomyces cerevisie.

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    Colombo, Sonia; Paiardi, Chiara; Pardons, Katrien; Winderickx, Joris; Martegani, Enzo

    2014-05-01

    Data in literature suggest that budding yeast adenylate cyclase forms a membrane-associated complex with the upstream components of the cAMP/PKA pathway. Here we provide evidences that adenylate cyclase (Cyr1p) acts as a scaffold protein keeping Ras2 available for its regulatory factors. We show that in a strain with deletion of the CYR1 gene (cyr1Δ pde2Δ msn2Δ msn4Δ) the basal Ras2-GTP level is very high and this is independent on the lack of feedback inhibition that could result from the absence of adenylate cyclase activity. Moreover, strains effected either in the intrinsic adenylate cyclase activity (fil1 strain) or in the stimulation of adenylate cyclase activity by active G-proteins (lcr1 strain) had a normal basal and glucose-induced Ras2-GTP level, indicating that adenylate cyclase activity does not influence the Ras2 activation state and suggesting that Cyr1 protein is required for the proper interaction between Ras2 and the Ira proteins. We also provide evidence that the two Ras-binding sites mapped on Cyr1p are required for the signalling complex assembly. In fact, we show that the cyr1Δ strain expressing CYR1 alleles lacking either the LRR region or the C-terminal domain still have a high basal and glucose-induced Ras2-GTP level. In contrast, a mutant expressing a Cyr1 protein only missing the N-terminal domain showed a normal Ras2 activation pattern. Likewise, the Ras2-GTP levels are comparable in the wild type strain and the srv2Δ strain, supporting the hypothesis that Cap is not essential for the Ras-adenylate cyclase interaction.

  12. Adenylate cyclase regulates elongation of mammalian primary cilia

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    Ou, Young; Ruan, Yibing; Cheng, Min; Moser, Joanna J. [Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1 (Canada); Rattner, Jerome B. [Department of Cell Biology and Anatomy, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1 (Canada); Hoorn, Frans A. van der, E-mail: fvdhoorn@ucalgary.ca [Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1 (Canada)

    2009-10-01

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

  13. Neurohypophyseal hormone-responsive renal adenylate cyclase. IV. A random-hit matrix model for coupline in a hormone-sensitive adenylate cyclase system.

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    Bergman, R N; Hechter, O

    1978-05-10

    A "random-hit" matrix model is proposed to account for the dynamic and steady state relationship between occupation of bovine renal medullary membrane receptors by [Lys8]vasopressin (LVP) and neurohypophyseal hormones (NHH) and the associated activation of membrane-bound adenylate cyclase. The model was developed by systematic introduction of specific rules concerning receptor coupling into a general structural model which consists of two square matrices of identical size, one composed of homogeneous R ("receptor") units, the second of homogeneous C ("cyclase") units. R units are either occupied (RO) or unoccupied (RU); C units are either active (CA) or inactive (CI). Hormone molecules are envisioned to "collide" with R units randomly; collision with RU leads to "binding", and occupation is maintained for a characteristic mean occupancy time, TO. In this structure, each R unit has an "interaction field" which consists of the "twin" unit in the "C" matrix, and the 4 nearest neighbor C units surrounding the twin. Occupation of an R unit leads to activation of all CI units in the interaction field of that R; CA units in the interaction field are refractory. Thus binding at a given R may "recruit" a variable number of inactive neighboring C units (5, 4, 3, 2, 1, or 0). The model requires that there be individual coupling delays between the moment of binding at a given R and subsequent activation of CI units (mean coupling delay (Td) approximately 10% To). Activation of C units persists as long as the "parent" R is occupied and is maintained for an additional short time interval (Tp) after RO reverts to RU, corresponding to hormone dissociation from receptor. The model accounts for the following previously demonstrated relations between LVP occupation of receptors and adenylate cyclase activation in bovine renal medullary membranes: 1) the shape of the nonlinear steady state relation between normalized (percentage maximal) receptor occupation (O) and cyclase activation

  14. [The aspects of adenylate cyclase activity regulation in myocardium cell membranes during hypokinesia].

    Science.gov (United States)

    Bulanova, K Ia; Komar, E S; Lobanok, L M

    1999-01-01

    Nonstimulated and isoproterenol, GTF, GITF, NaF stimulated activities of the adenylate cyclase in sarcolemma in white rats' myocardium was studied after two weeks of hypokinesia. As was established, in restrained animals the sensitivity of adenylate cyclase to the specified agents was increased and transition to the bimodal GTF regulation took place. It is hypothesised that involvement of membrane-bound Gi-proteins in the adrenergic effects on cardiomyocytes is one of mechanisms of the cardiotropic effects of restraint and heart distresses.

  15. Regulation of adenylate cyclase of Dictyostelium discoideum by divalent cations and adenosine analogs

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    Khachatrian, L.; Howlett, A.; Klein, C.

    1986-03-05

    Cyclic AMP is synthesized and secreted in a pulsatile fashion as a chemotactic signaling system intrinsic to the differentiation program of D. discoideum. They examined the regulation of D. dischoideum adenylate cyclase using a membrane fraction which exhibits high specific activity enzyme. When Mn-ATP was used as substrate, increasing Mn/sup 2 +/ concentrations activated the enzyme 3 to 8 fold. In contrast, Mg ion increased the adenylate cyclase activity by only 60%. These results suggested an activation of the catalytic subunit by Mn/sup 2 +/. Inhibition of activity was observed in response to adenosine and its analogs. P-site agonist, 2',5'-Dideoxy-adenosine, inhibited activity by about 25% in the presence of Mg/sup 2 +/, and about 80% in presence of Mn/sup 2 +/. This inhibition was not dependent on guanine nucleotides. The data are in agreement with characteristics of P-site regulation of the catalytic subunit of eukaryotic systems. Kinetic analysis of previously reported inhibition of D. discoideum adenylate cyclase by guanine nucleotides revealed that guanine nucleotides do not compete for the substrate binding site. Further, the enzyme activity cannot be accounted for by guanylate cyclase. Their data suggest that regulation of adenylate cyclase may exist not only at the catalytic subunit but also via inhibitory G protein, N/sub i/.

  16. Receptor binding and adenylate cyclase activities of glucagon analogues modified in the N-terminal region

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    McKee, R.L.; Pelton, J.T.; Trivedi, D.; Johnson, D.G.; Coy, D.H.; Sueiras-Diaz, J.; Hruby, V.J.

    1986-04-08

    In this study, we determined the ability of four N-terminally modified derivatives of glucagon, (3-Me-His1,Arg12)-, (Phe1,Arg12)-, (D-Ala4,Arg12)-, and (D-Phe4)glucagon, to compete with 125I-glucagon for binding sites specific for glucagon in hepatic plasma membranes and to activate the hepatic adenylate cyclase system, the second step involved in producing many of the physiological effects of glucagon. Relative to the native hormone, (3-Me-His1,Arg12)glucagon binds approximately twofold greater to hepatic plasma membranes but is fivefold less potent in the adenylate cyclase assay. (Phe1,Arg12)glucagon binds threefold weaker and is also approximately fivefold less potent in adenylate cyclase activity. In addition, both analogues are partial agonists with respect to adenylate cyclase. These results support the critical role of the N-terminal histidine residue in eliciting maximal transduction of the hormonal message. (D-Ala4,Arg12)glucagon and (D-Phe4)glucagon, analogues designed to examine the possible importance of a beta-bend conformation in the N-terminal region of glucagon for binding and biological activities, have binding potencies relative to glucagon of 31% and 69%, respectively. (D-Ala4,Arg12)glucagon is a partial agonist in the adenylate cyclase assay system having a fourfold reduction in potency, while the (D-Phe4) derivative is a full agonist essentially equipotent with the native hormone. These results do not necessarily support the role of an N-terminal beta-bend in glucagon receptor recognition. With respect to in vivo glycogenolysis activities, all of the analogues have previously been reported to be full agonists.

  17. Age-associated alterations in hepatic. beta. -adrenergic receptor/adenylate cyclase complex

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    Graham, S.M.; Herring, P.A.; Arinze, I.J.

    1987-09-01

    The effect of age on catecholamine regulation of hepatic glycogenolysis and on hepatic adenylate cyclase was studied in male rats up to 24 mo of age. Epinephrine and norepinephrine stimulated glycogenolysis in isolated hepatocytes at all age groups studied. Isoproterenol, however, stimulated glycogenolysis only at 24 mo. In isolated liver membranes, usual activators of adenylate cyclase increased the activity of the enzyme considerably more in membranes from 24-mo-old rats than in membranes from either 3- or 22-mo-old rats. The Mn/sup 2 +/-dependent activity of the cyclase was increased by 2.9-fold in 3-mo-old animals and approx. 5.7-fold in 24-mo-old rats, indicating a substantial age-dependent increase in the intrinsic activity of the catalytic unit. The density of the ..beta..-adrenergic receptor, as measured by the binding of (/sup 125/I)-iodocyanopindolol to plasma membranes, was 5-8 fmol/mg protein in rats aged 3-12 mo but increased to 19 fmol/mg protein in 24-mo-old rats. Computer-aided analysis of isoproterenol competition of the binding indicated a small age-dependent increase in the proportion of ..beta..-receptors in the high-affinity state. These observations suggest that ..beta..-receptor-mediated hepatic glycogenolysis in the aged rat is predicated upon increases in the density of ..beta..-receptors as well as increased intrinsic activity of the catalytic unit of adenylate cyclase.

  18. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) in the circulation after sumatriptan

    DEFF Research Database (Denmark)

    Hansen, Jakob Møller; Fahrenkrug, Jan; Petersen, Jesper Troensegaard;

    2013-01-01

    The origin of migraine pain is still elusive, but increasingly researchers focus on the neuropeptides in the perivascular space of cranial vessels as important mediators of nociceptive input during migraine attacks. The parasympathetic neurotransmitters, pituitary adenylate cyclase activating...

  19. The role of transcriptional regulation in maintaining the availability of mycobacterial adenylate cyclases

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    Sarah J. Casey

    2014-03-01

    Full Text Available Mycobacterium species have a complex cAMP regulatory network indicated by the high number of adenylate cyclases annotated in their genomes. However the need for a high level of redundancy in adenylate cyclase genes remains unknown. We have used semiquantitiative RT-PCR to examine the expression of eight Mycobacterium smegmatis cyclases with orthologs in the human pathogen Mycobacterium tuberculosis, where cAMP has recently been shown to be important for virulence. All eight cyclases were transcribed in all environments tested, and only four demonstrated environmental-mediated changes in transcription. M. smegmatis genes MSMEG_0545 and MSMEG_4279 were upregulated during starvation conditions while MSMEG_0545 and MSMEG_4924 were downregulated in H2O2 and MSMEG_3780 was downregulated in low pH and starvation. Promoter fusion constructs containing M. tuberculosis H37Rv promoters showed consistent regulation compared to their M. smegmatis orthologs. Overall our findings indicate that while low levels of transcriptional regulation occur, regulation at the mRNA level does not play a major role in controlling cellular cyclase availability in a given environment.

  20. Catecholamine-sensitive adenylate cyclase of caudate nucleus and cerebral cortex. Effects of guanine nucleotides.

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    Sulakhe, P V; Leung, N L; Arbus, A T; Sulakhe, S J; Jan, S H; Narayanan, N

    1977-01-01

    1. GTP and GMP-P(NH)P (guanyl-5'-yl imidodiphosphate) were observed to increase the stimulation of neural adenylate cyclase by dopamine (3,4-dihydroxyphenethylamine) and noradrenaline. 2. GMP-P(NH)P had a biphasic effect on the enzyme activity. 3. Preincubation of membranes with GMP-P(NH)P activated the enzyme by a process dependent on time and temperature. Catecholamines increased the speed and the extent of this activation. 4. Membrane fractions contained high- and low-affinity sites for GMP-P(NH)P binding: this binding was due to protein(s) of the membrane preparations. 5. Low-affinity-site binding of GMP-P(NH)P appeared to be related to the stimulatory effect on the adenylate cyclase activity. PMID:18147

  1. Muscarinic receptor binding and muscarinic receptor-mediated inhibition of adenylate cyclase in rat brain myelin

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    Larocca, J.N.; Ledeen, R.W.; Dvorkin, B.; Makman, M.H.

    1987-12-01

    High-affinity muscarinic cholinergic receptors were detected in myelin purified from rat brain stem with use of the radioligands /sup 3/H-N-methylscopolamine (/sup 3/H-NMS), /sup 3/H-quinuclidinyl benzilate (/sup 3/H-QNB), and /sup 3/H-pirenzepine. /sup 3/H-NMS binding was also present in myelin isolated from corpus callosum. In contrast, several other receptor types, including alpha 1- and alpha 2-adrenergic receptors, present in the starting brain stem, were not detected in myelin. Based on Bmax values from Scatchard analyses, /sup 3/H-pirenzepine, a putative M1 selective ligand, bound to about 25% of the sites in myelin labeled by /sup 3/H-NMS, a nonselective ligand that binds to both M1 and M2 receptor subtypes. Agonist affinity for /sup 3/H-NMS binding sites in myelin was markedly decreased by Gpp(NH)p, indicating that a major portion of these receptors may be linked to a second messenger system via a guanine-nucleotide regulatory protein. Purified myelin also contained adenylate cyclase activity; this activity was stimulated several fold by forskolin and to small but significant extents by prostaglandin E1 and the beta-adrenergic agonist isoproterenol. Myelin adenylate cyclase activity was inhibited by carbachol and other muscarinic agonists; this inhibition was blocked by the antagonist atropine. Levels in myelin of muscarinic receptors were 20-25% and those of forskolin-stimulated adenylate cyclase 10% of the values for total particulate fraction of whole brain stem. These levels in myelin are appreciably greater than would be predicted on the basis of contamination. Also, additional receptors and adenylate cyclase, added by mixing nonmyelin tissue with whole brain stem, were quantitatively removed during the purification procedure.

  2. Stimulatory and inhibitory effects of forskolin on adenylate cyclase in rat normal hepatocytes and hepatoma cells.

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    Miyamoto, K; Sanae, F; Koshiura, R; Matsunaga, T; Takagi, K; Satake, T; Hasegawa, T

    1989-02-01

    Forskolin synergistically potentiated adenosine 3',5'-cyclic monophosphate formation by prostaglandin E1 (PGE1) in rat normal hepatocytes freshly prepared by collagenase digestion and rat ascites hepatoma AH66 cells, but dose-dependently inhibited the accumulation by PGE1 in AH66F cells. Forskolin activated adenylate cyclase in a dose-dependent manner in homogenates of all cell lines. In normal hepatocytes and AH66 cells, simultaneous addition of forskolin and other adenylate cyclase activators [isoproterenol (IPN), PGE1, guanosine 5'-triphosphate sodium salt (GTP), 5'-guanylylimidodiphosphate sodium salt (Gpp (NH)p), NaF, cholera toxin, islet activating protein and MnCl2] gave greater than additive responses. On the other hand, in AH66F cells, the effect of forskolin on adenylate cyclase was hardly influenced by GTP, but forskolin diminished the activities induced by high concentrations of GTP to that by the diterpene alone. Forskolin also significantly inhibited the PGE1-stimulated and the guanine nucleotide binding regulatory protein-stimulated activities. Because AH66F cells were insensitive to IPN, the combination with forskolin and IPN gave similar activity to that obtained with the diterpene alone. The effect of forskolin on the activation by manganese ion was neither synergistic nor inhibitory but was additive in AH66F cells. These results suggest that forskolin promotes the interaction between the stimulatory guanine nucleotide binding regulatory protein and the catalytic unit in normal hepatocytes and AH66 cells, but in AH66F cells forskolin interferes with the coupling of the two components of adenylate cyclase.

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

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    Ma’ayan Israeli

    2016-08-01

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

  4. Adenylate cyclase toxin promotes internalisation of integrins and raft components and decreases macrophage adhesion capacity.

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    César Martín

    Full Text Available Bordetella pertussis, the bacterium that causes whooping cough, secretes an adenylate cyclase toxin (ACT that must be post-translationally palmitoylated in the bacterium cytosol to be active. The toxin targets phagocytes expressing the CD11b/CD18 integrin receptor. It delivers a catalytic adenylate cyclase domain into the target cell cytosol producing a rapid increase of intracellular cAMP concentration that suppresses bactericidal functions of the phagocyte. ACT also induces calcium fluxes into target cells. Biochemical, biophysical and cell biology approaches have been applied here to show evidence that ACT and integrin molecules, along with other raft components, are rapidly internalized by the macrophages in a toxin-induced calcium rise-dependent process. The toxin-triggered internalisation events occur through two different routes of entry, chlorpromazine-sensitive receptor-mediated endocytosis and clathrin-independent internalisation, maybe acting in parallel. ACT locates into raft-like domains, and is internalised, also in cells devoid of receptor. Altogether our results suggest that adenylate cyclase toxin, and maybe other homologous pathogenic toxins from the RTX (Repeats in Toxin family to which ACT belongs, may be endowed with an intrinsic capacity to, directly and efficiently, insert into raft-like domains, promoting there its multiple activities. One direct consequence of the integrin removal from the cell surface of the macrophages is the hampering of their adhesion ability, a fundamental property in the immune response of the leukocytes that could be instrumental in the pathogenesis of Bordetella pertussis.

  5. Adenylate cyclase activity along the rabbit nephron as measured in single isolated segments.

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    Imbert, M; Chabardès, D; Montégut, M; Clique, A; Morel, F

    1975-01-01

    A method is described, which allows adenylate cyclase activity measurement in single pieces of various nephron segments. Tubular samples of 0.5 to 2 mm length were isolated by microdissection from collagenase treated slices of rabbit kidney. A photograph of each piece was taken in order to measure its length. After a permeabilisation treatment involving preincubation in a hypoosmotic medium and a freezing step, each sample was incubated for 30 mm at 30 degrees C in a medium containing high specific (alpha-32-P)-ATP 3-10-4 M, final volume 2.5 mu 1. The (32P)-cAMP formed was separated from the other labelled nucleotides by filtering the incubate on a dry aluminium oxide microcolumn, 3H cAMP was added as a tracer for measuring cAMP recovery. The sensitivity of the method was found to be a few fentomoles (10-15 M) cAMP. cAMP generation increased linearly as a function of the incubation time up to more than 30 min, and as a function of the length of the segment used. Control and fluoride (5 mM) stimulated adenvlate cyclase activities were measured in the following segments of the nephron: early proximal convoluted tubule (PCT), pars recta of the proximal tubule (PR), thin descending limb of the loop (TDL), cortical portion of the thick ascending limb (CAL), distal convoluted tubule (dct), first branched portion of the collecting tubule (BCT), further cortical (CCT) and medullary (MCT) portions of the collecting tubule. Mean control adenylate cyclase activity varied from 7 (PR) to 75 (BCT) fmoles/mm/30 min. Flouride addition resulted in a 10 (BCT) to 50 (PR) fold increase in enzyme activity. Series of replicates gave a scatter equal to plus or minus 20% (S.D. as a per cent of the mean). The method described appears to be suitable to determine which nephron segments contain hormone-dependent adenylate cyclase.

  6. Effect of mitomycin C on the activation of adenylate cyclase in rat ascites hepatoma AH130 cells.

    Science.gov (United States)

    Miyamoto, K; Matsunaga, T; Sanae, F; Koshiura, R

    1986-09-01

    Isoproterenol (IPN)-stimulated activity of adenylate cyclase was enhanced in a dose-dependent manner by exposure of AH130 cells to mitomycin C (MMC). The enhancement was also observed in prostaglandin E1-, guanine nucleotide analog-, NaF-, cholera toxin- and forskolin-stimulated activities of the enzyme but not in manganese-stimulated activity. In addition, even when the cells pretreated with islet-activating protein were exposed to MMC, IPN-stimulated activity of adenylate cyclase was enhanced. Anaerobic exposure of AH130 cells to MMC somewhat inhibited IPN-stimulated activity of adenylate cyclase in contrast with aerobic exposure. Exposure of cells to adriamycin also caused enhancement of IPN-stimulated activity of adenylate cyclase but exposure to nitrogen mustard inhibited the enzyme stimulation by IPN. The enhancing effect of MMC was lost by the combined treatment with alpha-tocopherol. From these results, it was shown that MMC modulated the activity of adenylate cyclase, probably through alterations in membrane structure.

  7. Identification of Adenyl Cyclase Activity in a Disease Resistance Protein in Arabidopsis thaliana

    KAUST Repository

    Hussein, Rana

    2012-11-01

    Cyclic nucleotide, cAMP, is an important signaling molecule in animals and plants. However, in plants the enzymes that synthesize this second messenger, adenyl cyclases (ACs), remain elusive. Given the physiological importance of cAMP in signaling, particularly in response to biotic and abiotic stresses, it is thus important to identify and characterize ACs in higher plants. Using computational approaches, a disease resistance protein from Arabidopsis thaliana, At3g04220 was found to have an AC catalytic center motif. In an attempt to prove that this candidate has adenyl cyclases activity in vitro, the coding sequence of the putative AC catalytic domain of this protein was cloned and expressed in E. coli and the recombinant protein was purified. The nucleotide cyclase activity of the recombinant protein was examined using cyclic nucleotide enzyme immunoassays. In parallel, the expression of At3g04220 was measured in leaves under three different stress conditions in order to determine under which conditions the disease resistance protein could function. Results show that the purified recombinant protein has Mn2+ dependent AC activity in vitro, and the expression analysis supports a role for At3g04220 and cAMP in plant defense.

  8. Bordetella adenylate cyclase toxin: a unique combination of a pore-forming moiety with a cell-invading adenylate cyclase enzyme.

    Science.gov (United States)

    Masin, Jiri; Osicka, Radim; Bumba, Ladislav; Sebo, Peter

    2015-11-01

    The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) is a key virulence factor of the whooping cough agent Bordetella pertussis. CyaA targets myeloid phagocytes expressing the complement receptor 3 (CR3, known as αMβ2 integrin CD11b/CD18 or Mac-1) and translocates by a poorly understood mechanism directly across the cytoplasmic membrane into cell cytosol of phagocytes an adenylyl cyclase(AC) enzyme. This binds intracellular calmodulin and catalyzes unregulated conversion of cytosolic ATP into cAMP. Among other effects, this yields activation of the tyrosine phosphatase SHP-1, BimEL accumulation and phagocyte apoptosis induction. In parallel, CyaA acts as a cytolysin that forms cation-selective pores in target membranes. Direct penetration of CyaA into the cytosol of professional antigen-presenting cells allows the use of an enzymatically inactive CyaA toxoid as a tool for delivery of passenger antigens into the cytosolic pathway of processing and MHC class I-restricted presentation, which can be exploited for induction of antigen-specific CD8(+) cytotoxic T-lymphocyte immune responses.

  9. Cyclic 3', 5'-AMP relay in Dictyostelium discoideum: adaptation is independent of activation of adenylate cyclase

    OpenAIRE

    1983-01-01

    In Dictyostelium discoideum, binding of cAMP to high affinity surface receptors leads to a rapid activation of adenylate cyclase followed by subsequent adaptation within several minutes. The rate of secretion of [ 3H ]cAMP, which reflects the state of activation of the enzyme, was measured. Caffeine noncompetitively inhibited the response to cAMP. Inhibition was rapidly reversible and pretreatment of cells with caffeine for up to 22 min had little effect on the subsequent responsiveness to cA...

  10. Delivery of Bordetella pertussis adenylate cyclase toxin to target cells via outer membrane vesicles

    OpenAIRE

    Donato, Gina M.; Goldsmith, Cynthia S.; Paddock, Christopher D.; Eby, Joshua C.; Gray, Mary C.; Hewlett, Erik L.

    2012-01-01

    B.pertussis adenylate cyclase toxin (ACT) intoxicates cells by producing intracellular cAMP. B.pertussis outer membrane vesicles (OMV) contain ACT on their surface (OMV-ACT), but the properties of OMV-ACT were previously unknown. We found that B.pertussis in the lung from a fatal pertussis case contains OMV, suggesting an involvement in pathogenesis. OMV-ACT and ACT intoxicate cells with and without the toxin’s receptor CD11b/CD18. Intoxication by ACT is blocked by antitoxin and anti-CD11b an...

  11. Differential Effects of Temperature on cAMP-induced Excitation, Adaptation, and Deadaptation of Adenylate and Guanylate Cyclase in Dictyostelium discoideum

    NARCIS (Netherlands)

    Haastert, Peter J.M. van

    1987-01-01

    Extracellular cAMP induces excitation of adenylate and guanylate cyclase in Dictyostelium discoideum. Continuous stimulation with cAMP leads to adaptation, while cells deadapt upon removal of the cAMP stimulus. Excitation of guanylate cyclase by cAMP has a lag time of ~1 s; excitation of adenylate c

  12. Structure-Function Relationships Underlying the Capacity of Bordetella Adenylate Cyclase Toxin to Disarm Host Phagocytes.

    Science.gov (United States)

    Novak, Jakub; Cerny, Ondrej; Osickova, Adriana; Linhartova, Irena; Masin, Jiri; Bumba, Ladislav; Sebo, Peter; Osicka, Radim

    2017-09-24

    Bordetellae, pathogenic to mammals, produce an immunomodulatory adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) that enables them to overcome the innate immune defense of the host. CyaA subverts host phagocytic cells by an orchestrated action of its functional domains, where an extremely catalytically active adenylyl cyclase enzyme is delivered into phagocyte cytosol by a pore-forming repeat-in-toxin (RTX) cytolysin moiety. By targeting sentinel cells expressing the complement receptor 3, known as the CD11b/CD18 (αMβ₂) integrin, CyaA compromises the bactericidal functions of host phagocytes and supports infection of host airways by Bordetellae. Here, we review the state of knowledge on structural and functional aspects of CyaA toxin action, placing particular emphasis on signaling mechanisms by which the toxin-produced 3',5'-cyclic adenosine monophosphate (cAMP) subverts the physiology of phagocytic cells.

  13. Cyclic AMP intoxication of macrophages by a Mycobacterium tuberculosis adenylate cyclase.

    Science.gov (United States)

    Agarwal, Nisheeth; Lamichhane, Gyanu; Gupta, Radhika; Nolan, Scott; Bishai, William R

    2009-07-02

    With 8.9 million new cases and 1.7 million deaths per year, tuberculosis is a leading global killer that has not been effectively controlled. The causative agent, Mycobacterium tuberculosis, proliferates within host macrophages where it modifies both its intracellular and local tissue environment, resulting in caseous granulomas with incomplete bacterial sterilization. Although infection by various mycobacterial species produces a cyclic AMP burst within macrophages that influences cell signalling, the underlying mechanism for the cAMP burst remains unclear. Here we show that among the 17 adenylate cyclase genes present in M. tuberculosis, at least one (Rv0386) is required for virulence. Furthermore, we demonstrate that the Rv0386 adenylate cyclase facilitates delivery of bacterial-derived cAMP into the macrophage cytoplasm. Loss of Rv0386 and the intramacrophage cAMP it delivers results in reductions in TNF-alpha production via the protein kinase A and cAMP response-element-binding protein pathway, decreased immunopathology in animal tissues, and diminished bacterial survival. Direct intoxication of host cells by bacterial-derived cAMP may enable M. tuberculosis to modify both its intracellular and tissue environments to facilitate its long-term survival.

  14. Adenylate Cyclase AcyA Regulates Development, Aflatoxin Biosynthesis and Fungal Virulence in Aspergillus flavus

    Science.gov (United States)

    Yang, Kunlong; Qin, Qiuping; Liu, Yinghang; Zhang, Limei; Liang, Linlin; Lan, Huahui; Chen, Chihao; You, Yunchao; Zhang, Feng; Wang, Shihua

    2016-01-01

    Aspergillus flavus is one of the most important opportunistic pathogens of crops and animals. The carcinogenic mycotoxin, aflatoxins produced by this pathogen cause a health problem to human and animals. Since cyclic AMP signaling controls a range of physiological processes, like fungal development and infection when responding to extracellular stimuli in fungal pathogens, in this study, we investigated the function of adenylate cyclase, a core component of cAMP signaling, in aflatoxins biosynthesis and virulence on plant seeds in A. flavus. A gene replacement strategy was used to generate the deletion mutant of acyA that encodes the adenylate cyclase. Severe defects in fungal growth, sporulation and sclerotia formation were observed in the acyA deletion mutant. The defect in radical growth could be partially rescued by exogenous cAMP analog. The acyA mutant was also significantly reduced in aflatoxins production and virulence. Similar to the former studies in other fungi, The acyA mutant showed enhancing tolerance to oxidative stress, but more sensitive to heat stress. Overall, the pleiotropic defects of the acyA deletion mutant indicates that the cAMP-PKA pathway is involved in fungal development, aflatoxins biosynthesis and plant seed invasion in A. flavus. PMID:28066725

  15. Adenylate-cyclase activity in platelets of patients with obsessive-compulsive disorder

    Directory of Open Access Journals (Sweden)

    D Marazziti, S Baroni

    2009-07-01

    Full Text Available D Marazziti, S Baroni, L Palego, I Masala, G Consoli, M Catena Dell’Osso, G Giannaccini, A LucacchiniDipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Università di Pisa, Pisa, ItalyAbstract: Although the main biological hypothesis on the pathophysiology of obsessive-compulsive disorder (OCD is centered on the serotonin system, indications are available that other neurotransmitters, and even second messengers, particularly the cyclic adenosine monophosphate (cAMP signaling, may be involved, though effective data are few. Therefore, the aim of the present study was to evaluate and compare the basal and isoprenaline (ISO-stimulated velocity of adenylate-cyclase (AC in human platelet membranes of patients with OCD and healthy control subjects. The results showed that the basal and ISO-stimulated AC activity, as well as the dose-response curves of ISO by using agonist concentrations ranging between 0.1 nM and 10 µM, were not different in the two groups. However, OCD patients showed lower EC50 and higher Emax values than healthy subjects. These findings suggest the presence of supersensitive β-adrenergic receptors in platelets of OCD patients.Keywords: obsessive-compulsive disorder, norepinephrine, second messengers, adenylate-cyclase, platelets, isoprenaline, β-adrenergic receptors

  16. BIOTIC STRESS IMPACT ON ACTIVITY OF VARIOUS FORMS OF ADENYLATE CYCLASE IN ORGANELLES OF POTATO PLANT CELLS

    Directory of Open Access Journals (Sweden)

    Lomovatskaya L.A.

    2006-12-01

    Full Text Available Notwithstanding significant interest towards study of adenylate cyclase plant signal system, there is still no complete picture of functioning and regulation mechanisms of this signal system in plants under biotic stress. With this in view, our study was aimed at identification of various forms of adenylate cyclase (transmembrane and “soluble” in the nucleus and chloroplasts of potato cells and modulation of their activity under the impact of exopolysaсcharides ofpotato ring rot pathogen. The investigations conducted allowed to conclude that two forms of adenylate cyclase function in nuclei and chloroplasts of potato plants: transmembrane and “soluble”. Activity of these forms of the enzyme extracted from plant cells of the two potato varieties contrasted by resistance to potato ring rot pathogen Clavibacter michiganensis subsp. sepedonicus, changed in the reverse manner with the mediated impact of exopolysaсcharides secreted by virulent and mucinous strain of bacterial pathogen: in the plants of resistant сultivar it increased, in the plants of sensitive сultivar it was oppressed. It was concluded that activity of both forms of adenylate cyclase directly depended on the degree of resistance of a particular potato variety to given pathogen.

  17. Distribution and protective function of pituitary adenylate cyclase-activating polypeptide (PACAP in the retina

    Directory of Open Access Journals (Sweden)

    Tomoya eNakamachi

    2012-11-01

    Full Text Available Pituitary adenylate cyclase-activating polypeptide (PACAP, which is found in 27- or 38-amino acid forms, belongs to the VIP/glucagon/secretin family. PACAP and its three receptor subtypes are expressed in neural tissues, with PACAP known to exert a protective effect against several types of neural damage. The retina is considered to be part of the central nervous system, and retinopathy is a common cause of profound and intractable loss of vision. This review will examine the expression and morphological distribution of PACAP and its receptors in the retina, and will summarize the current state of knowledge regarding the protective effect of PACAP against different kinds of retinal damage, such as that identified in association with diabetes, ultraviolet light, hypoxia, optic nerve transection, and toxins. This article will also address PACAP-mediated protective pathways involving retinal glial cells.

  18. The effect of adenylate cyclase stimulation on endocochlear potential in the guinea pig.

    Science.gov (United States)

    Doi, K; Mori, N; Matsunaga, T

    1990-01-01

    Forskolin, a diterpene extracted from Coleus forskohlii, is potentially an important tool for studying the modulation of ionic currents by cAMP because it stimulates adenylate cyclase in a variety of cells. We studied the effect of forskolin on cochlear potentials and found that its perfusion of the scala vestibuli (SV) to a concentration more than 10(-5) M and the scala tympani (ST) to more than 10(-4) M produced a reversible elevation of the endocochlear potential (EP) in a dose-dependent manner. The cochlear microphonics recorded simultaneously with the EP was not depressed during the EP elevation. A large negative EP was induced by anoxia following the SV perfusion with forskolin (2 X 10(-4) M). The results suggest that the EP elevation produced by forskolin does not result from the decrease in the negative component of EP but from the increase in the positive component of EP.

  19. Adenylate cyclase 5 is required for melanophore and male pattern development in the guppy (Poecilia reticulata).

    Science.gov (United States)

    Kottler, Verena A; Künstner, Axel; Koch, Iris; Flötenmeyer, Matthias; Langenecker, Tobias; Hoffmann, Margarete; Sharma, Eshita; Weigel, Detlef; Dreyer, Christine

    2015-09-01

    Guppies (Poecilia reticulata) are colorful fish that have attracted the attention of pigmentation researchers for almost a century. Here, we report that the blond phenotype of the guppy is caused by a spontaneous mutation in the guppy ortholog of adenylate cyclase 5 (adcy5). Using double digest restriction site-associated DNA sequencing (ddRADseq) and quantitative trait locus (QTL) mapping, we linked the blond phenotype to a candidate region of 118 kb, in which we subsequently identified a 2-bp deletion in adcy5 that alters splicing and leads to a premature stop codon. We show that adcy5, which affects life span and melanoma growth in mouse, is required for melanophore development and formation of male orange pigmentation traits in the guppy. We find that some components of the male orange pattern are particularly sensitive to loss of Adcy5 function. Our work thus reveals a function for Adcy5 in patterning of fish color ornaments.

  20. Pituitary adenylate cyclase-activating polypeptide: occurrence and relaxant effect in female genital tract

    DEFF Research Database (Denmark)

    Steenstrup, B R; Alm, P; Hannibal, J

    1995-01-01

    that PACAP was located in delicate varicose nerve fibers that were most abundant in the internal cervical os, where they mainly seemed to innervate blood vessels and smooth muscle cells. PACAP-38 and PACAP-27 (10(-10)-10(-6) M) caused a concentration-dependent relaxation of the spontaneous activity......The distribution, localization, and smooth muscle effects of pituitary adenylate cyclase-activating polypeptide (PACAP) were studied in the human female genital tract. The concentrations of PACAP-38 and PACAP-27 were measured by radioimmunoassays, and both peptides were found throughout the genital...... of the nonvascular smooth muscle strips from fallopian tube and myometrium in vitro. Likewise, both peptides (10(-10)-10(-6) M) caused relaxation of nonrepinephrine (10(-6) M)-precontracted intramyometrial arteries. No effect of the PACAP sequences, PACAP-(6-27), PACAP-(16-38), and PACAP-(18-27), on fallopian tube...

  1. Delivery of Bordetella pertussis adenylate cyclase toxin to target cells via outer membrane vesicles.

    Science.gov (United States)

    Donato, Gina M; Goldsmith, Cynthia S; Paddock, Christopher D; Eby, Joshua C; Gray, Mary C; Hewlett, Erik L

    2012-02-17

    Bordetella pertussis adenylate cyclase toxin (ACT) intoxicates cells by producing intracellular cAMP. B. pertussis outer membrane vesicles (OMV) contain ACT on their surface (OMV-ACT), but the properties of OMV-ACT were previously unknown. We found that B. pertussis in the lung from a fatal pertussis case contains OMV, suggesting an involvement in pathogenesis. OMV-ACT and ACT intoxicate cells with and without the toxin's receptor CD11b/CD18. Intoxication by ACT is blocked by antitoxin and anti-CD11b antibodies, but not by cytochalasin-D; in contrast, OMV-ACT is unaffected by either antibody and blocked by cytochalasin-D. Thus OMV-ACT can deliver ACT by processes distinct from those of ACT alone. Copyright © 2012 Federation of European Biochemical Societies. All rights reserved.

  2. Forskolin inhibits the Gs-stimulated adenylate cyclase in rat ascites hepatoma AH66F cells.

    Science.gov (United States)

    Miyamoto, K; Sanae, F; Koshiura, R; Matsunaga, T; Hasegawa, T; Takagi, K; Satake, T

    1989-09-01

    Forskolin increased intracellular cyclic AMP and augmented cyclic AMP formation by prostaglandin E1 (PGE1) in normal rat hepatocytes and ascites hepatoma AH66 cells. However, in AH66F cells which were derived from the AH66 cell line, the diterpene only slightly increased the cyclic AMP level, and dose-dependently inhibited the accumulation caused by PGE1. Forskolin dose-dependently activated adenylate cyclase in these membranes, and the magnitude of activation by forskolin was largest in the following order: hepatocytes, AH66 cells, and AH66F cells. This difference may be based on the number of forskolin-binding sites. The binding affinity of forskolin for each cell membrane was similar. The number and affinity of forskolin-binding sites in these cells were not influenced by 5'-guanylylimidodiphosphate [Gpp(NH)p]. In hepatocytes and AH66 cells, forskolin and other adenylate cyclase activators such as PGE1, GTP, Gpp(NH)p, F-, and Mn2+ synergistically increased the enzyme activity. In AH66F cells, the forskolin-stimulated activity was hardly influenced by the GTP analog, and forskolin diminished the activities induced by the GTP analog in a manner similar to that of diterpene alone. Forskolin (10 microM) also significantly inhibited the activities induced by PGE1, GTP, and F-. The effect of forskolin with Mn2+ was additive in AH66F cells. The data suggest that forskolin promotes the interaction between the stimulatory guanine nucleotide-binding protein and the catalytic unit in the membrane of normal hepatocytes and AH66 cells, but it interferes with the coupling in AH66F cells.

  3. Adenyl cyclases and cAMP in plant signaling - Past and present

    KAUST Repository

    Gehring, Christoph A.

    2010-06-25

    In lower eukaryotes and animals 3\\'-5\\'-cyclic adenosine monophosphate (cAMP) and adenyl cyclases (ACs), enzymes that catalyse the formation of cAMP from ATP, have long been established as key components and second messengers in many signaling pathways. In contrast, in plants, both the presence and biological role of cAMP have been a matter of ongoing debate and some controversy. Here we shall focus firstly on the discovery of cellular cAMP in plants and evidence for a role of this second messenger in plant signal transduction. Secondly, we shall review current evidence of plant ACs, analyse aspects of their domain organisations and the biological roles of candidate molecules. In addition, we shall assess different approaches based on search motifs consisting of functionally assigned amino acids in the catalytic centre of annotated and/or experimentally tested nucleotide cyclases that can contribute to the identification of novel candidate molecules with AC activity such as F-box and TIR proteins. 2010 Gehring; licensee BioMed Central Ltd.

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

    Science.gov (United States)

    Wang, Xianzhe; Maynard, Jennifer A.

    2015-01-01

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

  5. Responsiveness of adenylate cyclase to pituitary gonadotropins and evidence of a hormone-induced desensitization in the lizard ovary.

    Science.gov (United States)

    Borrelli, L; De Stasio, R; Bovenzi, V; Parisi, E; Filosa, S

    1997-07-01

    Gonadotropins (FSH and LH) affect several mammalian gonadal functions. In particular, FSH stimulates oogonial proliferation and oocyte growth, while LH regulates ovulation and progesterone secretion. In lacertilian reptiles, gonadal function is also regulated by pituitary gonadotropins, but which hormone controls ovarian activities and the mechanisms of action are unknown. The present study aimed to clarify mechanisms of action of pituitary gonadotropins on the ovary of Podarcis sicula (Lacertilia). The data demonstrate that mammalian gonadotropins FSH and LH produce a threefold stimulation of adenylate cyclase activity in follicular membranes, while hCG and TSH are less effective, causing a twofold increase in adenylate cyclase activity. Neurotransmitters such as dopamine, serotonin, and catecholamines have no effect on enzyme activity. The action of mammalian FSH and LH on the ovary mimics the effect of homologous hormones: in lizard ovaries incubated in vitro in the presence of isolated homologous pituitary glands, the intracellular cAMP level increased by 50% with respect to control ovaries. Mammalian gonadotropins appear homologous to lizard gonadotropin(s): Southern blot analyses show that the lizard genome contains nucleotide sequences homologous to those encoding for mammalian beta FSH and beta LH. Both homologous and heterologous desensitization of adenylate cyclase activity occurs in the lizard ovary. In fact, responsiveness of adenylate cyclase to gonadotropin stimulation is abolished in animals 2 hr after in vivo treatment with FSH. Sensitivity to gonadotropin stimulation is restored 2 weeks after the beginning of the in vivo treatment. Desensitization was also observed in ovaries incubated in vitro with mammalian FSH or with isolated pituitary glands.

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

    Energy Technology Data Exchange (ETDEWEB)

    Conner, D.A.

    1988-01-01

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

  7. Overexpression of adenylate cyclase-associated protein 2 is a novel prognostic marker in malignant melanoma.

    Science.gov (United States)

    Masugi, Yohei; Tanese, Keiji; Emoto, Katsura; Yamazaki, Ken; Effendi, Kathryn; Funakoshi, Takeru; Mori, Mariko; Sakamoto, Michiie

    2015-12-01

    Malignant melanoma is one of the lethal malignant tumors worldwide. Previously we reported that adenylate cyclase-associated protein 2 (CAP2), which is a well-conserved actin regulator, was overexpressed in hepatocellular carcinoma; however, CAP2 expression in other clinical cancers remains unclear. The aim of the current study was to clarify the clinicopathological significance of CAP2 overexpression in malignant melanoma. Immunohistochemical analyses revealed that many melanoma cells exhibited diffuse cytoplasmic expression of CAP2, whereas no normal melanocytes showed detectable immunostaining for CAP2. A high level of CAP2 expression was seen in 14 of 50 melanomas and was significantly correlated with greater tumor thickness and nodular melanoma subtypes. In addition, a high level of CAP2 expression was associated with poor overall survival in univariate and multivariate analyses. For 13 patients, samples of primary and metastatic melanoma tissue were available: four patients exhibited higher levels of CAP2 expression in metastatic tumor compared to the primary site, whereas no patient showed lower levels of CAP2 expression in metastatic melanomas. Our findings show that CAP2 overexpression is a novel prognostic marker in malignant melanoma and that CAP2 expression seems to increase stepwise during tumor progression, suggesting the involvement of CAP2 in the aggressive behavior of malignant melanoma.

  8. Adenylate cyclase-associated protein 1 overexpressed in pancreatic cancers is involved in cancer cell motility.

    Science.gov (United States)

    Yamazaki, Ken; Takamura, Masaaki; Masugi, Yohei; Mori, Taisuke; Du, Wenlin; Hibi, Taizo; Hiraoka, Nobuyoshi; Ohta, Tsutomu; Ohki, Misao; Hirohashi, Setsuo; Sakamoto, Michiie

    2009-04-01

    Pancreatic cancer has the worst prognosis among cancers due to the difficulty of early diagnosis and its aggressive behavior. To characterize the aggressiveness of pancreatic cancers on gene expression, pancreatic cancer xenografts transplanted into severe combined immunodeficient mice served as a panel for gene-expression profiling. As a result of profiling, the adenylate cyclase-associated protein 1 (CAP1) gene was shown to be overexpressed in all of the xenografts. The expression of CAP1 protein in all 73 cases of pancreatic cancer was recognized by immunohistochemical analyses. The ratio of CAP1-positive tumor cells in clinical specimens was correlated with the presence of lymph node metastasis and neural invasion, and also with the poor prognosis of patients. Immunocytochemical analyses in pancreatic cancer cells demonstrated that CAP1 colocalized to the leading edge of lamellipodia with actin. Knockdown of CAP1 by RNA interference resulted in the reduction of lamellipodium formation, motility, and invasion of pancreatic cancer cells. This is the first report demonstrating the overexpression of CAP1 in pancreatic cancers and suggesting the involvement of CAP1 in the aggressive behavior of pancreatic cancer cells.

  9. Pituitary adenylate cyclase activating peptide (PACAP participates in adipogenesis by activating ERK signaling pathway.

    Directory of Open Access Journals (Sweden)

    Tatjana Arsenijevic

    Full Text Available Pituitary adenylate cyclase activating peptide (PACAP belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP family. Its action can be mediated by three different receptor subtypes: PAC1, which has exclusive affinity for PACAP, and VPAC1 and VPAC2 which have equal affinity for PACAP and VIP. We showed that all three receptors are expressed in 3T3-L1 cells throughout their differentiation into adipocytes. We established the activity of these receptors by cAMP accumulation upon induction by PACAP. Together with insulin and dexamethasone, PACAP induced adipogenesis in 3T3-L1 cell line. PACAP increased cAMP production within 15 min upon stimulation and targeted the expression and phosphorylation of MAPK (ERK1/2, strengthened by the ERK1/2 phosphorylation being partially or completely abolished by different combinations of PACAP receptors antagonists. We therefore speculate that ERK1/2 activation is crucial for the activation of CCAAT/enhancer- binding protein β (C/EBPβ.

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

    Directory of Open Access Journals (Sweden)

    Mounira Tlili

    2015-01-01

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

  11. Adenylate Cyclase Type III Is Not a Ubiquitous Marker for All Primary Cilia during Development

    Science.gov (United States)

    Antal, Maria Cristina; Bénardais, Karelle; Samama, Brigitte; Auger, Cyril; Schini-Kerth, Valérie; Ghandour, Said; Boehm, Nelly

    2017-01-01

    Adenylate cyclase type III (AC3) is localized in plasma membrane of neuronal primary cilium and can be used as a marker of this cilium. AC3 has also been detected in some other primary cilia such as those of fibroblasts, synoviocytes or astrocytes. Despite the presence of a cilium in almost all cell types, we show that AC3 is not a common marker of all primary cilia of different human and mouse tissues during development. In peripheral organs, AC3 is present mainly in primary cilia in cells of the mesenchymal lineage (fibroblasts, chondroblasts, osteoblasts-osteocytes, odontoblasts, muscle cells and endothelial cells). In epithelia, the apical cilium of renal and pancreatic tubules and of ductal plate in liver is AC3-negative whereas the cilium of basal cells of stratified epithelia is AC3-positive. Using fibroblasts cell culture, we show that AC3 appears at the plasma membrane of the primary cilium as soon as this organelle develops. The functional significance of AC3 localization at the cilium membrane in some cells but not others has to be investigated in relationship with cell physiology and expression at the cilium plasma membrane of specific upstream receptors. PMID:28122017

  12. How adenylate cyclase choreographs the pas de deux of the receptors heteromerization dance.

    Science.gov (United States)

    Woods, A S; Jackson, S N

    2013-05-15

    Our work suggests that heteromer formation, mainly involves linear motifs (LMs) found in disordered regions of proteins. Local disorder imparts plasticity to LMs. Most molecular recognition of proteins occurs between short linear segments, known as LMs. Interaction of short continuous epitopes is not constrained by sequence and has the advantage of resulting in interactions with micromolar affinities which suit transient, reversible complexes such as receptor heteromers. Electrostatic interactions between epitopes of the G-protein coupled receptors (GPCR) involved, are the key step in driving heteromer formation forward. The first step in heteromerization, involves phosphorylating Ser/Thr in an epitope containing a casein kinase 1/2-consensus site. Our data suggest that dopaminergic neurotransmission, through cAMP-dependent protein kinase A (PKA) slows down heteromerization. The negative charge, acquired by the phosphorylation of a Ser/Thr in a PKA consensus site in the Arg-rich epitope, affects the activity of the receptors involved in heteromerization by causing allosteric conformational changes, due to the repulsive effect generated by the negatively charged phosphate. In addition to modulating heteromerization, it affects the stability of the heteromers' interactions and their binding affinity. So here we have an instance where phosphorylation is not just an on/off switch, instead by weakening the noncovalent bond, heteromerization acts like a rheostat that controls the stability of the heteromer through activation or inhibition of adenylate cyclase by the neurotransmitter Dopamine depending on which Dopamine receptor it docks at. Published by Elsevier Ltd.

  13. Cytochemical localization of adenylate cyclase activity in heart tissue with cerium.

    Science.gov (United States)

    Schulze, W; Will-Shahab, L; Küttner, I

    1986-01-01

    Adenylate cyclase (AC) activity showed a doses depending inactivation of the basal activity and of the sodium fluoride stimulation by cerium in homogenates of unfixed and fixed guinea pig hearts. The isoproterenol and guanine nucleotide stimulation was not more than two times of the basal activity in glutaraldehyde-prefixed heart homogenates in the presence of 2 mmol/l CeCl3. The inactivation of the AC (activity) by cerium was less than in the presence of lead. Test tube experiments showed no differences in the precipitation of imidodiphosphate in comparison with inorganic phosphate. The substrate AMP-PNP was not spontaneously hydrolysed by 2 mmol/l CeCl3. Ultrastructural analysis of cytochemical incubation of glutaraldehyde-fixed slices and small pieces of guinea pig heart tissue showed fine-amorphous precipitations of reaction products localized along the plasma membrane of the sarcolemma, the nexuses of the intercalated discs and the T-tubule membranes. No precipitates were found neither on the junctional nor on other SR membranes. Nonspecific coarse and clumped precipitates have been detected in the intercellular space on components of the basal membranes. It was not able to demonstrate cytochemically stimulation of AC by hormones or by sodium fluoride. The localization of the basal AC activity in heart tissue seems to be better with cerium as capture agent than with lead. However, differences in the localization of the AC activity in heart tissue were not observed.

  14. Cellular localization of pituitary adenylate cyclase-activating peptide (PACAP) following traumatic brain injury in humans.

    Science.gov (United States)

    van Landeghem, Frank K H; Weiss, Thorsten; Oehmichen, Manfred; von Deimling, Andreas

    2007-06-01

    The pituitary adenylate cyclase-activating peptide (PACAP) is involved in many processes of the developing and mature central nervous system, such as proliferation, differentiation, apoptosis, neurotransmission, inflammation and neuroprotection. Alternative posttranslational processing of PACAP results in two biologically active, amidated 27- and 38-amino acid peptides termed PACAP27 and PACAP38. In the present study, we examined whether traumatic brain injury (TBI) affects cellular immunopositivity for PACAP27 and PACAP38. Patients (n = 55) were classified into three groups dependent on their survival time (under 24 h, between 24 h and 7 days and between 7 days and 99 days postinjury). PACAP27 and PACAP38 were expressed by neurons and glial cells in normal human neocortex (n = 10). Following TBI, the total number of PACAP27- and PACAP38-positive cells was significantly decreased for a prolonged survival period within the traumatized neocortex. In the pericontusional cortex, the number of cells expressing PACAP27 and PACAP38 was significantly increased at all survival times examined. Triple immunofluorescence examinations revealed a significant increase in the absolute numbers of GFAP-positive reactive astrocytes as well as a decrease in the CNP-positive oligodendrocytes, each coexpressing PACAP27 or PACAP38 in the contusional and pericontusional cortex. We hypothesize that the increase of glial PACAP immunoreactivity may be interpreted as part of a complex endogenous neuroprotective response in the pericontusional regions, but the precise role of PACAP following TBI is yet to be determined.

  15. Molecular cloning and expression of a chicken pituitary adenylate cyclase-activating polypeptide receptor.

    Science.gov (United States)

    Peeters, K; Gerets, H H; Princen, K; Vandesande, F

    1999-08-25

    Although, since the isolation of pituitary adenylate cyclase-activating polypeptide (PACAP), a wealth of literature has been published describing its localization, binding sites, and biological activities in a variety of mammalian tissues, only very little is known about PACAP in avian species. Therefore, in order to find out the sites of actions of PACAP and to elucidate its physiological significance in birds, we identified a chicken PACAP receptor homologue of the mammalian type I receptors (PAC(1)-Rs). The chicken PACAP type I cDNA sequence was obtained using reverse transcriptase-polymerase chain reaction (RT-PCR) in combination with 3'- and 5'-RACE PCR. This cDNA encodes a 471 amino acid precursor protein, sharing 81-83% sequence identity with mammalian analogs and 76% amino acid identity with the goldfish type I PACAP receptor. Northern blot analysis of chicken brain poly(A)(+)-rich RNA revealed the presence of a 5.5 kb and 7.5 kb PAC(1) receptor transcript. RT-PCR revealed that the chicken PACAP receptor is mainly expressed in the brain and gonads. A smaller amount of the receptor mRNA was found in pituitary, adrenal gland, kidney, intestine, pancreas, lung, and heart tissue. In situ hybridization with specific antisense oligodeoxynucleotide probes showed a widespread distribution of PAC(1) receptor mRNA in the chicken brain, with the highest expression being found in the dorsal telencephalon, olfactory bulb, hypothalamus, optic tectum, and cerebellar cortex. These findings suggest that PACAP affect a variety of functions both in the brain and peripheral tissues of the chicken.

  16. Pituitary adenylate cyclase activating polypeptide modulates catecholamine storage and exocytosis in PC12 cells.

    Directory of Open Access Journals (Sweden)

    Yan Dong

    Full Text Available A number of efforts have been made to understand how pituitary adenylate cyclase activating polypeptide (PACAP functions as a neurotrophic and neuroprotective factor in Parkinson's disease (PD. Recently its effects on neurotransmission and underlying mechanisms have generated interest. In the present study, we investigate the effects of PACAP on catecholamine storage and secretion in PC12 cells with amperometry and transmission electron microscopy (TEM. PACAP increases quantal release induced by high K+ without significantly regulating the frequency of vesicle fusion events. TEM data indicate that the increased volume of the vesicle is mainly the result of enlargement of the fluidic space around the dense core. Moreover, the number of docked vesicles isn't modulated by PACAP. When cells are acutely treated with L-DOPA, the vesicular volume and quantal release both increase dramatically. It is likely that the characteristics of amperometric spikes from L-DOPA treated cells are associated with increased volume of individual vesicles rather than a direct effect on the mechanics of exocytosis. Treatment with PACAP versus L-DOPA results in different profiles of the dynamics of exocytosis. Release via the fusion pore prior to full exocytosis was observed with the same frequency following treatment with PACAP and L-DOPA. However, release events have a shorter duration and higher average current after PACAP treatment compared to L-DOPA. Furthermore, PACAP reduced the proportion of spikes having rapid decay time and shortened the decay time of both fast and slow spikes. In contrast, the distributions of the amperometric spike decay for both fast and slow spikes were shifted to longer time following L-DOPA treatment. Compared to L-DOPA, PACAP may produce multiple favorable effects on dopaminergic neurons, including protecting dopaminergic neurons against neurodegeneration and potentially regulating dopamine storage and release, making it a promising

  17. Comprehensive behavioral analysis of pituitary adenylate cyclase-activating polypeptide (PACAP) knockout mice.

    Science.gov (United States)

    Hattori, Satoko; Takao, Keizo; Tanda, Koichi; Toyama, Keiko; Shintani, Norihito; Baba, Akemichi; Hashimoto, Hitoshi; Miyakawa, Tsuyoshi

    2012-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. PACAP is widely expressed throughout the brain and exerts its functions through the PACAP-specific receptor (PAC(1)). Recent studies reveal that genetic variants of the PACAP and PAC(1) genes are associated with mental disorders, and several behavioral abnormalities of PACAP knockout (KO) mice are reported. However, an insufficient number of backcrosses was made using PACAP KO mice on the C57BL/6J background due to their postnatal mortality. To elucidate the effects of PACAP on neuropsychiatric function, the PACAP gene was knocked out in F1 hybrid mice (C57BL/6J × 129SvEv) for appropriate control of the genetic background. The PACAP KO mice were then subjected to a behavioral test battery. PACAP deficiency had no significant effects on neurological screen. As shown previously, the mice exhibited significantly increased locomotor activity in a novel environment and abnormal anxiety-like behavior, while no obvious differences between genotypes were shown in home cage (HC) activity. In contrast to previous reports, the PACAP KO mice showed normal prepulse inhibition (PPI) and slightly decreased depression-like behavior. Previous study demonstrates that the social interaction (SI) in a resident-intruder test was decreased in PACAP KO mice. On the other hand, we showed that PACAP KO mice exhibited increased SI in Crawley's three-chamber social approach test, although PACAP KO had no significant impact on SI in a HC. PACAP KO mice also exhibited mild performance deficit in working memory in an eight-arm radial maze (RM) and the T-maze (TM), while they did not show any significant abnormalities in the left-right discrimination task in the TM. These results suggest that PACAP has an important role in the regulation of locomotor activity, social behavior, anxiety-like behavior and, potentially, working memory.

  18. Comprehensive behavioral analysis of pituitary adenylate cyclase-activating polypeptide (PACAP knockout mice

    Directory of Open Access Journals (Sweden)

    Satoko eHattori

    2012-10-01

    Full Text Available Pituitary adenylate cyclase-activating polypeptide (PACAP is a neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. PACAP is widely expressed throughout the brain and exerts its functions through the PACAP-specific receptor (PAC1. Recent studies reveal that genetic variants of the PACAP and PAC1 genes are associated with mental disorders, and several behavioral abnormalities of PACAP knockout (KO mice are reported. However, an insufficient number of backcrosses was made using PACAP KO mice on the C57BL/6J background due to their postnatal mortality. To elucidate the effects of PACAP on neuropsychiatric function, the PACAP gene was knocked out in F1 hybrid mice (C57BL/6J x 129SvEv for appropriate control of the genetic background. The PACAP KO mice were then subjected to a behavioral test battery. PACAP deficiency had no significant effects on neurological screen. As shown previously, the mice exhibited significantly increased locomotor activity in a novel environment and abnormal anxiety-like behavior, while no obvious differences between genotypes were shown in home cage activity. In contrast to previous reports, the PACAP KO mice showed normal prepulse inhibition and slightly decreased depression-like behavior. Previous study demonstrates that the social interaction in a resident-intruder test was decreased in PACAP KO mice. On the other hand, we showed that PACAP KO mice exhibited increased social interaction in Crawley’s three-chamber social approach test, although PACAP KO had no significant impact on social interaction in a home cage. PACAP KO mice also exhibited mild performance deficit in working memory in an eight-arm radial maze and the T-maze, while they did not show any significant abnormalities in the left-right discrimination task in the T-maze. These results suggest that PACAP has an important role in the regulation of locomotor activity, social behavior, anxiety-like behavior and, potentially

  19. Pituitary adenylate cyclase-activating polypeptide is a sympathoadrenal neurotransmitter involved in catecholamine regulation and glucohomeostasis.

    Science.gov (United States)

    Hamelink, Carol; Tjurmina, Olga; Damadzic, Ruslan; Young, W Scott; Weihe, Eberhard; Lee, Hyeon-Woo; Eiden, Lee E

    2002-01-08

    The adrenal gland is important for homeostatic responses to metabolic stress: hypoglycemia stimulates the splanchnic nerve, epinephrine is released from adrenomedullary chromaffin cells, and compensatory glucogenesis ensues. Acetylcholine is the primary neurotransmitter mediating catecholamine secretion from the adrenal medulla. Accumulating evidence suggests that a secretin-related neuropeptide also may function as a transmitter at the adrenomedullary synapse. Costaining with highly specific antibodies against the secretin-related neuropeptide pituitary adenylate cyclase-activating peptide (PACAP) and the vesicular acetylcholine transporter (VAChT) revealed that PACAP is found in nerve terminals at all mouse adrenomedullary cholinergic synapses. Mice with a targeted deletion of the PACAP gene had otherwise normal cholinergic innervation and morphology of the adrenal medulla, normal adrenal catecholamine and blood glucose levels, and an intact initial catecholamine secretory response to insulin-induced hypoglycemia. However, insulin-induced hypoglycemia was more profound and longer-lasting in PACAP knock-outs, and was associated with a dose-related lethality absent in wild-type mice. Failure of PACAP-deficient mice to adequately counterregulate plasma glucose levels could be accounted for by impaired long-term secretion of epinephrine, secondary to a lack of induction of tyrosine hydroxylase, normally occurring after insulin hypoglycemia in wild-type mice, and a consequent depletion of adrenomedullary epinephrine stores. Thus, PACAP is needed to couple epinephrine biosynthesis to secretion during metabolic stress. PACAP appears to function as an "emergency response" cotransmitter in the sympathoadrenal axis, where the primary secretory response is controlled by a classical neurotransmitter but sustained under paraphysiological conditions by a neuropeptide.

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

    Energy Technology Data Exchange (ETDEWEB)

    Springer, Tzvia I.; Goebel, Erich; Hariraju, Dinesh [Department of Microbiology, Miami University, Oxford, OH 45056 (United States); Finley, Natosha L., E-mail: finleynl@miamioh.edu [Department of Microbiology, Miami University, Oxford, OH 45056 (United States); Cell, Molecular, and Structural Biology Program, Miami University, Oxford, OH 45056 (United States)

    2014-10-10

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

  1. Effect of Cardiopulmonary Bypass on Beta Adrenergic ReceptorAdenylate Cyclase System on Surfaces of Peripheral Lymphocytes

    Institute of Scientific and Technical Information of China (English)

    LUO Ailin; TIAN Yuke; JIN Shiao

    2000-01-01

    The experimental results showed that the level of CAMP, the ratio of cAPM to cGMP,IL-2R expression and IL-2 production in vitro in lymphocytes immediate and 2 weeks after cardiopulmonary bypass (CPB) were significantly lower than those before anesthetics in the patients undergoing cardiac surgery with CPB. These findings suggested that CPB could cause serious damage to adrenergic beta receptor-adenylate cyclase system on circulating lymphocytes surfaces,which might be one of the mechanisms resulting in immunosuppression after open heart surgery with CPB.

  2. The Arabidopsis thalianaK+-uptake permease 7 (AtKUP7) contains a functional cytosolic adenylate cyclase catalytic centre

    KAUST Repository

    Al-Younis, Inas

    2015-11-27

    Adenylate Cyclases (ACs) catalyze the formation of the second messenger cyclic adenosine 3′, 5′-monophosphate (cAMP) from adenosine 5’-triphosphate (ATP). Although cAMP is increasingly recognized as an important signaling molecule in higher plants, ACs have remained somewhat elusive. Here we used a search motif derived from experimentally tested guanylyl cyclases (GCs), substituted the residues essential for substrate specificity and identified the Arabidopsis thaliana K+-uptake permease 7 (AtKUP7) as one of several candidate ACs. Firstly, we show that a recombinant N-terminal, cytosolic domain of AtKUP71-100 is able to complement the AC-deficient mutant cyaA in Escherichia coli and thus restoring the fermentation of lactose, and secondly, we demonstrate with both enzyme immunoassays and mass spectrometry that a recombinant AtKUP71-100 generates cAMP in vitro.

  3. Morphine-induced changes of adenylate and guanylate cyclase in locus ceruleus, periaqueductal gray, and substantia nigra in rats.

    Science.gov (United States)

    Shijun, Hong; Liping, Zhao; Yongqiang, Qu; Zhen, Li; Yonghe, Zhao; Lihua, Li

    2009-01-01

    To observe the changes of adenylate cyclase (AC) and guanylate cyclase (GC) in the cerebral regions including the locus ceruleus, periaqueductal gray, and substantia nigra in rats that were physiologically dependent on morphine. We also investigated the relationship of enzymatic changes in these cerebral regions to the mechanism of morphine dependence. A morphine-dependent rat model was established and withdrawal symptoms evaluated. Enzyme histochemistry was used to detect the variations of AC and GC in cerebral regions. Compared to controls, AC and GC significantly increased in morphine-dependent groups. Comparisons of four different morphine-dependent groups also showed AC and GC significantly differed at weeks 1, 2, 4, and 8. Results found that the content of AC and GC increased in these cerebral regions in rats that demonstrated morphine dependence and appeared to be closely linked to increases in AC and GC activity.

  4. Studies on cell migration, adenylate cyclase and membrane-coating granules in the buccal epithelium of the zinc-deficient rabbit, including the influence of isoproterenol.

    Science.gov (United States)

    Chen, S Y

    1988-01-01

    Cell migration was slightly increased; cytochemical reaction deposits of adenylate cyclase and the area density of membrane-coating granules (MCG) were significantly increased. Upon isoproterenol stimulation, the MCG area density was significantly increased, whereas the cell migration rate was unchanged. Thus in zinc deficiency, there may be a simultaneous increase in the production and secretion of MCGs, in adenylate cyclase activity, and in cell migration. The non-significantly increased cell migration rate may not keep pace with the significantly increased cell-production rate, resulting in thickening of the epithelium.

  5. Inhibition of adenylate cyclase by delta 9-tetrahydrocannabinol in mouse spleen cells: a potential mechanism for cannabinoid-mediated immunosuppression.

    Science.gov (United States)

    Schatz, A R; Kessler, F K; Kaminski, N E

    1992-01-01

    The ability of delta 9-Tetrahydrocannabinol (delta 9-THC) to modulate adenylate cyclase activity in mouse spleen cells was investigated. These studies were prompted by the recent identification and cloning of a G-protein coupled cannabinoid receptor localized in certain regions of the brain and the potential for a common mechanism between cannabinoid-mediated CNS effects and immunosuppression. Temporal addition studies were initially performed to identify the period of time when spleen cells in culture were most susceptible to the inhibitory effects of delta 9-THC, as measured by the day 5 IgM antibody forming cell response. delta 9-THC was only inhibitory when added to spleen cell cultures during the first 2 hr following antigen sensitization. In light of this time course, adenylate cyclase activity was measured in spleen cells incubated in the presence of 22 microM delta 9-THC for 5 min and subsequently stimulated with forskolin. delta 9-THC treated spleen cells demonstrated a 33% inhibition and a 66% inhibition in intracellular cAMP after a 5 or 15 min stimulation with forskolin, respectively. These studies suggest that inhibition of immune function by delta 9-THC may be mediated through the inhibition of intracellular cAMP early after antigen stimulation.

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

    Directory of Open Access Journals (Sweden)

    Kepa B Uribe

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

  7. Changes of Adenylate Cyclase and Guanylate Cyclase in the Frontal Cortex, Lenticula, Corpus Amygdaloideum, and Hippocampus in Morphine-dependent Rats

    Directory of Open Access Journals (Sweden)

    Shijun Hong

    2016-01-01

    Full Text Available To detect the changes of adenylate cyclase (AC and guanylate cyclase (GC in the four cerebral regions that are concerned with psychogenic dependence of morphine in rats, including the frontal cortex, lenticula, corpus amygdaloideum, and hippocampus. To discuss the relation between the expressions of AC and GC with the psychogenic dependence on morphine. Different periods of morphine-dependent rat models were established, and enzyme histochemistry was used to detect the variations of AC and GC in four cerebral regions. Compared with the control group, AC and GC in all the morphine-dependent groups increased. The data indicated that the amounts of AC and GC were significantly different between the morphine-dependent groups and the control group when tested at periods of 1 week, 2 weeks, 4 weeks, and 8 weeks (P ˂ 0.05 or P ˂ 0.01. There were significant differences when comparing the 1-week group with the 2-week, 4-week, and 8-week groups (P ˂ 0.05 or P ˂ 0.01. There were significant differences when comparing the 2-week dependent group with the 4-week dependent group or the 8-week dependent group (P ˂ 0.05 or P ˂ 0.01. The activities of AC and GC increased in four cerebral regions of morphine-dependent rats. The psychogenic dependence on morphine appears to be closely linked to the upgrade of AC and GC.

  8. Knocking down the expression of adenylate cyclase-associated protein 1 inhibits the proliferation and migration of breast cancer cells.

    Science.gov (United States)

    Yu, Xia-Fei; Ni, Qi-Chao; Chen, Jin-Peng; Xu, Jun-Fei; Jiang, Ying; Yang, Shu-Yun; Ma, Jing; Gu, Xiao-Ling; Wang, Hua; Wang, Ying-Ying

    2014-04-01

    Adenylate cyclase-associated protein 1 (CAP1) is a conserved protein that was found to be up-regulated in breast cancer and related to the migration of breast cancer. We verified its roles in breast cancer specimens and cell lines. In our results, 71 of 100 specimens of breast cancer showed high levels of CAP1 by immunohistochemistry. Associated with statistical analysis, we saw that CAP1 was related to the grade of breast cancer. In MDA-MB-231, the expression of CAP1 was the highest and by knocking down the expression of CAP1 in MDA-MB-231, its ability for proliferating and migrating apparently decreased and induced changes in morphology, which were related to the arrangement of F-actin. Therefore, CAP1 might be a potential molecular targeted therapy for surgery and immune treatment.

  9. Effects of cimetidine on adenylate cyclase activity of guinea pig gastric mucosa stimulated by histamine, sodium fluoride and 5'-guanylylimidodiphosphate.

    Science.gov (United States)

    Anttila, P; Westermann, E

    1976-08-01

    Cimetidine, a recently developed histamine H2-receptor blocking agent has been shown to be a potent inhibitor of histamine-stimulated gastric acid secretion in rat, cat, dog and man. To study the mode of action of cimetidine the modification of stimulatory effects of histamine, sodium flouride and 5'-guanylylimidodiphosphate by cimetidine on the adenylate cyclase activity of guinea pig gastric mucosa was studied. The effect of cimetidine was also compared to that of metiamide, an older histamine H2-receptor antagonist. The effect of cimetidine was qualitatively similar to that of metiamide, i.e. a selective blockade of histamine H2-receptors. Quantitatively cimetidine was about 10-fold more potent than metiamide.

  10. Adenylate cyclase toxin-mediated delivery of the S1 subunit of pertussis toxin into mammalian cells.

    Science.gov (United States)

    Iwaki, Masaaki; Konda, Toshifumi

    2016-02-01

    The adenylate cyclase toxin (ACT) of Bordetella pertussis internalizes its catalytic domain into target cells. ACT can function as a tool for delivering foreign protein antigen moieties into immune effector cells to induce a cytotoxic T lymphocyte response. In this study, we replaced the catalytic domain of ACT with an enzymatically active protein moiety, the S1 (ADP-ribosyltransferase) subunit of pertussis toxin (PT). The S1 moiety was successfully internalized independent of endocytosis into sheep erythrocytes. The introduced polypeptide exhibited ADP-ribosyltransferase activity in CHO cells and induced clustering typical to PT. The results indicate that ACT can act as a vehicle for not only epitopes but also enzymatically active peptides to mammalian cells.

  11. The adenylate cyclase gene MaAC is required for virulence and multi-stress tolerance of Metarhizium acridum

    Directory of Open Access Journals (Sweden)

    Liu Shuyang

    2012-08-01

    Full Text Available Abstract Background The efficacy of entomopathogenic fungi in pest control is mainly affected by various adverse environmental factors, such as heat shock and UV-B radiation, and by responses of the host insect, such as oxidative stress, osmotic stress and fever. In this study, an adenylate cyclase gene (MaAC was cloned from the locust-specific entomopathogenic fungus, Metarhizium acridum, which is homologous to various fungal adenylate cyclase genes. RNA silencing was adapted to analyze the role of MaAC in virulence and tolerance to adverse environmental and host insect factors. Results Compared with the wild type, the vegetative growth of the RNAi mutant was decreased in PD (potato dextrose medium, Czapek-dox and PDA plates, respectively, demonstrating that MaAC affected vegetative growth. The cAMP levels were also reduced in PD liquid culture, and exogenous cAMP restored the growth of RNAi mutants. These findings suggested that MaAC is involved in cAMP synthesis. The knockdown of MaAC by RNAi led to a reduction in virulence after injection or topical inoculation. Furthermore, the RNAi mutant grew much slower than the wild type in the haemolymph of locust in vitro and in vivo, thus demonstrating that MaAC affects the virulence of M. acridum via fungal growth inside the host locust. A plate assay indicated that the tolerances of the MaAC RNAi mutant under oxidative stress, osmotic stress, heat shock and UV-B radiation was decreased compared with the wild type. Conclusion MaAC is required for virulence and tolerance to oxidative stress, osmotic stress, heat shock and UV-B radiation. MaAC affects fungal virulence via vegetative growth inside the insect and tolerance against oxidative stress, osmotic stress and locust fever.

  12. Calcium influx rescues adenylate cyclase-hemolysin from rapid cell membrane removal and enables phagocyte permeabilization by toxin pores.

    Directory of Open Access Journals (Sweden)

    Radovan Fiser

    Full Text Available Bordetella adenylate cyclase toxin-hemolysin (CyaA penetrates the cytoplasmic membrane of phagocytes and employs two distinct conformers to exert its multiple activities. One conformer forms cation-selective pores that permeabilize phagocyte membrane for efflux of cytosolic potassium. The other conformer conducts extracellular calcium ions across cytoplasmic membrane of cells, relocates into lipid rafts, translocates the adenylate cyclase enzyme (AC domain into cells and converts cytosolic ATP to cAMP. We show that the calcium-conducting activity of CyaA controls the path and kinetics of endocytic removal of toxin pores from phagocyte membrane. The enzymatically inactive but calcium-conducting CyaA-AC⁻ toxoid was endocytosed via a clathrin-dependent pathway. In contrast, a doubly mutated (E570K+E581P toxoid, unable to conduct Ca²⁺ into cells, was rapidly internalized by membrane macropinocytosis, unless rescued by Ca²⁺ influx promoted in trans by ionomycin or intact toxoid. Moreover, a fully pore-forming CyaA-ΔAC hemolysin failed to permeabilize phagocytes, unless endocytic removal of its pores from cell membrane was decelerated through Ca²⁺ influx promoted by molecules locked in a Ca²⁺-conducting conformation by the 3D1 antibody. Inhibition of endocytosis also enabled the native B. pertussis-produced CyaA to induce lysis of J774A.1 macrophages at concentrations starting from 100 ng/ml. Hence, by mediating calcium influx into cells, the translocating conformer of CyaA controls the removal of bystander toxin pores from phagocyte membrane. This triggers a positive feedback loop of exacerbated cell permeabilization, where the efflux of cellular potassium yields further decreased toxin pore removal from cell membrane and this further enhances cell permeabilization and potassium efflux.

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

    Science.gov (United States)

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

  14. High expression of adenylate cyclase-associated protein 1 accelerates the proliferation, migration and invasion of neural glioma cells.

    Science.gov (United States)

    Bao, Zhen; Qiu, Xiaojun; Wang, Donglin; Ban, Na; Fan, Shaochen; Chen, Wenjuan; Sun, Jie; Xing, Weikang; Wang, Yunfeng; Cui, Gang

    2016-04-01

    Adenylate cyclase-associated protein 1 (CAP1), a conserved member of cyclase-associated proteins was reported to be associated with the proliferation, migration or invasion of the tumors of pancreas, breast and liver, and was involved in astrocyte proliferation after acute Traumatic Brain Injury (TBI). In this study, we sought to investigate the character of CAP1 in the pathological process of human glioma by detecting human glioma specimens and cell lines. 43 of 100 specimens showed high expression of CAP1 via immunohistochemistry. With statistics analysis, we found out the expression level of CAP1 was correlated with the WHO grades of human glioma and was great positively related to Ki-67 (p<0.01). In vitro, silencing CAP1 in U251 and U87MG, the glioma cell lines with the relatively higher expression of CAP1, induced the proliferation of the cells significantly retarded, migration and invasion as well. Obviously, our results indicated that CAP1 participated in the molecular pathological process of glioma indeed, and in a certain sense, CAP1 might be a potential and promising molecular target for glioma diagnosis and therapies in the future.

  15. Studies on responsiveness of hepatoma cells to catecholamines. III. Difference between the receptor-adenylate cyclase regulating systems in AH130 cells and cultured normal rat liver cells.

    Science.gov (United States)

    Sanae, F; Matsunaga, T; Miyamoto, K; Koshiura, R

    1986-10-01

    The responsiveness to three beta-adrenergic agonists, isoproterenol (IPN), epinephrine (Epi) and norepinephrine (NE) in AH13O cells was examined compared with that in normal rat liver cells which were cultured for 24 hr after collagenase digestion. As regards to the activation of adenylate cyclase in the cell homogenates, the relative affinity of the three agonists was in order of IPN greater than NE greater than Epi in AH130 cells and IPN greater than Epi greater than NE in cultured normal liver cells. While the efficacies of the three agonists were similar in cultured liver cells, those of NE and Epi were markedly lower than that of IPN in AH13O cells and were increased to the similar level of IPN by pretreatment with phentolamine, but not with prazosin. Clonidine inhibited the activation of adenylate cyclase by IPN in AH13O cells. When cells were preincubated with islet-activating protein (IAP), the activity of adenylate cyclase in the presence or absence of agonist in both cell lines increased. In IAP-treated AH13O cells, the efficacies of NE and Epi became close to that of IPN. Adenylate cyclase in IAP-treated AH13O cells was activated by GTP in a dose-dependent manner, but that in IAP-treated cultured liver cells was not. In the presence of IPN, biphasic (activatory and inhibitory) effects of GTP on the cyclase were observed, and the inhibitory phase was eliminated by the IAP-treatment in both cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. A novel antithrombotic effect of sulforaphane via activation of platelet adenylate cyclase: ex vivo and in vivo studies.

    Science.gov (United States)

    Jayakumar, Thanasekaran; Chen, Wei-Fan; Lu, Wan-Jung; Chou, Duen-Suey; Hsiao, George; Hsu, Chung-Yi; Sheu, Joen-Rong; Hsieh, Cheng-Ying

    2013-06-01

    Sulforaphane is a naturally occurring isothiocyanate, which can be found in cruciferous vegetables such as broccoli and cabbage. Sulforaphane was found to have very potent inhibitory effects on tumor growth through regulation of diverse mechanisms. However, no data are available concerning the effects of sulforaphane on platelet activation and its relative issues. Activation of platelets caused by arterial thrombosis is relevant to a variety of cardiovascular diseases. Hence, the aim of this study was to examine the in vivo antithrombotic effects of sulforaphane and its possible mechanisms in platelet activation. Sulforaphane (0.125 and 0.25 mg/kg) was effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice. Other in vivo studies also revealed that sulforaphane (0.25 mg/kg) significantly prolonged platelet plug formation in mice. In addition, sulforaphane (15-75 μM) exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen. Sulforaphane inhibited platelet activation accompanied by inhibiting relative Ca(2+) mobilization; phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs) and Akt; and hydroxyl radical (OH(●)) formation. Sulforaphane markedly increased cyclic (c)AMP, but not cyclic (c)GMP levels, and stimulated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, but not ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxal in-1-one), an inhibitor of guanylate cyclase, obviously reversed the sulforaphane-mediated effects on platelet aggregation; PKC activation, p38 MAPK, Akt and VASP phosphorylation; and OH(●) formation. Furthermore, a PI3-kinase inhibitor (LY294002) and a p38 MAPK inhibitor (SB203580) both significantly diminished PKC activation and p38 MAPK and Akt phosphorylation; in contrast, a PKC inhibitor (RO318220) did not diminish p38 MAPK or Akt phosphorylation stimulated by collagen. This

  17. Bi-directional effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on fear-related behavior and c-Fos expression after fear conditioning in rats

    OpenAIRE

    Meloni, Edward G.; Venkataraman, Archana; Donahue, Rachel J.; Carlezon, William A.

    2015-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is implicated in stress regulation and learning and memory. PACAP has neuromodulatory actions on brain structures within the limbic system that could contribute to its acute and persistent effects in animal models of stress and anxiety-like behavior. Here, male Sprague-Dawley rats were implanted with intracerebroventricular (ICV) cannula for infusion of PACAP-38 (0.5, 1, or 1.5 ug) or vehicle followed 30 min later by fear conditioning...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-03-01

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

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

    Science.gov (United States)

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

    2014-10-10

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

  20. Stress tolerance of the Saccharomyces cerevisiae adenylate cyclase fil1 (CYR1) mutant depends on Hsp26.

    Science.gov (United States)

    Vianna, Cristina R; Ferreira, Mariana C; Silva, Carol L C; Tanghe, An; Neves, Maria J; Thevelein, Johan M; Rosa, Carlos A; Van Dijck, Patrick

    2010-01-01

    Fermentation-induced loss of stress resistance in yeast is an important phenotype from an industrial point of view. It hampers optimal use of frozen dough applications as well as high gravity brewing fermentations because these applications require stress-tolerant yeast strains during active fermentation. Different mutants (e.g. fil1, an adenylate cyclase mutant CYR1(lys1682)) that are affected in this loss of stress resistance have been isolated, but so far the identification of the target genes important for the increased tolerance has failed. Previously we have shown that neither trehalose nor Hsp104 nor STRE-controlled genes are involved in the higher stress tolerance of the fil1 mutant. The contribution of other putative downstream factors of the PKA pathway was investigated and here we show that the small heat-shock protein Hsp26 is required for the high heat stress tolerance of the fil1 mutant, both in stationary phase cells as well as during active fermentation.

  1. The Pseudomonas aeruginosa Chp chemosensory system regulates intracellular cAMP levels by modulating adenylate cyclase activity.

    Science.gov (United States)

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

    2010-05-01

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

  2. The complete amino acid sequence of ubiquitin, an adenylate cyclase stimulating polypeptide probably universal in living cells.

    Science.gov (United States)

    Schlesinger, D H; Goldstein, G; Niall, H D

    1975-05-20

    The complete amino acid sequence was determined for bovine ubiquitin, and adenylate cyclase stimulating polypeptide, which is probably represented universally in living cells. Ubiquitin has a molecular weight of 8451 and consists of a single polypeptide chain containing 74 amino acid residues. It contains four arginine residues but no cysteine or trytophan residues. The first 61 amino acid residues were obtained by automated Edman degradations. Tryptic digestion of maleated ubiquitin yielded four peptide fragments that were resolved by molecular sieve chromatography and coded in order of decreasing chain length (MT-1, MT-2, MT-3, and MT-4). The automated sequenator determinations on native ubiquintin provided overlapping sequence data for three of these fragments that gave an order of MT-1, MT-3, and then MT-2; Peptide MT-4, a dipeptide, was therefore assigned to the C terminus, and the placement of peptide MT-2 was corroborated by analysis of data from carboxypeptidase digestions of maleated ubiquitin. Peptide MT-2 was domaleated and sequenced by manual Edman degradations through a single lysine residue. It was cleaved at this residue with trypsin, and the two resultant peptides were separated by ion-exchange chromatography. Manual sequencing of the C-terminal demaleated tryptic peptide of MT-2 completed the sequence of MT-2 and that of native ubiquitin. The sequence of ubiquitin was further confirmed and supported by amino acid and parital sequence anlysis of fragments obtained by digestion of maleated ubiquitin with chymotrypsin or staphylococcal protease.

  3. Plant-activated bacterial receptor adenylate cyclases modulate epidermal infection in the Sinorhizobium meliloti-Medicago symbiosis.

    Science.gov (United States)

    Tian, Chang Fu; Garnerone, Anne-Marie; Mathieu-Demazière, Céline; Masson-Boivin, Catherine; Batut, Jacques

    2012-04-24

    Legumes and soil bacteria called rhizobia have coevolved a facultative nitrogen-fixing symbiosis. Establishment of the symbiosis requires bacterial entry via root hair infection threads and, in parallel, organogenesis of nodules that subsequently are invaded by bacteria. Tight control of nodulation and infection is required to maintain the mutualistic character of the interaction. Available evidence supports a passive bacterial role in nodulation and infection after the microsymbiont has triggered the symbiotic plant developmental program. Here we identify in Sinorhizobium meliloti, the Medicago symbiont, a cAMP-signaling regulatory cascade consisting of three receptor-like adenylate cyclases, a Crp-like regulator, and a target gene of unknown function. The cascade is activated specifically by a plant signal during nodule organogenesis. Cascade inactivation results in a hyperinfection phenotype consisting of abortive epidermal infection events uncoupled from nodulation. These findings show that, in response to a plant signal, rhizobia play an active role in the control of infection. We suggest that rhizobia may modulate the plant's susceptibility to infection. This regulatory loop likely aims at optimizing legume infection.

  4. Rapid, semi-automated, and inexpensive radioimmunoassay of cAMP: application in GPCR-mediated adenylate cyclase assays.

    Science.gov (United States)

    Brown, Justin T; Kant, Andrew; Mailman, Richard B

    2009-03-15

    Cyclic AMP (cAMP) is an important signal transduction second messenger that is commonly used as a functional mirror on the actions of G protein-coupled receptors that can activate or inhibit adenylate cyclases. A radioimmunoassay for cAMP with femtomole sensitivity was first reported by Steiner more than 30 years ago, and there have been several subsequent modifications that have improved this assay in various ways. Here we describe additional improvement to existing methods that markedly improve speed and reduce cost without sacrificing sensitivity, and is also adaptable to analysis of cGMP. The primary antibody is coupled directly to magnetic beads that are then separated from unbound marker using filtration on microplates. This eliminates the need for a secondary antibody, and markedly increases throughput. In addition, we report a simple, reproducible, and inexpensive method to make the radiomarker used for this assay. Although still requiring the use of radioactivity, the resulting method retains a high degree of accuracy and precision, and is suitable for low-cost high throughput screening. Use of aspects of this method can also improve throughput in other radioimmunoassays.

  5. Pituitary adenylate cyclase-activating polypeptide type 1 (PAC1) receptor is expressed during embryonic development of the earthworm.

    Science.gov (United States)

    Boros, Akos; Somogyi, Ildikó; Engelmann, Péter; Lubics, Andrea; Reglodi, Dóra; Pollák, Edit; Molnár, László

    2010-03-01

    Pituitary adenylate cyclase activating polypeptide (PACAP)-like molecules have been shown to be present in cocoon albumin and in Eisenia fetida embryos at an early developmental stage (E1) by immunocytochemistry and radioimmunoassay. Here, we focus on detecting the stage at which PAC1 receptor (PAC1R)-like immunoreactivity first appears in germinal layers and structures, e.g., various parts of the central nervous system (CNS), in developing earthworm embryos. PAC1R-like immunoreactivity was revealed by Western blot and Far Western blot as early as the E2 developmental stage, occurring in the ectoderm and later in specific neurons of the developing CNS. Labeled CNS neurons were first seen in the supraesophageal ganglion (brain) and subsequently in the subesophageal and ventral nerve cord ganglia. Ultrastructurally, PAC1Rs were located mainly on plasma membranes and intracellular membranes, especially on cisternae of the endoplasmic reticulum. Therefore, PACAP-like compounds probably influence the differentiation of germinal layers (at least the ectoderm) and of some neurons and might act as signaling molecules during earthworm embryonic development.

  6. PPARgamma-dependent regulation of adenylate cyclase 6 amplifies the stimulatory effect of cAMP on renin gene expression.

    Science.gov (United States)

    Desch, Michael; Schubert, Thomas; Schreiber, Andrea; Mayer, Sandra; Friedrich, Björn; Artunc, Ferruh; Todorov, Vladimir T

    2010-11-01

    The second messenger cAMP plays an important role in the regulation of renin gene expression. Nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) is known to stimulate renin gene transcription acting through PPARγ-binding sequences in renin promoter. We show now that activation of PPARγ by unsaturated fatty acids or thiazolidinediones drastically augments the cAMP-dependent increase of renin mRNA in the human renin-producing cell line Calu-6. The underlying mechanism involves potentiation of agonist-induced cAMP increase and up-regulation of adenylate cyclase 6 (AC6) gene expression. We identified a palindromic element with a 3-bp spacer (Pal3) in AC6 intron 1 (AC6Pal3). AC6Pal3 bound PPARγ and mediated trans-activation by PPARγ agonist. AC6 knockdown decreased basal renin mRNA level and attenuated the maximal PPARγ-dependent stimulation of the cAMP-induced renin gene expression. AC6Pal3 decoy oligonucleotide abrogated the PPARγ-dependent potentiation of cAMP-induced renin gene expression. Treatment of mice with PPARγ agonist increased AC6 mRNA kidney levels. Our data suggest that in addition to its direct effect on renin gene transcription, PPARγ "sensitizes" renin gene to cAMP via trans-activation of AC6 gene. AC6 has been identified as PPARγ target gene with a functional Pal3 sequence.

  7. Pituitary adenylate cyclase activating peptide (PACAP) immunoreactivity and mRNA expression in the duck gastrointestinal tract.

    Science.gov (United States)

    Mirabella, N; Squillacioti, C; Colitti, M; Germano, G; Pelagalli, A; Paino, G

    2002-06-01

    The presence and distribution of pituitary adenylate cyclase activating peptide (PACAP) immunoreactivity were studied in the duck gastrointestinal tract using immunohistochemistry and radioimmunoassays. Expression and distribution of PACAP mRNA were also studied using reverse transcriptase polymerase chain reaction (RT-PCR) and hybridization techniques. In addition, a partial coding sequence (cds) of the duck growth hormone-releasing hormone (GRF)/PACAP gene was identified. The presence of both PACAP-38 and PACAP-27 was demonstrated, the former being the predominant form. PACAP immunoreactivity was found in neurons and fibers of the enteric nervous system (ENS), in endocrine cells and in the gut associated lymphoid tissue (GALT). Double immunostaining showed that PACAP is almost completely colocalized with vasoactive intestinal peptide (VIP) in the ENS. Moreover, PACAP was also found in nitric oxide synthase (NOS)-containing neurons and nerve fibers. Radioimmunoassay (RIA) performed on denervated gut showed that more than one-half of the duodenal PACAP is extrinsic in origin. RT-PCR, Northern blot analysis and in situ hybridization confirmed the immunohistochemical data. The findings of the present study suggest that, in birds, PACAP may have multiple roles in regulating gastrointestinal functions.

  8. Amidate prodrugs of 9-[2-(phosphonomethoxy)ethyl]adenine as inhibitors of adenylate cyclase toxin from Bordetella pertussis.

    Science.gov (United States)

    Šmídková, Markéta; Dvoráková, Alexandra; Tloust'ová, Eva; Česnek, Michal; Janeba, Zlatko; Mertlíková-Kaiserová, Helena

    2014-01-01

    Adenylate cyclase toxin (ACT) is the key virulence factor of Bordetella pertussis that facilitates its invasion into the mammalian body. 9-[2-(Phosphonomethoxy)ethyl]adenine diphosphate (PMEApp), the active metabolite of the antiviral drug bis(POM)PMEA (adefovir dipivoxil), has been shown to inhibit ACT. The objective of this study was to evaluate six novel amidate prodrugs of PMEA, both phenyloxy phosphonamidates and phosphonodiamidates, for their ability to inhibit ACT activity in the J774A.1 macrophage cell line. The two phenyloxy phosphonamidate prodrugs exhibited greater inhibitory activity (50% inhibitory concentration [IC50] = 22 and 46 nM) than the phosphonodiamidates (IC50 = 84 to 3,960 nM). The inhibitory activity of the prodrugs correlated with their lipophilicity and the degree of their hydrolysis into free PMEA in J774A.1 cells. Although the prodrugs did not inhibit ACT as effectively as bis(POM)PMEA (IC50 = 6 nM), they were significantly less cytotoxic. Moreover, they all reduced apoptotic effects of ACT and prevented an ACT-induced elevation of intracellular [Ca(2+)]i. The amidate prodrugs were less susceptible to degradation in Caco-2 cells compared to bis(POM)PMEA, while they exerted good transepithelial permeability in this assay. As a consequence, a large amount of intact amidate prodrug is expected to be available to target macrophages in vivo. This feature makes nontoxic amidate prodrugs attractive candidates for further investigation as novel antimicrobial agents.

  9. The effects of isatin (indole-2, 3-dione on pituitary adenylate cyclase-activating polypeptide-induced hyperthermia in rats

    Directory of Open Access Journals (Sweden)

    Tóth Gábor

    2002-02-01

    Full Text Available Abstract Background Previous studies have demonstrated that centrally administered natriuretic peptides and pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38 have hyperthermic properties. Isatin (indole-2, 3-dione is an endogenous indole that has previously been found to inhibit hyperthermic effects of natriuretic peptides. In this study the aim was to investigate the effects of isatin on thermoregulatory actions of PACAP-38, in rats. Results One μg intracerebroventricular (icv. injection of PACAP-38 had hyperthermic effect in male, Wistar rats, with an onset of the effect at 2 h and a decline by the 6th h after administration. Intraperitoneal (ip. injection of different doses of isatin (25-50 mg/kg significantly decreased the hyperthermic effect of 1 μg PACAP-38 (icv., whereas 12.5 mg/kg isatin (ip. had no inhibiting effect. Isatin alone did not modify the body temperature of the animals. Conclusion The mechanisms that participate in the mediation of the PACAP-38-induced hyperthermia may be modified by isatin. The capability of isatin to antagonize the hyperthermia induced by all members of the natriuretic peptide family and by PACAP-38 makes it unlikely to be acting directly on receptors for natriuretic peptides or on those for PACAP in these hyperthermic processes.

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

    Science.gov (United States)

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

    2016-02-27

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

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

    Science.gov (United States)

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

    2016-05-01

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

  12. Augmented cystine-glutamate exchange by pituitary adenylate cyclase-activating polypeptide signaling via the VPAC1 receptor

    Science.gov (United States)

    Resch, Jon M.; Albano, Rebecca; Liu, XiaoQian; Hjelmhaug, Julie; Lobner, Doug; Baker, David A.; Choi, SuJean

    2014-01-01

    In the central nervous system, cystine import in exchange for glutamate through system xc− is critical for the production of the antioxidant glutathione by astrocytes, as well as the maintenance of extracellular glutamate. Therefore, regulation of system xc− activity affects multiple aspects of cellular physiology and may contribute to disease states. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuronally-derived peptide that has already been demonstrated to modulate multiple aspects of glutamate signaling suggesting PACAP may also target activity of cystine-glutamate exchange via system xc−. In the current study, 24-hour treatment of primary cortical cultures containing neurons and glia with PACAP concentration-dependently increased system xc− function as measured by radiolabeled cystine uptake. Furthermore, the increase in cystine uptake was completely abolished by the system xc− inhibitor, (S)-4-carboxyphenylglycine (CPG), attributing increases in cystine uptake specifically to system xc− activity. Time course and quantitative PCR results indicate that PACAP signaling may increase cystine-glutamate exchange by increasing expression of xCT, the catalytic subunit of system xc−. Furthermore, the potentiation of system xc− activity by PACAP occurs via a PKA-dependent pathway that is not mediated by the PAC1R, but rather the shared vasoactive intestinal polypeptide receptor VPAC1R. Finally, assessment of neuronal, astrocytic, and microglial-enriched cultures demonstrated that only astrocyte-enriched cultures exhibit enhanced cystine uptake following both PACAP and VIP treatment. These data introduce a novel mechanism by which both PACAP and VIP regulate system xc− activity. PMID:25066643

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

    Science.gov (United States)

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

    2016-01-25

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

  14. Impaired nocifensive behaviours and mechanical hyperalgesia, but enhanced thermal allodynia in pituitary adenylate cyclase-activating polypeptide deficient mice.

    Science.gov (United States)

    Sándor, K; Kormos, V; Botz, B; Imreh, A; Bölcskei, K; Gaszner, B; Markovics, A; Szolcsányi, J; Shintani, N; Hashimoto, H; Baba, A; Reglodi, D; Helyes, Z

    2010-10-01

    Pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38) and its receptors (PAC1 and VPAC) have been shown in the spinal dorsal horn, dorsal root ganglia and sensory nerve terminals. Data concerning the role of PACAP in central pain transmission are controversial and we have recently published its divergent peripheral effects on nociceptive processes. The aim of the present study was to investigate acute somatic and visceral nocifensive behaviours, partial sciatic nerve ligation-evoked chronic neuropathic, as well as resiniferatoxin-induced inflammatory thermal and mechanical hyperalgesia in PACAP deficient (PACAP(-/-)) mice to elucidate its overall function in pain transmission. Neuronal activation was investigated with c-Fos immunohistochemistry. Paw lickings in the early (0-5 min) and late (20-45 min) phases of the formalin test were markedly reduced in PACAP(-/-) mice. Acetic acid-evoked abdominal contractions referring to acute visceral chemonociception was also significantly attenuated in PACAP knockout animals. In both models, the excitatory role of PACAP was supported by markedly greater c-Fos expression in the periaqueductal grey and the somatosensory cortex. In PACAP-deficient animals neuropathic mechanical hyperalgesia was absent, while c-Fos immunopositivity 20 days after the operation was significantly higher. In this chronic model, these neurons are likely to indicate the activation of secondary inhibitory pathways. Intraplantarly injected resiniferatoxin-evoked mechanical hyperalgesia involving both peripheral and central processes was decreased, but thermal allodynia mediated by only peripheral mechanisms was increased in PACAP(-/-) mice. These data clearly demonstrate an overall excitatory role of PACAP in pain transmission originating from both exteroceptive and interoceptive areas, it is also involved in central sensitization. This can be explained by the signal transduction mechanisms of its identified receptors, both PAC1 and VPAC

  15. The circadian neuropeptide PDF signals preferentially through a specific adenylate cyclase isoform AC3 in M pacemakers of Drosophila.

    Science.gov (United States)

    Duvall, Laura B; Taghert, Paul H

    2012-01-01

    The neuropeptide Pigment Dispersing Factor (PDF) is essential for normal circadian function in Drosophila. It synchronizes the phases of M pacemakers, while in E pacemakers it decelerates their cycling and supports their amplitude. The PDF receptor (PDF-R) is present in both M and subsets of E cells. Activation of PDF-R stimulates cAMP increases in vitro and in M cells in vivo. The present study asks: What is the identity of downstream signaling components that are associated with PDF receptor in specific circadian pacemaker neurons? Using live imaging of intact fly brains and transgenic RNAi, we show that adenylate cyclase AC3 underlies PDF signaling in M cells. Genetic disruptions of AC3 specifically disrupt PDF responses: they do not affect other Gs-coupled GPCR signaling in M cells, they can be rescued, and they do not represent developmental alterations. Knockdown of the Drosophila AKAP-like scaffolding protein Nervy also reduces PDF responses. Flies with AC3 alterations show behavioral syndromes consistent with known roles of M pacemakers as mediated by PDF. Surprisingly, disruption of AC3 does not alter PDF responses in E cells--the PDF-R(+) LNd. Within M pacemakers, PDF-R couples preferentially to a single AC, but PDF-R association with a different AC(s) is needed to explain PDF signaling in the E pacemakers. Thus critical pathways of circadian synchronization are mediated by highly specific second messenger components. These findings support a hypothesis that PDF signaling components within target cells are sequestered into "circadian signalosomes," whose compositions differ between E and M pacemaker cell types.

  16. Protective Effects of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Against Oxidative Stress in Zebrafish Hair Cells.

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    Kasica, Natalia; Podlasz, Piotr; Sundvik, Maria; Tamas, Andrea; Reglodi, Dora; Kaleczyc, Jerzy

    2016-11-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide, with known antiapoptotic functions. Our previous in vitro study has demonstrated the ameliorative role of PACAP-38 in chicken hair cells under oxidative stress conditions, but its effects on living hair cells is now yet known. Therefore, the aim of the present study was to investigate in vivo the protective role of PACAP-38 in hair cells found in zebrafish (Danio rerio) sense organs-neuromasts. To induce oxidative stress the 5-day postfertilization (dpf) zebrafish larvae were exposed to 1.5 mM H2O2 for 15 min or 1 h. This resulted in an increase in caspase-3 and p-38 MAPK level in the hair cells as well as in an impairment of the larvae basic behavior. To investigate the ameliorative role of PACAP-38, the larvae were incubated with a mixture of 1.5 mM H2O2 and 100 nM PACAP-38 following 1 h preincubation with 100 nM PACAP-38 only. PACAP-38 abilities to prevent hair cells from apoptosis were investigated. Whole-mount immunohistochemistry and confocal microscopy analyses revealed that PACAP-38 treatment decreased the cleaved caspase-3 level in the hair cells, but had no influence on p-38 MAPK. The analyses of basic locomotor activity supported the protective role of PACAP-38 by demonstrating the improvement of the fish behavior after PACAP-38 treatment. In summary, our in vivo findings demonstrate that PACAP-38 protects zebrafish hair cells from oxidative stress by attenuating oxidative stress-induced apoptosis.

  17. Stability, structural and functional properties of a monomeric, calcium–loaded adenylate cyclase toxin, CyaA, from Bordetella pertussis

    Science.gov (United States)

    Cannella, Sara E.; Ntsogo Enguéné, Véronique Yvette; Davi, Marilyne; Malosse, Christian; Sotomayor Pérez, Ana Cristina; Chamot-Rooke, Julia; Vachette, Patrice; Durand, Dominique; Ladant, Daniel; Chenal, Alexandre

    2017-01-01

    Bordetella pertussis, the causative agent of whooping cough, secretes an adenylate cyclase toxin, CyaA, which invades eukaryotic cells and alters their physiology by cAMP overproduction. Calcium is an essential cofactor of CyaA, as it is the case for most members of the Repeat-in-ToXins (RTX) family. We show that the calcium-bound, monomeric form of CyaA, hCyaAm, conserves its permeabilization and haemolytic activities, even in a fully calcium-free environment. In contrast, hCyaAm requires sub-millimolar calcium in solution for cell invasion, indicating that free calcium in solution is involved in the CyaA toxin translocation process. We further report the first in solution structural characterization of hCyaAm, as deduced from SAXS, mass spectrometry and hydrodynamic studies. We show that hCyaAm adopts a compact and stable state that can transiently conserve its conformation even in a fully calcium-free environment. Our results therefore suggest that in hCyaAm, the C-terminal RTX-domain is stabilized in a high-affinity calcium-binding state by the N-terminal domains while, conversely, calcium binding to the C-terminal RTX-domain strongly stabilizes the N-terminal regions. Hence, the different regions of hCyaAm appear tightly connected, leading to stabilization effects between domains. The hysteretic behaviour of CyaA in response to calcium is likely shared by other RTX cytolysins. PMID:28186111

  18. Pituitary Adenylate Cyclase-Activating Polypeptide Disrupts Motivation, Social Interaction, and Attention in Male Sprague Dawley Rats.

    Science.gov (United States)

    Donahue, Rachel J; Venkataraman, Archana; Carroll, F Ivy; Meloni, Edward G; Carlezon, William A

    2016-12-15

    Severe or prolonged stress can trigger psychiatric illnesses including mood and anxiety disorders. Recent work indicates that pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in regulating stress effects. In rodents, exogenous PACAP administration can produce persistent elevations in the acoustic startle response, which may reflect anxiety-like signs including hypervigilance. We investigated whether PACAP causes acute or persistent alterations in behaviors that reflect other core features of mood and anxiety disorders (motivation, social interaction, and attention). Using male Sprague Dawley rats, we examined if PACAP (.25-1.0 µg, intracerebroventricular infusion) affects motivation as measured in the intracranial self-stimulation test. We also examined if PACAP alters interactions with a conspecific in the social interaction test. Finally, we examined if PACAP affects performance in the 5-choice serial reaction time task, which quantifies attention and error processing. Dose-dependent disruptions in motivation, social interaction, and attention were produced by PACAP, as reflected by increases in reward thresholds, decreases in social behaviors, and decreases in correct responses and alterations in posterror accuracy. Behavior normalized quickly in the intracranial self-stimulation and 5-choice serial reaction time task tests but remained dysregulated in the social interaction test. Effects on attention were attenuated by the corticotropin-releasing factor receptor-1 antagonist antalarmin but not the κ opioid receptor antagonist JDTic. Our findings suggest that PACAP affects numerous domains often dysregulated in mood and anxiety disorders, but that individual signs depend on brain substrates that are at least partially independent. This work may help to devise therapeutics that mitigate specific signs of these disorders. Copyright © 2015 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  19. The circadian neuropeptide PDF signals preferentially through a specific adenylate cyclase isoform AC3 in M pacemakers of Drosophila.

    Directory of Open Access Journals (Sweden)

    Laura B Duvall

    Full Text Available The neuropeptide Pigment Dispersing Factor (PDF is essential for normal circadian function in Drosophila. It synchronizes the phases of M pacemakers, while in E pacemakers it decelerates their cycling and supports their amplitude. The PDF receptor (PDF-R is present in both M and subsets of E cells. Activation of PDF-R stimulates cAMP increases in vitro and in M cells in vivo. The present study asks: What is the identity of downstream signaling components that are associated with PDF receptor in specific circadian pacemaker neurons? Using live imaging of intact fly brains and transgenic RNAi, we show that adenylate cyclase AC3 underlies PDF signaling in M cells. Genetic disruptions of AC3 specifically disrupt PDF responses: they do not affect other Gs-coupled GPCR signaling in M cells, they can be rescued, and they do not represent developmental alterations. Knockdown of the Drosophila AKAP-like scaffolding protein Nervy also reduces PDF responses. Flies with AC3 alterations show behavioral syndromes consistent with known roles of M pacemakers as mediated by PDF. Surprisingly, disruption of AC3 does not alter PDF responses in E cells--the PDF-R(+ LNd. Within M pacemakers, PDF-R couples preferentially to a single AC, but PDF-R association with a different AC(s is needed to explain PDF signaling in the E pacemakers. Thus critical pathways of circadian synchronization are mediated by highly specific second messenger components. These findings support a hypothesis that PDF signaling components within target cells are sequestered into "circadian signalosomes," whose compositions differ between E and M pacemaker cell types.

  20. Presence of pituitary adenylate cyclase-activating polypeptide (PACAP) in the plasma and milk of ruminant animals.

    Science.gov (United States)

    Czegledi, Levente; Tamas, Andrea; Borzsei, Rita; Bagoly, Terez; Kiss, Peter; Horvath, Gabriella; Brubel, Reka; Nemeth, Jozsef; Szalontai, Balint; Szabadfi, Krisztina; Javor, Andras; Reglodi, Dora; Helyes, Zsuzsanna

    2011-05-15

    Milk contains a variety of proteins and peptides that possess biological activity. Growth factors, such as growth hormone, insulin-like, epidermal and nerve growth factors are important milk components which may regulate growth and differentiation in various neonatal tissues and also those of the mammary gland itself. We have recently shown that pituitary adenylate cyclase-activating polypeptide (PACAP), an important neuropeptide with neurotrophic actions, is present in the human milk in much higher concentration than in the plasma of lactating women. Investigation of growth factors in the milk of domestic animals is of utmost importance for their nutritional values and agricultural significance. Therefore, the aim of the present study was to determine the presence and concentration of PACAP in the plasma and milk of three ruminant animal species. Furthermore, the presence of PACAP and its specific PAC1 receptor were investigated in the mammary glands. Radioimmunoassay measurements revealed that PACAP was present in the plasma and the milk of the sheep, goat and the cow in a similar concentration to that measured previously in humans. PACAP38-like immunoreactivity (PACAP38-LI) was 5-20-fold higher in the milk than in the plasma samples of the respective animals, a similar serum/milk ratio was found in all the three species. The levels did not show significant changes within the examined 3-month-period of lactation after delivery. Similar PACAP38-LI was measured in the homogenates of the sheep mammary gland samples taken 7 and 30 days after delivery. PAC1 receptor expression was detected in these udder biopsies by fluorescent immunohistochemistry suggesting that this peptide might have an effect on the mammary glands themselves. These data show that PACAP is present in the milk of various ruminant domestic animal species at high concentrations, the physiological implications of which awaits further investigation.

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

    Science.gov (United States)

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

    2015-03-01

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

  2. Effects of combinatorial treatment with pituitary adenylate cyclase activating peptide and human mesenchymal stem cells on spinal cord tissue repair.

    Directory of Open Access Journals (Sweden)

    Kuan-Min Fang

    Full Text Available The aim of this study is to understand if human mesenchymal stem cells (hMSCs and neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP have synergistic protective effect that promotes functional recovery in rats with severe spinal cord injury (SCI. To evaluate the effect of delayed combinatorial therapy of PACAP and hMSCs on spinal cord tissue repair, we used the immortalized hMSCs that retain their potential of neuronal differentiation under the stimulation of neurogenic factors and possess the properties for the production of several growth factors beneficial for neural cell survival. The results indicated that delayed treatment with PACAP and hMSCs at day 7 post SCI increased the remaining neuronal fibers in the injured spinal cord, leading to better locomotor functional recovery in SCI rats when compared to treatment only with PACAP or hMSCs. Western blotting also showed that the levels of antioxidant enzymes, Mn-superoxide dismutase (MnSOD and peroxiredoxin-1/6 (Prx-1 and Prx-6, were increased at the lesion center 1 week after the delayed treatment with the combinatorial therapy when compared to that observed in the vehicle-treated control. Furthermore, in vitro studies showed that co-culture with hMSCs in the presence of PACAP not only increased a subpopulation of microglia expressing galectin-3, but also enhanced the ability of astrocytes to uptake extracellular glutamate. In summary, our in vivo and in vitro studies reveal that delayed transplantation of hMSCs combined with PACAP provides trophic molecules to promote neuronal cell survival, which also foster beneficial microenvironment for endogenous glia to increase their neuroprotective effect on the repair of injured spinal cord tissue.

  3. Overexpression of adenylate cyclase-associated protein 1 is associated with metastasis of lung cancer.

    Science.gov (United States)

    Tan, Min; Song, Xiaolian; Zhang, Guoliang; Peng, Aimei; Li, Xuan; Li, Ming; Liu, Yang; Wang, Changhui

    2013-10-01

    Lung cancer ranks first in both prevalence and mortality rates among all types of cancer. Metastasis is the main cause of treatment failure. Biomarkers are critical to early diagnosis and prediction and monitoring of progressive lesions. Several biomarkers have been identified for lung cancer but none have been routinely used clinically. The present study assessed the diagnostic and prognostic value of cyclase-associated protein 1 (CAP1) for lung cancer. CAP1 mRNA abundance and protein content were determined by real-time PCR and western blot analysis and/or immunostaining in biopsy specimens (24 neoplastic and 6 non-neoplastic) freshly collected at surgical lung resection, in 82 pathologically banked lung cancer specimens and in cultured non-invasive (95-C) and invasive (95-D) lung cancer cells. Multivariate regression analysis was performed to correlate immunoreactive CAP1 signal with cancer type and stage. In vitro cell migration was performed to determine the effect of RNA interference-mediated CAP1 gene silencing on invasiveness of 95-D cells. These analyses collectively demonstrated that: i) both CAP1 mRNA abundance and protein content were significantly higher in neoplastic compared to non-neoplastic specimens and in metastatic compared to non-metastatic specimens but not different between adenocarcinoma and squamous cell carcinoma; ii) immunoreactive CAP1 signal was significantly stronger in metastatic specimens and 95-D cells compared to non-metastatic specimens and 95-C cells; and iii) RNA interference-mediated CAP1 gene silencing adequately attenuated the invasive capacity of 95-D cells in vitro. These findings suggest that overexpression of CAP1 in lung cancer cells, particularly at the metastatic stage, may have significant clinical implications as a diagnostic/prognostic factor for lung cancer.

  4. Adenylate cyclase 5 coordinates the action of ADP, P2Y1, P2Y13 and ATP-gated P2X7 receptors on axonal elongation.

    Science.gov (United States)

    del Puerto, Ana; Díaz-Hernández, Juan-Ignacio; Tapia, Mónica; Gomez-Villafuertes, Rosa; Benitez, María José; Zhang, Jin; Miras-Portugal, María Teresa; Wandosell, Francisco; Díaz-Hernández, Miguel; Garrido, Juan José

    2012-01-01

    In adult brains, ionotropic or metabotropic purinergic receptors are widely expressed in neurons and glial cells. They play an essential role in inflammation and neurotransmission in response to purines secreted to the extracellular medium. Recent studies have demonstrated a role for purinergic receptors in proliferation and differentiation of neural stem cells although little is known about their role in regulating the initial neuronal development and axon elongation. The objective of our study was to investigate the role of some different types of purinergic receptors, P2Y1, P2Y13 and P2X7, which are activated by ADP or ATP. To study the role and crosstalk of P2Y1, P2Y13 and P2X7 purinergic receptors in axonal elongation, we treated neurons with specific agonists and antagonists, and we nucleofected neurons with expression or shRNA plasmids. ADP and P2Y1-GFP expression improved axonal elongation; conversely, P2Y13 and ATP-gated P2X7 receptors halted axonal elongation. Signaling through each of these receptor types was coordinated by adenylate cyclase 5. In neurons nucleofected with a cAMP FRET biosensor (ICUE3), addition of ADP or Blue Brilliant G, a P2X7 antagonist, increased cAMP levels in the distal region of the axon. Adenylate cyclase 5 inhibition or suppression impaired these cAMP increments. In conclusion, our results demonstrate a crosstalk between two metabotropic and one ionotropic purinergic receptor that regulates cAMP levels through adenylate cyclase 5 and modulates axonal elongation triggered by neurotropic factors and the PI3K-Akt-GSK3 pathway.

  5. ASP-56, a new actin sequestering protein from pig platelets with homology to CAP, an adenylate cyclase-associated protein from yeast.

    Science.gov (United States)

    Gieselmann, R; Mann, K

    1992-02-24

    A new 56 kDa actin-binding protein (ASP-56) was isolated from pig platelet lysate. In falling ball viscosimetry it caused a reduction in viscosity that could be attributed to a decrease in the concentration of polymeric actin. Fluorescence measurements with NBD-labelled actin showed reduction of polymeric actin, too. These results could be explained by sequestering of actin in a non-polymerizable 1:1 ASP-56/actin complex. Sequencing of about 20 tryptic peptides of ASP-56 and comparison with known sequences revealed about 60% homology to the adenylate cyclase-associated protein (CAP) from yeast.

  6. Suppression of the humoral immune response by cannabinoids is partially mediated through inhibition of adenylate cyclase by a pertussis toxin-sensitive G-protein coupled mechanism.

    Science.gov (United States)

    Kaminski, N E; Koh, W S; Yang, K H; Lee, M; Kessler, F K

    1994-11-16

    Cannabinoid compounds, including the major psychoactive component of marihuana, delta 9-tetrahydrocannabinol (delta 9-THC), have been widely established as being inhibitory on a broad array of humoral and cell-mediated immune responses. The presence of cannabinoid receptors has been identified recently on mouse spleen cells, which possess structural and functional characteristics similar to those of the G-protein coupled cannabinoid receptor originally identified in rat brain. These findings, together with those demonstrating that delta 9-THC inhibits adenylate cyclase in splenocytes, strongly suggest that certain aspects of immune inhibition by cannabinoids may be mediated through a cannabinoid receptor-associated mechanism. The objective of the present studies was to determine whether inhibition of adenylate cyclase is relevant to mouse spleen cell immune function and, if so, whether this inhibition is mediated through a Gi-protein coupled mechanism as previously described in neuronal tissue. Spleen cell activation by the phorbol ester phorbol-12-myristate-13-acetate (PMA), plus the calcium ionophore ionomycin, produced a rapid but transient increase in cytosolic cAMP, which was inhibited completely by immunosuppressive concentrations of delta 9-THC (22 microM) and the synthetic bicyclic cannabinoid CP-55940 (5.2 microM), which produced no effect on cell viability. Inhibition by cannabinoids of lymphocyte proliferative responses to PMA plus ionomycin and sheep erythrocyte (sRBC) IgM antibody-forming cell (AFC) response, was abrogated completely by low concentrations of dibutyryl-cAMP (10-100 microM). Inhibition of the sRBC AFC response by both delta 9-THC (22 microM) and CP-55940 (5.2 microM) was also abrogated by preincubation of splenocytes for 24 hr with pertussis toxin (0.1-100 ng/mL). Pertussis toxin pretreatment of spleen cells was also found to directly abrogate cannabinoid inhibition of adenylate cyclase, as measured by forskolin-stimulated accumulation

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

    Science.gov (United States)

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

    2013-09-01

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

  8. Absorption and fluorescence characteristics of photo-activated adenylate cyclase nano-clusters from the amoeboflagellate Naegleria gruberi NEG-M strain

    Energy Technology Data Exchange (ETDEWEB)

    Penzkofer, A., E-mail: alfons.penzkofer@physik.uni-regensburg.de [Fakultaet fuer Physik, Universitaet Regensburg, Universitaetsstrasse 31, D-93053 Regensburg (Germany); Stierl, M.; Hegemann, P. [Institut fuer Biologie/Experimentelle Biophysik, Humboldt Universitaet zu Berlin, Invalidenstrasse 42, D-10115 Berlin (Germany); Kateriya, S. [Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021 (India)

    2012-01-02

    Graphical abstract: Protein color center emissions were observed in the wavelength range from 340 nm to 900 nm from nano-clusters of the photo-activated adenylate cyclase (nPAC) from the amoeboflagellate Naegleria gruberi. Highlights: Black-Right-Pointing-Pointer Adenylyl cyclase nPAC in aqueous pH 7.5 buffer dissolved only to nano-clusters. Black-Right-Pointing-Pointer Nano-cluster size was determined by light attenuation (scattering) measurements. Black-Right-Pointing-Pointer The size of the nano-clusters was growing by coalescing during observation period. Black-Right-Pointing-Pointer In nPAC nano-clusters color centers were present in emission range of 360-900 nm. Black-Right-Pointing-Pointer The nPAC color center emission is compared with fluorescent protein emission. - Abstract: The spectroscopic characteristics of BLUF (BLUF = sensor of blue light using flavin) domain containing soluble adenylate cyclase (nPAC = Naegleria photo-activated cyclase) samples from the amoeboflagellate Naegleria gruberi NEG-M strain is studied at room temperature. The absorption and fluorescence spectroscopic development in the dark was investigated over two weeks. Attenuation coefficient spectra, fluorescence quantum distributions, fluorescence quantum yields, and fluorescence excitation distributions were measured. Thawing of frozen nPAC samples gave solutions with varying protein nano-cluster size and varying flavin, tyrosine, tryptophan, and protein color-center emission. Protein color-center emission was observed in the wavelength range of 360-900 nm with narrow emission bands of small Stokes shift and broad emission bands of large Stokes shift. The emission spectra evolved in time with protein nano-cluster aging.

  9. Characterization of adenylate cyclase toxin from a mutant of Bordetella pertussis defective in the activator gene, cyaC.

    Science.gov (United States)

    Hewlett, E L; Gray, M C; Ehrmann, I E; Maloney, N J; Otero, A S; Gray, L; Allietta, M; Szabo, G; Weiss, A A; Barry, E M

    1993-04-15

    Bordetella pertussis adenylate cyclase (AC) toxin has the abilities to 1) enter target cells where it catalyzes cyclic AMP production and 2) lyse sheep erythrocytes, and these abilities require post-translational modification by the product of an accessory gene cyaC (Barry, E. M., Weiss, A. A., Ehrmann, E. E., Gray, M. C., Hewlett, E. L., and Goodwin, M. St. M. (1991) J. Bacteriol. 173, 720-726). In the present study, AC toxin has been purified from an organism with a mutation in cyaC, BPDE386, and evaluated for its physical and functional properties in order to determine the basis for its lack of toxin and hemolytic activities. AC toxin from BPDE386 is indistinguishable from wild-type toxin in enzymatic activity, migration on SDS-polyacrylamide gel electrophoresis, ability to bind calcium, and calcium-dependent conformational change. Although unable to elicit cAMP accumulation, AC toxin from BPDE386 exhibits binding to the surface of Jurkat cells which is comparable to that of wild-type toxin. This target cell interaction is qualitatively different, however, in that 99% of the mutant toxin remains sensitive to trypsin, whereas approximately 20% of cell-associated wild-type toxin enters a trypsin-resistant compartment. To evaluate the ability of this mutant AC toxin to function at its intracellular site of action, the cAMP-stimulated L-type calcium current in frog atrial myocytes was used. Extracellular addition of wild-type toxin results in cAMP-dependent events that include activation of calcium channels and enhancement of calcium current. In contrast, there is no response to externally applied toxin from BPDE386. When injected into the cell interior, however, the AC toxin from BPDE386 is able to produce increases in the calcium current comparable to those observed with wild-type toxin. Although AC toxin from BPDE386 is unaffected in its enzymatic activity, calcium binding, and calcium-dependent conformational change, the mutation in cyaC does result in a toxin

  10. Pituitary adenylate cyclase activating-peptide and its receptor antagonists in development of acute pancreatitis in rats

    Institute of Scientific and Technical Information of China (English)

    You-Dai Chen; Zong-Guang Zhou; Zhao Wang; Hong-Kai Gao; Wen-Wei Yan; Cun Wang; Gao-Ping Zhao; Xiao-Hui Peng

    2005-01-01

    AIM: Pituitary adenylate cyclase activating-peptide (PACAP) is a late member of the secretin/glucagon/vasoactive intestinal peptide (VIP) family of brain-gut peptides. It is unknown whether PACAP takes part in the development of acute pancreatitis and whether PACAP or its antagonists can be used to suppress the progression of acute pancreatitis.We investigated the actions of PACAP and its receptor antagonists in acute pancreatitis on rats.METHODS: Acute pancreatitis was induced in rats with caerulein or 3.5% sodium taurocholate. The rats were continuously infused with 5-30 μg/kg PACAP via jugular vein within the first 90 min, while 10-100 μg/kg PACAP6-27 and (4-Cl-D-Phe6, Leu17) VIP (PACAP receptor antagonists) were intravenously infused for 1 h. Biochemical and histopathological assessments were made at 4 h after infusion. Pancreatic and duodenal PACAP concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Chinese ink-perfused pancreas was fixed, sectioned and cleared for counting the functional capillary density.RESULTS: PACAP augmented caerulein-induced pancreatitis and failed to ameliorate sodium taurocholate-induced pancreatitis. ELISA revealed that relative concentrations of PACAP in pancreas and duodenum were significantly increased in both sodium taurocholate- and caeruleininduced pancreatitis compared with those in normal controls.Unexpectedly, PACAP6-27 and (4-Cl-DPhe6, Leu17) VIP could induce mild acute pancreatitis and aggravate caeruleininduced pancreatitis with characteristic manifestations of acute hemorrhagic/necrotizing pancreatitis. Functional capillary density of pancreas was interpreted in the context of pancreatic edema, and calibrated functional capillary density (calibrated FCD), which combined measurement of functional capillary density with dry weight/wet weight ratio, was introduced. Hyperemia or congestion, rather than ischemia, characterized pancreatic microcirculatory changes in acute pancreatitis

  11. Identification of a prostacyclin receptor coupled to the adenylate cyclase system via a stimulatory GTP-binding protein in mouse mastocytoma P-815 cells

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    Hashimoto, H.; Negishi, M.; Ichikawa, A. (Kyoto Univ. (Japan))

    1990-11-01

    A stable analogue of prostacyclin, iloprost, specifically bound to 30,000 x g pellet (the membrane fraction) prepared from mouse mastocytoma P-815 cells. The binding was dependent on time, temperature and pH, and absolutely required a divalent cation. The equilibrium dissociation constant and the maximal concentration of the binding site as determined by Scatchard plot analysis were 10.4 nM and 1.12 pmol/mg of protein, respectively. The Hill coefficient was 1.0, indicating a single entity of binding site and no cooperativity. The binding site was highly specific for iloprost among PGs tested (iloprost much greater than PGE1 greater than carbacyclin greater than PGE2). In contrast, the membrane fraction had the binding site specific for PGE2 and PGE1, which was distinct from the prostacyclin receptor. The dissociation of bound (3H)iloprost from the membrane fraction was specifically enhanced by guanine nucleotides. Furthermore, iloprost dose-dependently enhanced the activity of adenylate cyclase in a GTP-dependent manner. These results indicate that a specific prostacyclin receptor is coupled to the adenylate cyclase system via a stimulatory GTP-binding protein in mastocytoma cells.

  12. Reduced early and late phase insulin response to glucose in isolated spiny mouse (Acomys cahirinus) islets: a defective link between glycolysis and adenylate cyclase.

    Science.gov (United States)

    Nesher, R; Abramovitch, E; Cerasi, E

    1989-09-01

    The spiny mouse (Acomys cahirinus) exhibits low insulin responsiveness to glucose with a nearly absent early phase release. The alternative fuel-secretagogue glyceraldehyde (10 mmol/l) produced a maximal early insulin response in rat islets but failed to affect early response in Acomys; however, it potentiated the late insulin response in both species alike. Glucagon (1.5 mumol/l) potentiated the early insulin response to intermediate (8.3 mmol/l) glucose in rat and Acomys islets by two- and four-fold, respectively. Glucose doubled cyclic AMP levels in rat islets but no significant response was noted in Acomys islets. Isobutylmethylxanthine (0.1 mmol/l) and forskolin (25 mumol/l) caused a significant rise in islet cyclic AMP levels in both types of islets; however, neither agent restored the glucose stimulation of cyclic AMP in spiny mouse islets. Forskolin and isobutylmethylxanthine potentiated early and late phase insulin release in both species; however, neither augmented the early response in the Acomys to the degree observed in rat islets. Thus: (1) A deficient link exists in Acomys between glycolysis and subsequent signals. (2) These islets contain a glucose-insensitive adenylate cyclase. (3) The early insulin response may be potentiated by direct activation of adenylate cyclase. (4) The glucose effects on early and late phase insulin release are probably mediated by distinct pathways. (5) In the spiny mouse the signals mediating the early response are deranged to a greater extent than those activating the late phase insulin release.

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

    Energy Technology Data Exchange (ETDEWEB)

    Bliddal, H. (Frederiksberg Hospital, Copenhagen, Denmark); Bech, K.; Feldt-Rasmussen, U.; Thomsen, M.; Ryder, L.P.; Hansen, J.M.; Siersbaek-Nielsen, K.; Friis, T.

    1982-11-01

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

  14. Bisamidate Prodrugs of 2-Substituted 9-[2-(Phosphonomethoxy)ethyl]adenine (PMEA, adefovir) as Selective Inhibitors of Adenylate Cyclase Toxin from Bordetella pertussis.

    Science.gov (United States)

    Česnek, Michal; Jansa, Petr; Šmídková, Markéta; Mertlíková-Kaiserová, Helena; Dračínský, Martin; Brust, Tarsis F; Pávek, Petr; Trejtnar, František; Watts, Val J; Janeba, Zlatko

    2015-08-01

    Novel small-molecule agents to treat Bordetella pertussis infections are highly desirable, as pertussis (whooping cough) remains a serious health threat worldwide. In this study, a series of 2-substituted derivatives of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA, adefovir), in their isopropyl ester bis(L-phenylalanine) prodrug form, were designed and synthesized as potent inhibitors of adenylate cyclase toxin (ACT) isolated from B. pertussis. The series consists of PMEA analogues bearing either a linear or branched aliphatic chain or a heteroatom at the C2 position of the purine moiety. Compounds with a small C2 substituent showed high potency against ACT without cytotoxic effects as well as good selectivity over human adenylate cyclase isoforms AC1, AC2, and AC5. The most potent ACT inhibitor was found to be the bisamidate prodrug of the 2-fluoro PMEA derivative (IC50 =0.145 μM). Although the bisamidate prodrugs reported herein exhibit overall lower activity than the bis(pivaloyloxymethyl) prodrug (adefovir dipivoxil), their toxicity and plasma stability profiles are superior. Furthermore, the bisamidate prodrug was shown to be more stable in plasma than in macrophage homogenate, indicating that the free phosphonate can be effectively distributed to target tissues, such as the lungs. Thus, ACT inhibitors based on acyclic nucleoside phosphonates may represent a new strategy to treat whooping cough.

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

    Science.gov (United States)

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

    2016-01-01

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

  16. Pituitary adenylate cyclase activating polypeptide induces vascular relaxation and inhibits non-vascular smooth muscle activity in the rabbit female genital tract

    DEFF Research Database (Denmark)

    Steenstrup, B R; Ottesen, B; Jørgensen, M

    1994-01-01

    a significant dose-related relaxation on the NA-precontracted vessels. However, pre-incubation of the vessels with 10(-7) M PACAP-38, PACAP-27 and vaso active intestinal polypeptide (VIP) did not induce a general rightward shift of the NA concentration-response curves, although a tendency to inhibition......In vitro effects of two bioactive forms of pituitary adenylate cyclase activating polypeptide (PACAP): PACAP-38 and PACAP-27 were studied on rabbit vascular and non-vascular smooth muscle. Segments of the ovarian artery and muscle strips from the fallopian tube were used. Two series of experiments...... in the low-dose interval was observed. The peptides caused a significant, dose-dependent inhibition of both frequency and amplitude on the fallopian tube smooth muscle activity. The effects of the three peptides on longitudinally as well as transversally cut specimens were alike....

  17. X-linked recessive congenital muscle fiber hypotrophy with central nuclei: abnormalities of growth and adenylate cyclase in muscle tissue cultures.

    Science.gov (United States)

    Askanas, V; Engel, W K; Reddy, N B; Barth, P G; Bethlem, J; Krauss, D R; Hibberd, M E; Lawrence, J V; Carter, L S

    1979-10-01

    Muscle cells in cultures established from biopsy specimens of two children with an infantile-fatal form of X-linked recessive muscle fiber smallness with central nuclei showed an unusual ability to proliferate through numerous passages. Ultrastructurally, the cultured muscle fibers appeared very immature even after several weeks. The nuclei were large, the number of ribosomes was greatly increased, the myofibrils remained unstriated, and glycogen was accumulated in large lakes. The plasmalemma bound concanavalin A, alpha-bungarotoxin, and ruthenium red normally, but with tannic acid it did not show the dark binding of mature fibers. Biochemically, in the cultured muscle fibers, beta-adrenergic receptors were quantitatively normal. The level of adenylate cyclase in membranes was less than in cultured normal muscle; this defect could be responsible for impaired control mechanisms resulting in the other abnormalities observed.

  18. A homolog of the vertebrate pituitary adenylate cyclase-activating polypeptide is both necessary and instructive for the rapid formation of associative memory in an invertebrate.

    Science.gov (United States)

    Pirger, Zsolt; László, Zita; Kemenes, Ildikó; Tóth, Gábor; Reglodi, Dóra; Kemenes, György

    2010-10-13

    Similar to other invertebrate and vertebrate animals, cAMP-dependent signaling cascades are key components of long-term memory (LTM) formation in the snail Lymnaea stagnalis, an established experimental model for studying evolutionarily conserved molecular mechanisms of long-term associative memory. Although a great deal is already known about the signaling cascades activated by cAMP, the molecules involved in the learning-induced activation of adenylate cyclase (AC) in Lymnaea remained unknown. Using matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy in combination with biochemical and immunohistochemical methods, recently we have obtained evidence for the existence of a Lymnaea homolog of the vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) and for the AC-activating effect of PACAP in the Lymnaea nervous system. Here we first tested the hypothesis that PACAP plays an important role in the formation of robust LTM after single-trial classical food-reward conditioning. Application of the PACAP receptor antagonist PACAP6-38 around the time of single-trial training with amyl acetate and sucrose blocked associative LTM, suggesting that in this "strong" food-reward conditioning paradigm the activation of AC by PACAP was necessary for LTM to form. We found that in a "weak" multitrial food-reward conditioning paradigm, lip touch paired with sucrose, memory formation was also dependent on PACAP. Significantly, systemic application of PACAP at the beginning of multitrial tactile conditioning accelerated the formation of transcription-dependent memory. Our findings provide the first evidence to show that in the same nervous system PACAP is both necessary and instructive for fast and robust memory formation after reward classical conditioning.

  19. CAP1, an adenylate cyclase-associated protein gene, regulates bud-hypha transitions, filamentous growth, and cyclic AMP levels and is required for virulence of Candida albicans.

    Science.gov (United States)

    Bahn, Y S; Sundstrom, P

    2001-05-01

    In response to a wide variety of environmental stimuli, the opportunistic fungal pathogen Candida albicans exits the budding cycle, producing germ tubes and hyphae concomitant with expression of virulence genes, such as that encoding hyphal wall protein 1 (HWP1). Biochemical studies implicate cyclic AMP (cAMP) increases in promoting bud-hypha transitions, but genetic evidence relating genes that control cAMP levels to bud-hypha transitions has not been reported. Adenylate cyclase-associated proteins (CAPs) of nonpathogenic fungi interact with Ras and adenylate cyclase to increase cAMP levels under specific environmental conditions. To initiate studies on the relationship between cAMP signaling and bud-hypha transitions in C. albicans, we identified, cloned, characterized, and disrupted the C. albicans CAP1 gene. C. albicans strains with inactivated CAP1 budded in conditions that led to germ tube formation in isogenic strains with CAP1. The addition of 10 mM cAMP and dibutyryl cAMP promoted bud-hypha transitions and filamentous growth in the cap1/cap1 mutant in liquid and solid media, respectively, showing clearly that cAMP promotes hypha formation in C. albicans. Increases in cytoplasmic cAMP preceding germ tube emergence in strains having CAP1 were markedly diminished in the budding cap1/cap1 mutant. C. albicans strains with deletions of both alleles of CAP1 were avirulent in a mouse model of systemic candidiasis. The avirulence of a germ tube-deficient cap1/cap1 mutant coupled with the role of Cap1 in regulating cAMP levels shows that the Cap1-mediated cAMP signaling pathway is required for bud-hypha transitions, filamentous growth, and the pathogenesis of candidiasis.

  20. Studies on responsiveness of hepatoma cells to catecholamines. VI. Characteristics of adrenoceptors and adenylate cyclase response in rat ascites hepatoma cells and human hepatoma cells.

    Science.gov (United States)

    Sanae, F; Kohei, K; Nomura, M; Miyamoto, K

    1992-06-01

    Alpha 1, alpha 2- and beta-Adrenoceptor densities and catecholamine responsiveness in established hepatoma cells, rat ascites hepatoma AH13, AH66, AH66F, AH109A, AH130 and AH7974 cells and human hepatocellular carcinoma HLF and HepG2 cells, were compared with those in normal rat hepatocytes and Chang liver cells. Alpha 1-Adrenoceptor densities measured by [3H]prazosin bindings were not detected in all hepatoma cell lines. Alpha 2-Adrenoceptor densities measured by [3H]clonidine bindings were also barely detected in hepatoma cell lines except for AH130 cells and HepG2 cells. Regarding beta-adrenoceptor, AH109A, AH130 and AH7974 cells had much more [125I]iodocyanopindolol binding sites than normal rat hepatocytes, although we could not detect the binding in HepG2 cells. Adenylate cyclase of normal rat hepatocyte and Chang liver cells were stimulated by beta 2-adrenergic agonist salbutamol, while the cyclase in hepatoma cells had no beta 2-adrenergic response but a beta 1-type response. These findings indicate that the characteristics of adrenergic response in hepatoma cell lines is very different from that in normal hepatocytes, suggesting a participation in the hepatocarcinogenesis and/or the autonomous proliferation of hepatoma cells.

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

    Energy Technology Data Exchange (ETDEWEB)

    Penzkofer, A., E-mail: alfons.penzkofer@physik.uni-regensburg.de [Fakultät für Physik, Universität Regensburg, Universitätsstrasse 31, D-93053 Regensburg (Germany); Tanwar, M.; Veetil, S.K.; Kateriya, S. [Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021 (India); Stierl, M.; Hegemann, P. [Institut für Biologie/Experimentelle Biophysik, Humboldt Universität zu Berlin, Invalidenstrasse 42, D-10115 Berlin (Germany)

    2013-09-23

    Highlights: • Lyophilizing of NgPAC2 from Naegleria gruberi caused loss of BLUF domain activity. • Photo-induced tyrosine to flavin electron transfer in lyophilized NgPAC2. • Photo-induced Tyr–Tyr cross-linking to o,o′-dityrosine in lyophilized NgPAC2. • Photo-induced partial flavin cofactor reduction in lyophilized NgPAC2. • Two NgPAC2 conformations with fast and slow photo-induced electron transfer. - Abstract: The absorption and emission spectroscopic behavior of lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain consisting of a BLUF domain (BLUF = Blue Light sensor Using Flavin) and a cyclase homology domain was studied in the dark, during blue-light exposure and after blue-light exposure at a temperature of 4 °C. The BLUF domain photo-cycle dynamics observed for snap-frozen NgPAC2 was lost by lyophilization (no signaling state formation with flavin absorption red-shift). Instead, blue-light photo-excitation of lyophilized NgPAC2 caused sterically restricted Tyr–Tyr cross-linking (o,o′-ditysosine formation) and partial flavin cofactor reduction.

  2. Effects of Yulangsan polysaccharide on monoamine neurotransmitters, adenylate cyclase activity and brain-derived neurotrophic factor expression in a mouse model of depression induced by unpredictable chronic mild stress

    Institute of Scientific and Technical Information of China (English)

    Shuang Liang; Renbin Huang; Xing Lin; Jianchun Huang; Zhongshi Huang; Huagang Liu

    2012-01-01

    The present study established a mouse model of depression induced by unpredictable chronic mild stress. The model mice were treated with Yulangsan polysaccharide (YLSPS; 150, 300 and 600 mg/kg) for 21 days, and compared with fluoxetine-treated and normal control groups. Enzyme-linked immunosorbent assay, radioimmunity and immunohistochemical staining showed that following treatment with YLSPS (300 and 600 mg/kg), monoamine neurotransmitter levels, prefrontal cortex adenylate cyclase activity and hippocampal brain-derived neurotrophic factor expression were significantly elevated, and depression-like behaviors were improved. Open-field and novelty-suppressed feeding tests showed that mouse activity levels were increased and feeding latency was shortened following treatment. Our results indicate that YLSPS inhibits depression by upregulating monoamine neurotransmitters, prefrontal cortex adenylate cyclase activity and hippocampal brain-derived neurotrophic factor expression.

  3. Photo-dynamics of the BLUF domain containing soluble adenylate cyclase (nPAC) from the amoeboflagellate Naegleria gruberi NEG-M strain

    Energy Technology Data Exchange (ETDEWEB)

    Penzkofer, A., E-mail: alfons.penzkofer@physik.uni-regensburg.de [Fakultaet fuer Physik, Universitaet Regensburg, Universitaetsstrasse 31, D-93053 Regensburg (Germany); Stierl, M.; Hegemann, P. [Institut fuer Biologie/Experimentelle Biophysik, Humboldt Universitaet zu Berlin, Invalidenstrasse 42, D-10115 Berlin (Germany); Kateriya, Suneel [Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021 (India)

    2011-08-25

    Graphical abstract: The photoactivated adenylyl cyclase (nPAC) from Naegleria gruberi was expressed heterologously in Escherichia coli and its photo-cycling dynamics was studied by optical absorption and fluorescence spectroscopy. Highlights: {yields} Photo-activated adenylyl cyclase (nPAC) from Naegleria gruberi NEG-M was expressed. {yields} Photodynamics of BLUF domain in BLUF sensor - cyclase actuator protein was studied. {yields} Photo-excitation caused BLUF photo-cycling and permanent protein re-conformation. {yields} Re-conformed protein enabled photo-induced flavin reduction by proton transfer. {yields} Fluorescence of flavin in dark- and light-adapted state of nPAC was characterized. - Abstract: The amoeboflagellate Naegleria gruberi NEG-M comprises a BLUF (blue light sensor using flavin) regulated adenylate cyclase (nPAC). The nPAC gene was expressed heterologously in Escherichia coli and the photo-dynamics of the nPAC protein was studied by optical absorption and fluorescence spectroscopy. Blue-light exposure of nPAC caused a typical BLUF-type photo-cycle behavior (spectral absorption red-shift, fluorescence quenching, absorption and fluorescence recovery in the dark). Additionally, time-delayed reversible photo-induced one-electron reduction of fully oxidized flavin (Fl{sub ox}) to semi-reduced flavin (FlH{sup {center_dot}}) occurred. Furthermore, photo-excitation of FlH{sup {center_dot}} caused irreversible electron transfer to fully reduced anionic flavin (FlH{sup -}). A photo-induced electron transfer from Tyr or Trp to flavin (Tyr{sup {center_dot}+}-Fl{sup {center_dot}-} or Trp{sup {center_dot}+}-Fl{sup {center_dot}-} radical ion-pair formation) is thought to cause H-bond restructuring responsible for BLUF-type photo-cycling and permanent protein re-conformation enabling photo-induced flavin reduction by proton transfer. Some photo-degradation of Fl{sub ox} to lumichrome was observed. A model of the photo-dynamics of nPAC is developed.

  4. Bordetella pertussis commits human dendritic cells to promote a Th1/Th17 response through the activity of adenylate cyclase toxin and MAPK-pathways.

    Directory of Open Access Journals (Sweden)

    Giorgio Fedele

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

  5. Bordetella adenylate cyclase toxin differentially modulates toll-like receptor-stimulated activation, migration and T cell stimulatory capacity of dendritic cells.

    Directory of Open Access Journals (Sweden)

    Irena Adkins

    Full Text Available Adenylate cyclase toxin (CyaA is a key virulence factor of the whooping cough agent Bordetella pertussis. The toxin targets CD11b-expressing phagocytes and delivers into their cytosol an adenylyl cyclase (AC enzyme that subverts cellular signaling by increasing cAMP levels. In the present study, we analyzed the modulatory effects of CyaA on adhesive, migratory and antigen presenting properties of Toll-like receptor (TLR-activated murine and human dendritic cells (DCs. cAMP signaling of CyaA enhanced TLR-induced dissolution of cell adhesive contacts and migration of DCs towards the lymph node-homing chemokines CCL19 and CCL21 in vitro. Moreover, we examined in detail the capacity of toxin-treated DCs to induce CD4(+ and CD8(+ T cell responses. Exposure to CyaA decreased the capacity of LPS-stimulated DCs to present soluble protein antigen to CD4+ T cells independently of modulation of co-stimulatory molecules and cytokine production, and enhanced their capacity to promote CD4(+CD25(+Foxp3(+ T regulatory cells in vitro. In addition, CyaA decreased the capacity of LPS-stimulated DCs to induce CD8(+ T cell proliferation and limited the induction of IFN-γ producing CD8(+ T cells while enhancing IL-10 and IL-17-production. These results indicate that through activation of cAMP signaling, the CyaA may be mobilizing DCs impaired in T cell stimulatory capacity and arrival of such DCs into draining lymph nodes may than contribute to delay and subversion of host immune responses during B. pertussis infection.

  6. Atomoxetine reverses locomotor hyperactivity, impaired novel object recognition, and prepulse inhibition impairment in mice lacking pituitary adenylate cyclase-activating polypeptide.

    Science.gov (United States)

    Shibasaki, Y; Hayata-Takano, A; Hazama, K; Nakazawa, T; Shintani, N; Kasai, A; Nagayasu, K; Hashimoto, R; Tanida, M; Katayama, T; Matsuzaki, S; Yamada, K; Taniike, M; Onaka, Y; Ago, Y; Waschek, J A; Köves, K; Reglődi, D; Tamas, A; Matsuda, T; Baba, A; Hashimoto, H

    2015-06-25

    Attention-deficit/hyperactivity disorder (ADHD) is a complex neurobehavioral disorder that is characterized by attention difficulties, impulsivity, and hyperactivity. A non-stimulant drug, atomoxetine (ATX), which is a selective noradrenaline reuptake inhibitor, is widely used for ADHD because it exhibits fewer adverse effects compared to conventional psychostimulants. However, little is known about the therapeutic mechanisms of ATX. ATX treatment significantly alleviated hyperactivity of pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient (PACAP(-/-)) mice with C57BL/6J and 129S6/SvEvTac hybrid background. ATX also improved impaired novel object recognition memory and prepulse inhibition in PACAP(-/-) mice with CD1 background. The ATX-induced increases in extracellular noradrenaline and dopamine levels were significantly higher in the prefrontal cortex of PACAP(-/-) mice compared to wild-type mice with C57BL/6J and 129S6/SvEvTac hybrid background. These results suggest that ATX treatment-induced increases in central monoamine metabolism may be involved in the rescue of ADHD-related abnormalities in PACAP(-/-) mice. Our current study suggests that PACAP(-/-) mice are an ideal rodent model with predictive validity for the study of ADHD etiology and drug development. Additionally, the potential effects of differences in genetic background of PACAP(-/-) mice on behaviors are discussed. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  7. First report of the pituitary adenylate cyclase activating polypeptide (PACAP) in crustaceans: conservation of its functions as growth promoting factor and immunomodulator in the white shrimp Litopenaeus vannamei.

    Science.gov (United States)

    Lugo, Juana María; Carpio, Yamila; Morales, Reynold; Rodríguez-Ramos, Tania; Ramos, Laida; Estrada, Mario Pablo

    2013-12-01

    The high conservation of the pituitary adenylate cyclase activating polypeptide (PACAP) sequence indicates that this peptide fulfills important biological functions in a broad spectrum of organisms. However, in invertebrates, little is known about its presence and its functions remain unclear. Up to now, in non-mammalian vertebrates, the majority of studies on PACAP have focused mainly on the localization, cloning and structural evolution of this peptide. As yet, little is known about its biological functions as growth factor and immunomodulator in lower vertebrates. Recently, we have shown that PACAP, apart from its neuroendocrine role, influences immune functions in larval and juvenile fish. In this work, we isolated for the first time the cDNA encoding the mature PACAP from a crustacean species, the white shrimp Litopenaeus vannamei, corroborating its high degree of sequence conservation, when compared to sequences reported from tunicates to mammalian vertebrates. Based on this, we have evaluated the effects of purified recombinant Clarias gariepinus PACAP administrated by immersion baths on white shrimp growth and immunity. We demonstrated that PACAP improves hemocyte count, superoxide dismutase, lectins and nitric oxide synthase derived metabolites in treated shrimp related with an increase in total protein concentration and growth performance. From our results, PACAP acts as a regulator of shrimp growth and immunity, suggesting that in crustaceans, as in vertebrate organisms, PACAP is an important molecule shared by both the endocrine and the immune systems.

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

    Science.gov (United States)

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

    2016-09-01

    Obesity is a worldwide pandemic associated with high incidence of cardiovascular disease. The mechanisms by which the obesity leads cardiac dysfunction are not fully elucidated and few studies have evaluated the relationship between obesity and proteins involved in myocardial β-adrenergic (βA) system. The purpose of this study was to evaluate the cardiac function and βA pathway components in myocardium of obese rats. Male Wistar rats were distributed into two groups: control (n = 17; standard diet) and obese (n = 17; saturated high-fat diet) fed for 33 weeks. Nutritional profile and comorbidities were assessed. Cardiac structure and function was evaluated by macroscopic postmortem, echocardiographic and isolated papillary muscle analyzes. Myocardial protein expression of β1- and β2-adrenergic receptors, Gαs protein, adenylate cyclase (AC) and protein kinase A (PKA) was performed by Western blot. Cardiac cyclic adenosine monophosphate (cAMP) levels and PKA activity were assessed by ELISA Obese rats showed increased adiposity index (P saturated high-fat diet-induced obesity was not effective in triggering cardiac dysfunction and impair the beta-adrenergic signaling. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

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

    Science.gov (United States)

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

    2016-04-01

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

  10. Alzheimer amyloid beta-peptide A-beta25-35 blocks adenylate cyclase-mediated forms of hippocampal long-term potentiation.

    Science.gov (United States)

    Bisel, Blaine E; Henkins, Kristen M; Parfitt, Karen D

    2007-02-01

    Progressive memory loss and deposition of amyloid beta (Abeta) peptides throughout cortical regions are hallmarks of Alzheimer's disease (AD). Several studies in mice and rats have shown that overexpression of amyloid precursor protein (APP) or pretreatment with Abeta peptide fragments results in the inhibition of hippocampal long-term potentiation (LTP) as well as impairments in learning and memory of hippocampal-dependent tasks. For these studies we have investigated the effects of the Abeta(25-35) peptide fragment on LTP induced by adenylate cyclase stimulation followed immediately by application of Mg(++)-free aCSF ("chemLTP"). Treatment of young adult slices with the Abeta(25-35) peptide had no significant effect on basal synaptic transmission in area CA1, but treatment with the peptide for 20 min before inducing chemLTP with isoproterenol (ISO; 1 microM) or forskolin (FSK;10 microM) + Mg(++)-free aCSF resulted in complete blockade of LTP. In contrast, normal ISO-chemLTP was observed after treatment with the control peptide Abeta(35-25). The ability of the Abeta(25-35) peptide fragment to block this and other forms of synaptic plasticity may help elucidate the mechanisms underlying hippocampal deficits observed in animal models of AD and/or AD individuals.

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

    Directory of Open Access Journals (Sweden)

    Tamás Juhász

    2015-07-01

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

  12. Reduced expression of brain-derived neurotrophic factor in mice deficient for pituitary adenylate cyclase activating polypeptide type-I-receptor.

    Science.gov (United States)

    Zink, Mathias; Otto, Christiane; Zörner, Björn; Zacher, Christiane; Schütz, Günther; Henn, Fritz A; Gass, Peter

    2004-04-22

    In vitro pituitary adenylate cyclase activating polypeptide (PACAP) induces the expression of brain-derived neurotrophic factor (BDNF) via its specific receptor PAC1. Since BDNF has been implicated in learning paradigms and mice lacking functional PAC1 have deficits in hippocampus-dependent associative learning, we investigated whether PAC1 mutants show alterations in hippocampal expression of BDNF and its receptor TrkB. Semi-quantitative in situ-hybridization using exon-specific BDNF-probes revealed significantly reduced expression of the exon-III and exon-V-specific transcripts within the hippocampal CA3 region in PAC1-deficient mice. A similar trend was observed for the exon-I-specific transcript. The expression of the exon-III-specific transcript was also reduced within the dentate gyrus, while Trk B-expression did not differ between genotypes. Our data demonstrate that even in vivo PAC1-mediated signaling seems to play a pivotal role for the transcriptional regulation of BDNF.

  13. Testosterone regulates levels of cystic fibrosis transmembrane regulator, adenylate cyclase, and cAMP in the seminal vesicles of orchidectomized rats.

    Science.gov (United States)

    Ramli, Nur Siti Khadijah; Giribabu, Nelli; Muniandy, Sekaran; Salleh, Naguib

    2016-01-15

    Secretions of chloride (Cl(-))- and bicarbonate (HCO3(-))-rich fluid by the seminal vesicles could involve cystic fibrosis transmembrane regulator (CFTR), which activity can be stimulated by cAMP generated from the reaction involving adenylate cyclase (AC). In this study, we investigated levels of CFTR, AC, and cAMP in the seminal vesicles under testosterone influence. Orchidectomized adult male rats received 7-day treatment with 125 or 250 μg/kg/day of testosterone with or without flutamide or finasteride. At the end of the treatment, animals were sacrificed and seminal vesicles were harvested for analyses of CFTR and AC protein expression level by Western blotting. Distribution of CFTR and AC in seminal vesicles was observed by immunohistochemistry. Levels of cAMP and dihydrotestosterone in seminal vesicle homogenates were measured by ELISA. Cystic fibrosis transmembrane regulator, AC, and cAMP levels increased with increasing doses of testosterone (P seminal vesicle lumen with higher expression levels observed in testosterone-treated rats than in non-treated orchidectomized rats (P seminal vesicle homogenates after treatment with 250 μg/kg/day than with 125 μg/kg/day of testosterone (P seminal vesicles might contribute toward an increase in Cl(-) and HCO3(-) concentrations in the seminal fluid as reported under testosterone influence.

  14. Calcium, acylation, and molecular confinement favor folding of Bordetella pertussis adenylate cyclase CyaA toxin into a monomeric and cytotoxic form.

    Science.gov (United States)

    Karst, Johanna C; Ntsogo Enguéné, V Yvette; Cannella, Sara E; Subrini, Orso; Hessel, Audrey; Debard, Sylvain; Ladant, Daniel; Chenal, Alexandre

    2014-10-31

    The adenylate cyclase (CyaA) toxin, a multidomain protein of 1706 amino acids, is one of the major virulence factors produced by Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic target cells in which it produces high levels of cAMP, thus altering the cellular physiology. Although CyaA has been extensively studied by various cellular and molecular approaches, the structural and functional states of the toxin remain poorly characterized. Indeed, CyaA is a large protein and exhibits a pronounced hydrophobic character, making it prone to aggregation into multimeric forms. As a result, CyaA has usually been extracted and stored in denaturing conditions. Here, we define the experimental conditions allowing CyaA folding into a monomeric and functional species. We found that CyaA forms mainly multimers when refolded by dialysis, dilution, or buffer exchange. However, a significant fraction of monomeric, folded protein could be obtained by exploiting molecular confinement on size exclusion chromatography. Folding of CyaA into a monomeric form was found to be critically dependent upon the presence of calcium and post-translational acylation of the protein. We further show that the monomeric preparation displayed hemolytic and cytotoxic activities suggesting that the monomer is the genuine, physiologically active form of the toxin. We hypothesize that the structural role of the post-translational acylation in CyaA folding may apply to other RTX toxins.

  15. Overexpression of adenylate cyclase-associated protein 1 may predict brain metastasis in non-small cell lung cancer.

    Science.gov (United States)

    Xie, Shuan-Shuan; Tan, Min; Lin, Hai-Yan; Xu, Lei; Shen, Chang-Xing; Yuan, Qing; Song, Xiao-Lian; Wang, Chang-Hui

    2015-01-01

    This study was designed to establish a biomarker risk model for predicting brain metastasis (BM) in non-small cell lung cancer (NSCLC). The model comprises 120 cases of NSCLC that were treated and followed up for 4 years. The patients were divided into the BM (n=50) and non-BM (other visceral metastasis and those without recurrence) (n=70) groups. Immunohistochemical and western blot analyses were performed in metastatic tissues of NSCLC. Multivariate regression analysis was performed to correlate the immunoreactive cyclase-associated protein 1 (CAP1) signal with BM. Survival analyses were performed by using the Kaplan-Meier method. CAP1 protein content and immunoreactivity were significantly increased in BM specimens compared to other-metastatic specimens. The survival analysis revealed that CAP1 overexpression was significantly associated with survival (P<0.05). The ROC test suggested that the area under the curve was 73.33% (P<0.001; 95% CI, 63.5-83.2%). When P=0.466, the sensitivity and specificity reached 79.5 and 67.1%, respectively. These findings suggested that CAP1 is involved in the BM of NSCLC, and that elevated levels of CAP1 expression may indicate a poor prognosis for patients with BM. The CAP1 molecular model may be useful in the prediction of the risk of BM in NSCLC.

  16. Pituitary adenylate cyclase-activating polypeptide (PACAP has a neuroprotective function in dopamine-based neurodegeneration in rat and snail parkinsonian models

    Directory of Open Access Journals (Sweden)

    Gabor Maasz

    2017-02-01

    Full Text Available Pituitary adenylate cyclase-activating polypeptide (PACAP rescues dopaminergic neurons from neurodegeneration and improves motor changes induced by 6-hydroxy-dopamine (6-OHDA in rat parkinsonian models. Recently, we investigated the molecular background of the neuroprotective effect of PACAP in dopamine (DA-based neurodegeneration using rotenone-induced snail and 6-OHDA-induced rat models of Parkinson's disease. Behavioural activity, monoamine (DA and serotonin, metabolic enzyme (S-COMT, MB-COMT and MAO-B and PARK7 protein concentrations were measured before and after PACAP treatment in both models. Locomotion and feeding activity were decreased in rotenone-treated snails, which corresponded well to findings obtained in 6-OHDA-induced rat experiments. PACAP was able to prevent the behavioural malfunctions caused by the toxins. Monoamine levels decreased in both models and the decreased DA level induced by toxins was attenuated by ∼50% in the PACAP-treated animals. In contrast, PACAP had no effect on the decreased serotonin (5HT levels. S-COMT metabolic enzyme was also reduced but a protective effect of PACAP was not observed in either of the models. Following toxin treatment, a significant increase in MB-COMT was observed in both models and was restored to normal levels by PACAP. A decrease in PARK7 was also observed in both toxin-induced models; however, PACAP had a beneficial effect only on 6-OHDA-treated animals. The neuroprotective effect of PACAP in different animal models of Parkinson's disease is thus well correlated with neurotransmitter, enzyme and protein levels. The models successfully mimic several, but not all etiological properties of the disease, allowing us to study the mechanisms of neurodegeneration as well as testing new drugs. The rotenone and 6-OHDA rat and snail in vivo parkinsonian models offer an alternative method for investigation of the molecular mechanisms of neuroprotective agents, including PACAP.

  17. Bi-directional effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on fear-related behavior and c-Fos expression after fear conditioning in rats.

    Science.gov (United States)

    Meloni, Edward G; Venkataraman, Archana; Donahue, Rachel J; Carlezon, William A

    2016-02-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is implicated in stress regulation and learning and memory. PACAP has neuromodulatory actions on brain structures within the limbic system that could contribute to its acute and persistent effects in animal models of stress and anxiety-like behavior. Here, male Sprague-Dawley rats were implanted with intracerebroventricular (ICV) cannula for infusion of PACAP-38 (0.5, 1, or 1.5 μg) or vehicle followed 30 min later by fear conditioning. Freezing was measured early (1, 4, and 7 days) or following a delay (7, 10, and 13 days) after conditioning. PACAP (1.5 μg) produced a bi-phasic response in freezing behavior across test days: relative to controls, PACAP-treated rats showed a reduction in freezing when tested 1 or 7 days after fear conditioning that evolved into a significant elevation in freezing by the third test session in the early, but not delayed, group. Corticosterone (CORT) levels were significantly elevated in PACAP-treated rats following fear conditioning, but not at the time of testing (Day 1). Brain c-Fos expression revealed PACAP-dependent alterations within, as well as outside of, areas typically implicated in fear conditioning. Our findings raise the possibility that PACAP disrupts fear memory consolidation by altering synaptic plasticity within neurocircuits normally responsible for encoding fear-related cues, producing a type of dissociation or peritraumatic amnesia often seen in people early after exposure to a traumatic event. However, fear memories are retained such that repeated testing and memory reactivation (e.g., re-experiencing) causes the freezing response to emerge and persist at elevated levels. PACAP systems may represent an axis on which stress and exposure to trauma converge to promote maladaptive behavioral responses characteristic of psychiatric illnesses such as post-traumatic stress disorder (PTSD). Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2014-01-01

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

  19. A membrane-associated adenylate cyclase modulates lactate dehydrogenase and creatine kinase activities required for bull sperm capacitation induced by hyaluronic acid.

    Science.gov (United States)

    Fernández, Silvina; Córdoba, Mariana

    2017-04-01

    Hyaluronic acid, as well as heparin, is a glycosaminoglycan present in the female genital tract of cattle. The aim of this study was to evaluate oxidative metabolism and intracellular signals mediated by a membrane-associated adenylate cyclase (mAC), in sperm capacitation with hyaluronic acid and heparin, in cryopreserved bull sperm. The mAC inhibitor, 2',5'-dideoxyadenosine, was used in the present study. Lactate dehydrogenase (LDH) and creatine kinase (CK) activities and lactate concentration were determined spectrophotometrically in the incubation medium. Capacitation and acrosome reaction were evaluated by chlortetracycline technique, while plasma membrane and acrosome integrity were determined by trypan blue stain/differential interference contrast microscopy. Heparin capacitated samples had a significant decrease in LDH and CK activities, while in hyaluronic acid capacitated samples LDH and CK activities both increased compared to control samples, in heparin and hyaluronic acid capacitation conditions, respectively. A significant increase in lactate concentration in the incubation medium occurred in hyaluronic acid-treated sperm samples compared to heparin treatment, indicating this energetic metabolite is produced during capacitation. The LDH and CK enzyme activities and lactate concentrations in the incubation medium were decreased with 2',5'-dideoxyadenosine treatment in hyaluronic acid samples. The mAC inhibitor significantly inhibited heparin-induced capacitation of sperm cells, but did not completely inhibit hyaluronic acid capacitation. Therefore, hyaluronic acid and heparin are physiological glycosaminoglycans capable of inducing in vitro capacitation in cryopreserved bull sperm, stimulating different enzymatic pathways and intracellular signals modulated by a mAC. Hyaluronic acid induces sperm capacitation involving LDH and CK activities, thereby reducing oxidative metabolism, and this process is mediated by mAC.

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

    Science.gov (United States)

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

    2014-06-01

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

  1. Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) regulate murine neural progenitor cell survival, proliferation, and differentiation.

    Science.gov (United States)

    Scharf, Eugene; May, Victor; Braas, Karen M; Shutz, Kristin C; Mao-Draayer, Yang

    2008-11-01

    Neural stem/progenitor cells (NPC) have gained wide interest over the last decade from their therapeutic potential, either through transplantation or endogenous replacement, after central nervous system (CNS) disease and damage. Whereas several growth factors and cytokines have been shown to promote NPC survival, proliferation, or differentiation, the identification of other regulators will provide much needed options for NPC self-renewal or lineage development. Although previous studies have shown that pituitary adenylate cyclase-activating polypeptide (PACAP)/vasoactive intestinal peptide (VIP) can regulate stem/progenitor cells, the responses appeared variable. To examine the direct roles of these peptides in NPCs, postnatal mouse NPC cultures were withdrawn from epidermal growth factor (EGF) and fibroblastic growth factor (FGF) and maintained under serum-free conditions in the presence or absence of PACAP27, PACAP38, or VIP. The NPCs expressed the PAC1(short)null receptor isoform, and the activation of these receptors decreased progenitor cell apoptosis more than 80% from TUNEL assays and facilitated proliferation more than fivefold from bromodeoxyuridine (BrdU) analyses. To evaluate cellular differentiation, replicate control and peptide-treated cultures were examined for cell fate marker protein and transcript expression. In contrast with previous work, PACAP peptides downregulated NPC differentiation, which appeared consistent with the proliferation status of the treated cells. Accordingly, these results demonstrate that PACAP signaling is trophic and can maintain NPCs in a multipotent state. With these attributes, PACAP may be able to promote endogenous NPC self-renewal in the adult CNS, which may be important for endogenous self-repair in disease and ageing processes.

  2. Pituitary adenylate cyclase-activating polypeptide (PACAP) has a neuroprotective function in dopamine-based neurodegeneration in rat and snail parkinsonian models

    Science.gov (United States)

    Kiss, Tibor; Jungling, Adel

    2017-01-01

    ABSTRACT Pituitary adenylate cyclase-activating polypeptide (PACAP) rescues dopaminergic neurons from neurodegeneration and improves motor changes induced by 6-hydroxy-dopamine (6-OHDA) in rat parkinsonian models. Recently, we investigated the molecular background of the neuroprotective effect of PACAP in dopamine (DA)-based neurodegeneration using rotenone-induced snail and 6-OHDA-induced rat models of Parkinson's disease. Behavioural activity, monoamine (DA and serotonin), metabolic enzyme (S-COMT, MB-COMT and MAO-B) and PARK7 protein concentrations were measured before and after PACAP treatment in both models. Locomotion and feeding activity were decreased in rotenone-treated snails, which corresponded well to findings obtained in 6-OHDA-induced rat experiments. PACAP was able to prevent the behavioural malfunctions caused by the toxins. Monoamine levels decreased in both models and the decreased DA level induced by toxins was attenuated by ∼50% in the PACAP-treated animals. In contrast, PACAP had no effect on the decreased serotonin (5HT) levels. S-COMT metabolic enzyme was also reduced but a protective effect of PACAP was not observed in either of the models. Following toxin treatment, a significant increase in MB-COMT was observed in both models and was restored to normal levels by PACAP. A decrease in PARK7 was also observed in both toxin-induced models; however, PACAP had a beneficial effect only on 6-OHDA-treated animals. The neuroprotective effect of PACAP in different animal models of Parkinson's disease is thus well correlated with neurotransmitter, enzyme and protein levels. The models successfully mimic several, but not all etiological properties of the disease, allowing us to study the mechanisms of neurodegeneration as well as testing new drugs. The rotenone and 6-OHDA rat and snail in vivo parkinsonian models offer an alternative method for investigation of the molecular mechanisms of neuroprotective agents, including PACAP. PMID:28067625

  3. Pituitary Adenylate Cyclase Activating Polypeptide, A Potential Therapeutic Agent for Diabetic Retinopathy in Rats: Focus on the Vertical Information Processing Pathway.

    Science.gov (United States)

    Szabadfi, K; Reglodi, D; Szabo, A; Szalontai, B; Valasek, A; Setalo, Gy; Kiss, P; Tamas, A; Wilhelm, M; Gabriel, R

    2016-04-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurotrophic and neuroprotective peptide that has been shown to exert protective effects in different neuronal injuries, such as retinal degenerations. Diabetic retinopathy (DR), the most common complication of diabetes, affects the microvasculature and neuronal architecture of the retina. We have proven earlier that PACAP is also protective in a rat model of DR. In this study, streptozotocin-induced DR was treated with intravitreal PACAP administration in order to further analyze the synaptic structure and proteins of PACAP-treated diabetic retinas, primarily in the vertical information processing pathway. Streptozotocin-treated Wistar rats received intravitreal PACAP injection three times into the right eye 2 weeks after the induction of diabetes. Morphological and molecular biological (qRT-PCR; Western blot) methods were used to analyze retinal synapses (ribbons, conventional) and related structures. Electron microscopic analysis revealed that retinal pigment epithelium, the ribbon synapses and other synaptic profiles suffered alterations in diabetes. However, in PACAP-treated diabetic retinas more bipolar ribbon synapses were found intact in the inner plexiform layer than in DR animals. The ribbon synapse was marked with C-terminal binding protein 2/Bassoon and formed horseshoe-shape ribbons, which were more retained in PACAP-treated diabetic retinas than in DR rats. These results are supported by molecular biological data. The selective degeneration of related structures such as bipolar and ganglion cells could be ameliorated by PACAP treatment. In summary, intravitreal administration of PACAP may have therapeutic potential in streptozotocin-induced DR through maintaining synapse integrity in the vertical pathway.

  4. Pituitary adenylate cyclase-activating polypeptide (PACAP) has a neuroprotective function in dopamine-based neurodegeneration in rat and snail parkinsonian models.

    Science.gov (United States)

    Maasz, Gabor; Zrinyi, Zita; Reglodi, Dora; Petrovics, Dora; Rivnyak, Adam; Kiss, Tibor; Jungling, Adel; Tamas, Andrea; Pirger, Zsolt

    2017-02-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) rescues dopaminergic neurons from neurodegeneration and improves motor changes induced by 6-hydroxy-dopamine (6-OHDA) in rat parkinsonian models. Recently, we investigated the molecular background of the neuroprotective effect of PACAP in dopamine (DA)-based neurodegeneration using rotenone-induced snail and 6-OHDA-induced rat models of Parkinson's disease. Behavioural activity, monoamine (DA and serotonin), metabolic enzyme (S-COMT, MB-COMT and MAO-B) and PARK7 protein concentrations were measured before and after PACAP treatment in both models. Locomotion and feeding activity were decreased in rotenone-treated snails, which corresponded well to findings obtained in 6-OHDA-induced rat experiments. PACAP was able to prevent the behavioural malfunctions caused by the toxins. Monoamine levels decreased in both models and the decreased DA level induced by toxins was attenuated by ∼50% in the PACAP-treated animals. In contrast, PACAP had no effect on the decreased serotonin (5HT) levels. S-COMT metabolic enzyme was also reduced but a protective effect of PACAP was not observed in either of the models. Following toxin treatment, a significant increase in MB-COMT was observed in both models and was restored to normal levels by PACAP. A decrease in PARK7 was also observed in both toxin-induced models; however, PACAP had a beneficial effect only on 6-OHDA-treated animals. The neuroprotective effect of PACAP in different animal models of Parkinson's disease is thus well correlated with neurotransmitter, enzyme and protein levels. The models successfully mimic several, but not all etiological properties of the disease, allowing us to study the mechanisms of neurodegeneration as well as testing new drugs. The rotenone and 6-OHDA rat and snail in vivo parkinsonian models offer an alternative method for investigation of the molecular mechanisms of neuroprotective agents, including PACAP.

  5. Optogenetic Modulation of an Adenylate Cyclase in Toxoplasma gondii Demonstrates a Requirement of the Parasite cAMP for Host-Cell Invasion and Stage Differentiation*

    Science.gov (United States)

    Hartmann, Anne; Arroyo-Olarte, Ruben Dario; Imkeller, Katharina; Hegemann, Peter; Lucius, Richard; Gupta, Nishith

    2013-01-01

    Successful infection and transmission of the obligate intracellular parasite Toxoplasma gondii depends on its ability to switch between fast-replicating tachyzoite (acute) and quiescent bradyzoite (chronic) stages. Induction of cAMP in the parasitized host cells has been proposed to influence parasite differentiation. It is not known whether the parasite or host cAMP is required to drive this phenomenon. Other putative roles of cAMP for the parasite biology also remain to be identified. Unequivocal research on cAMP-mediated signaling in such intertwined systems also requires a method for an efficient and spatial control of the cAMP pool in the pathogen or in the enclosing host cell. We have resolved these critical concerns by expressing a photoactivated adenylate cyclase that allows light-sensitive control of the parasite or host-cell cAMP. Using this method, we reveal multiple roles of the parasite-derived cAMP in host-cell invasion, stage-specific expression, and asexual differentiation. An optogenetic method provides many desired advantages such as: (i) rapid, transient, and efficient cAMP induction in extracellular/intracellular and acute/chronic stages; (ii) circumvention of the difficulties often faced in cultures, i.e. poor diffusion, premature degradation, steady activation, and/or pleiotropic effects of cAMP agonists and antagonists; (iii) genetically encoded enzyme expression, thus inheritable to the cell progeny; and (iv) conditional and spatiotemporal control of cAMP levels. Importantly, a successful optogenetic application in Toxoplasma also illustrates its wider utility to study cAMP-mediated signaling in other genetically amenable two-organism systems such as in symbiotic and pathogen-host models. PMID:23525100

  6. Correlation between oocyte number and follicular fluid concentration of pituitary adenylate cyclase-activating polypeptide (PACAP) in women after superovulation treatment.

    Science.gov (United States)

    Koppan, M; Varnagy, A; Reglodi, D; Brubel, R; Nemeth, J; Tamas, A; Mark, L; Bodis, J

    2012-11-01

    Follicular growth, ovulation, and luteinization are influenced by interactions of peptide and steroid hormone-signaling cascades in the ovary. Pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in the regulation of several endocrine processes and is present in ovarian follicular fluid (FF). However, little is known about PACAP in FF with regard to maturation, ovulation, fertilization, and successful pregnancy. The aim of this pilot study was to investigate whether there is a correlation between PACAP concentration in FF and ovarian response to superovulation treatment in infertile women, performed in volunteers (n = 132; aged between 20 and 35). After treatment, the number of harvested oocytes was recorded and PACAP immunoreactivity in FF was measured by radioimmunoassay. All the corresponding PACAP concentrations were below 290 fmol/ml in cases when the number of harvested oocytes exceeded 14 per patient, while in all cases above 290 fmol/ml, the number of oocytes was below 14. Using these cutoff values, we determined three study groups: high-PACAP concentration, high-oocyte number, and low-PACAP concentration-low-oocyte number groups. Median values of PACAP concentration in these groups were 411.2, 106.5, and 101.0 fmol/ml, respectively, while the median values of harvested oocytes were 5.5, 19.0, and 5.0, respectively. Differences were significant, indicating a correlation between concentration of PACAP in FF and the number of recruited oocytes. Higher concentrations of PACAP in FF might be associated with lower number of developing oocytes, while low concentrations of PACAP might correlate with a markedly higher number of ova retrieved, thus predicting a higher chance for ovarian hyperstimulation. Our present study is among the first few human clinical studies with direct conclusions drawn for possible clinical impact of PACAP.

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

    Science.gov (United States)

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

    2015-07-01

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

  8. Toxicity of Cry1A toxins from Bacillus thuringiensis to CF1 cells does not involve activation of adenylate cyclase/PKA signaling pathway.

    Science.gov (United States)

    Portugal, Leivi; Muñóz-Garay, Carlos; Martínez de Castro, Diana L; Soberón, Mario; Bravo, Alejandra

    2017-01-01

    Bacillus thuringiensis (Bt) bacteria produce Cry toxins that are able to kill insect pests. Different models explaining the mode of action of these toxins have been proposed. The pore formation model proposes that the toxin creates pores in the membrane of the larval midgut cells after interaction with different receptors such as cadherin, aminopeptidase N and alkaline phosphatase and that this pore formation activity is responsible for the toxicity of these proteins. The alternative model proposes that interaction with cadherin receptor triggers an intracellular cascade response involving protein G, adenylate cyclase (AC) and protein kinase A (PKA). In addition, it was shown that Cry toxins induce a defense response in the larvae involving the activation of mitogen-activated kinases such as MAPK p38 in different insect orders. Here we analyzed the mechanism of action of Cry1Ab and Cry1Ac toxins and a collection of mutants from these toxins in the insect cell line CF1 from Choristoneura fumiferana, that is naturally sensitive to these toxins. Our results show that both toxins induced permeability of K(+) ions into the cells. The initial response after intoxication with Cry1Ab and Cry1Ac toxins involves the activation of a defense response that involves the phosphorylation of MAPK p38. Analysis of activation of PKA and AC activities indicated that the signal transduction involving PKA, AC and cAMP was not activated during Cry1Ab or Cry1Ac intoxication. In contrast we show that Cry1Ab and Cry1Ac activate apoptosis. These data indicate that Cry toxins can induce an apoptotic death response not related with AC/PKA activation. Since Cry1Ab and Cry1Ac toxins affected K(+) ion permeability into the cells, and that mutant toxins affected in pore formation are not toxic to CF1, we propose that pore formation activity of the toxins is responsible of triggering cell death response in CF1cells.

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

    Science.gov (United States)

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

    2016-09-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  11. Racemic Salsolinol and its Enantiomers Act as Agonists of the μ-Opioid Receptor by Activating the Gi Protein-Adenylate Cyclase Pathway

    Science.gov (United States)

    Berríos-Cárcamo, Pablo; Quintanilla, María E.; Herrera-Marschitz, Mario; Vasiliou, Vasilis; Zapata-Torres, Gerald; Rivera-Meza, Mario

    2017-01-01

    Background: Several studies have shown that the ethanol-derived metabolite salsolinol (SAL) can activate the mesolimbic system, suggesting that SAL is the active molecule mediating the rewarding effects of ethanol. In vitro and in vivo studies suggest that SAL exerts its action on neuron excitability through a mechanism involving opioid neurotransmission. However, there is no direct pharmacologic evidence showing that SAL activates opioid receptors. Methods: The ability of racemic (R/S)-SAL, and its stereoisomers (R)-SAL and (S)-SAL, to activate the μ-opioid receptor was tested in cell-based (light-emitting) receptor assays. To further characterizing the interaction of SAL stereoisomers with the μ-opioid receptor, a molecular docking study was performed using the crystal structure of the μ-opioid receptor. Results: This study shows that SAL activates the μ-opioid receptor by the classical G protein-adenylate cyclase pathway with an half-maximal effective concentration (EC50) of 2 × 10−5 M. The agonist action of SAL was fully blocked by the μ-opioid antagonist naltrexone. The EC50 for the purified stereoisomers (R)-SAL and (S)-SAL were 6 × 10−4 M and 9 × 10−6 M respectively. It was found that the action of racemic SAL on the μ-opioid receptor did not promote the recruitment of β-arrestin. Molecular docking studies showed that the interaction of (R)- and (S)-SAL with the μ-opioid receptor is similar to that predicted for the agonist morphine. Conclusions: It is shown that (R)-SAL and (S)-SAL are agonists of the μ-opioid receptor. (S)-SAL is a more potent agonist than the (R)-SAL stereoisomer. In silico analysis predicts a morphine-like interaction between (R)- and (S)-SAL with the μ-opioid receptor. These results suggest that an opioid action of SAL or its enantiomers is involved in the rewarding effects of ethanol. PMID:28167903

  12. Multiple nickel-sensitive targets elicit cardiac arrhythmia in isolated mouse hearts after pituitary adenylate cyclase-activating polypeptide-mediated chronotropy.

    Science.gov (United States)

    Tevoufouet, Etienne E; Nembo, Erastus N; Distler, Fabian; Neumaier, Felix; Hescheler, Jürgen; Nguemo, Filomain; Schneider, Toni

    2017-03-01

    The pituitary adenylate cyclase-activating polypeptide (PACAP)-27 modulates various biological processes, from the cellular level to function specification. However, the cardiac actions of this neuropeptide are still under intense studies. Using control (+|+) and mice lacking (-|-) either R-type (Cav2.3) or T-type (Cav3.2) Ca(2+) channels, we investigated the effects of PACAP-27 on cardiac activity of spontaneously beating isolated perfused hearts. Superfusion of PACAP-27 (20nM) caused a significant increase of baseline heart frequency in Cav2.3(+|+) (156.9±10.8 to 239.4±23.4 bpm; p<0.01) and Cav2.3(-|-) (190.3±26.4 to 270.5±25.8 bpm; p<0.05) hearts. For Cav3.2, the heart rate was significantly increased in Cav3.2(-|-) (133.1±8.5 bpm to 204.6±27.9 bpm; p<0.05) compared to Cav3.2(+|+) hearts (185.7±11.2 bpm to 209.3±22.7 bpm). While the P wave duration and QTc interval were significantly increased in Cav2.3(+|+) and Cav2.3(-|-) hearts following PACAP-27 superfusion, there was no effect in Cav3.2(+|+) and Cav3.2(-|-) hearts. The positive chronotropic effects observed in the four study groups, as well as the effect on P wave duration and QTc interval were abolished in the presence of Ni(2+) (50μM) and PACAP-27 (20nM) in hearts from Cav2.3(+|+) and Cav2.3(-|-) mice. In addition to suppressing PACAP's response, Ni(2+) also induced conduction disturbances in investigated hearts. In conclusion, the most Ni(2+)-sensitive Ca(2+) channels (R- and T-type) may modulate the PACAP signaling cascade during cardiac excitation in isolated mouse hearts, albeit to a lesser extent than other Ni(2+)-sensitive targets.

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

    Institute of Scientific and Technical Information of China (English)

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

    2012-01-01

    The recombinant peptide,DBAYL,a promising therapeutic peptide for type 2 diabetes,is a new,potent,and highly selective agonist for VPAC2 generated through sitedirected mutagenesis based on sequence alignments of pituitary adenylate cyclase-activating peptide (PACAP),vasoactive intestinal peptide (VIP),and related analogs.The recombinant DBAYL was used to evaluate its effect and mechanism in blood glucose metabolism and utilization.As much as 28.9 mg recombinant DBAYL peptide with purity over 98% can be obtained from 1 I of Luria-Bertani medium culture by the method established in this study and the prepared DBAYL with four mutations (N10Q,V18L,N29Q,and M added to the N-terminal)were much more stable than BAY55-9837.The half-life of recombinant DBAYL was about 25 folds compared with that of BAY55-9837 in vitro.The bioactivity assay of DBAYL showed that it displaced [125I]PACAP38 and [125I]VIP from VPAC2 with a half-maximal inhibitory concentration of 48.4 ± 6.9 and 47.1 ± 4.9 nM,respectively,which were significantly lower than that of BAY55-9837,one established VPAC2 agonists.DBAYL enhances the cAMP accumulation in CHO cells expressing human VPAC2 with a half-maximal stimulatory concentration (EC5o) of 0.68 nM,whereas the receptor potency of DBAYL at human VPAC1 (ECso of 737 nM) was only 1/1083of that at human VPAC2,and DBAYL had no activity toward human PAC1 receptor.Western blot analysis of the key proteins of insulin receptor signaling pathway:insulin receptor substrate 1 (IRS-1) and glucose transporter 4(GLUT4) indicated that the DBAYL could significantly induce the insulin-stimulated IRS-1 and GLUT4 expression more efficiently than BAY55-9837 and VIP in adipocytes.Compared with BAY55-9837 and PACAP38,the recombinant peptide DBAYL can more efficiently promote insulin release and decrease plasma glucose level in Institute of Cancer Research (ICR) mice.These results suggested that DBAYL could efficiently improve glucose uptake and glucose-dependent insulin

  14. Adenylate cyclase stimulation and ocular hypertension inhibition by forskolin analogs%佛司可林类似物激动腺苷酸环化酶及抑制兔高眼压探讨

    Institute of Scientific and Technical Information of China (English)

    杨为民; 李新华; 陈植和; 聂玲辉; 王伯龄; 沈志强

    2001-01-01

    目的 从滇产毛喉鞘蕊花植物中提取到3个佛司可林类似物,即异佛司可林、去酰基佛司可林、1-乙酰基佛司可林,体外测定它们激动腺苷酸环化酶活性,并观察去酰基佛司可林及1-乙酰基佛司可林对水负荷兔高眼压的影响。 方法 体外测定激动腺苷酸环化酶活性用蛋白质结合放免法、气动式眼压计观察兔眼压。 结果 异佛司可林体外激动腺苷酸环化酶活性与佛司可林相当,去酰基佛司可林稍弱,1-乙酰基佛司可林无活性。1%去酰基佛司可林及1-乙酰基佛司可林滴眼液滴兔眼,能抑制水负荷造成的兔高眼压,抑制率最高分别达6.0%及10.9%,作用持续至少3h。 结论 研究表明异佛司可林及去酰基佛司可林具有体外激动腺苷酸环化酶活性的作用,而去酰基佛司可林及1-乙酰基佛司可林具有抑制水负荷兔高眼压的作用。%ObjectiveForskolin (FSK) analogs,isoforskolin (isoF),deacetylforskolin(deaF),and 1-acetylforskolin(1-aF),extracted from Coleus forskohlii native to Yunnan,were assayed for their adenylate cyclase stimulating activities in vitro and for effects of two analogs on ocular hypertension (OHT) in water-loaded rabbits.MethodsAdenylate cyclase stimulation was determined by protein-binding method of radioimmunoassay,and intraocular pressure was monitored by pneumatonometer.ResultsIt showed that isoforskolin and forskolin stimulated adenylate cyclase in vitro with almost equal activity,deacetylforskolin with milder activity,and 1-acetylforskolin with little activity in vitro.1% deaF and 1-aF suppressed rabbit OHT induced by water-loading for at least 3h,with the maximal inhibitory rates of 6.0,10.9% respectively.ConclusionThis study suggests that two foskolin analogs (isoforskolin,deacetylforskolin) possess adenylate cyclase stimulation activities in vitro;deacetylforskolin and 1

  15. Influence of N-phenyl-2-naphthylamine on the Activity of Adenylate Cyclase Signaling System and the Virulence of Clavibacter michiganensis subsp. sepedonicus

    Directory of Open Access Journals (Sweden)

    A.M. Goncharova

    2016-11-01

    Full Text Available The effect of N-phenyl-2-naphthylamine was obtained from exudates of pea root on growth, virulence and signaling-specific of potato phytopathogen Clavibacter michiganensis subsp. sepedonicus. It is shown that the compound in a physiological concentration of the peas 9 mkM had no effect on C. michiganensis subsp. sepedonicus, but when exposed to N-FNA in a concentration of 45 mkM was observed reduction in growth of planktonic culture C. michiganensis subsp. sepedonicus, as well as changes in the activity of adenylyl cyclase signaling system components in this phytopathogen.

  16. Changes in vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide and neuropeptide Y-ergic structures of the enteric nervous system in the carcinoma of the human large intestine.

    Directory of Open Access Journals (Sweden)

    Ireneusz Mirosław Łakomy

    2010-08-01

    Full Text Available This investigation was aimed at immunohistochemical analysis of potential changes in the enteric nervous system caused by cancer of the large intestine. In this purpose, neurons and nerve fibers of intestinal plexuses containing neuropeptides: vasoactive intestinal peptide (VIP, pituitary adenylate cyclase-activating polypeptide (PACAP and neuropeptide Y (NPY, in pathologically changed part of the large intestine were microscpically observed and compared. Samples were taken from patients operated due to cancer of the sigmoid colon and rectum. The number of neurons and density of nerve fibres containing neuropeptides found in sections with cancer tissues were compared to those observed in sections from the uninvolved intestinal wall. Changes relating to reductions in the number of NPY-ergic neurons and density of nerve fibres in submucous and myenteric plexuses in the sections with cancer tissues (pathological sections were statistically significant. A statistically similar presence of VIP-ergic and PACAP-ergic neurons in the submucosal and myenteric plexuses was observed in both the pathological and control sections. On the other hand, in the pathological sections, VIP-ergic nerve fibres in the myenteric plexuses and PACAP-ergic nerve fibres in the submucosal and myenteric plexuses were found to be less dense. Analysis revealed changes in pathologically affected part of the large intestine may caused disruption of proper intestinal function. Observed changes in the neural elements which are responsible for relaxation of the intestine may suggest dysfunction in the innervation of this part of the colon.

  17. Ca2+ influx and tyrosine kinases trigger Bordetella adenylate cyclase toxin (ACT endocytosis. Cell physiology and expression of the CD11b/CD18 integrin major determinants of the entry route.

    Directory of Open Access Journals (Sweden)

    Kepa B Uribe

    Full Text Available Humans infected with Bordetella pertussis, the whooping cough bacterium, show evidences of impaired host defenses. This pathogenic bacterium produces a unique adenylate cyclase toxin (ACT which enters human phagocytes and catalyzes the unregulated formation of cAMP, hampering important bactericidal functions of these immune cells that eventually cause cell death by apoptosis and/or necrosis. Additionally, ACT permeabilizes cells through pore formation in the target cell membrane. Recently, we demonstrated that ACT is internalised into macrophages together with other membrane components, such as the integrin CD11b/CD18 (CR3, its receptor in these immune cells, and GM1. The goal of this study was to determine whether ACT uptake is restricted to receptor-bearing macrophages or on the contrary may also take place into cells devoid of receptor and gain more insights on the signalling involved. Here, we show that ACT is rapidly eliminated from the cell membrane of either CR3-positive as negative cells, though through different entry routes, which depends in part, on the target cell physiology and characteristics. ACT-induced Ca(2+ influx and activation of non-receptor Tyr kinases into the target cell appear to be common master denominators in the different endocytic strategies activated by this toxin. Very importantly, we show that, upon incubation with ACT, target cells are capable of repairing the cell membrane, which suggests the mounting of an anti-toxin cell repair-response, very likely involving the toxin elimination from the cell surface.

  18. 斜带石斑鱼PACAP的原核表达及活性分析%The prokaryotic expression and biological activity of the pituitary adenylate cyclase activating polypeptide in groupers Epinephelus coioides

    Institute of Scientific and Technical Information of China (English)

    江湧; 李文笙; 林浩然

    2005-01-01

    自1989年从绵羊下丘脑提取物发现垂体腺苷酸环化酶激活多肽(Pituitary adenylate cyclase activating polypeptide,PACAP)以来(Miyata et al.,1989),已证明它能促进垂体激素释放,同时还具有神经递质、神经调质和神经营养等作用,使对PACAP的研究成为十分活跃的领域。PACAP属于血管活性肠肽(VIP)-胰高血糖素-生长激素释放因子-分泌素家族(Campbell and Scanes,1992)成员,已鉴别出包含27和38个氨基酸两种类型。对原索动物(McRory et al.,1997)、两栖类(蛙)(Alexandre et al.,2000)、爬行类(蜥蜴)(Pohland Wank,1998)、鸟类(鸡)(McRory et al.,1997),啮齿类(鼠)(Ghatei et al.,1993)等脊椎动物PACAP的研究多集中在结构与进化方面,对功能了解甚少。

  19. The expression and significance of adenylate cyclase-associated protein 2 in human hepatocellular carcinoma%腺苷酸环化酶相关蛋白2在肝癌组织中的表达及其意义

    Institute of Scientific and Technical Information of China (English)

    谢艳英; 徐秋霞; 韩双印; 张立达; 白阳秋; 杨玉秀

    2010-01-01

    @@ 有关腺苷酸环化酶相关蛋白(adenylate cyclase-associated protein,CAP)2在肝癌中的生物学作用机制尚不明确.本研究应用逆转录聚合酶链反应(RT-PCR)及免疫组织化学技术检测CAP2在人正常肝脏、肝硬化和肝癌组织中的表达,探讨CAP2在肝癌发生发展过程中的作用.

  20. The crystal structure of the catalytic domain of a eukaryotic guanylate cyclase

    Directory of Open Access Journals (Sweden)

    Marletta Michael A

    2008-10-01

    Full Text Available Abstract Background Soluble guanylate cyclases generate cyclic GMP when bound to nitric oxide, thereby linking nitric oxide levels to the control of processes such as vascular homeostasis and neurotransmission. The guanylate cyclase catalytic module, for which no structure has been determined at present, is a class III nucleotide cyclase domain that is also found in mammalian membrane-bound guanylate and adenylate cyclases. Results We have determined the crystal structure of the catalytic domain of a soluble guanylate cyclase from the green algae Chlamydomonas reinhardtii at 2.55 Å resolution, and show that it is a dimeric molecule. Conclusion Comparison of the structure of the guanylate cyclase domain with the known structures of adenylate cyclases confirms the close similarity in architecture between these two enzymes, as expected from their sequence similarity. The comparison also suggests that the crystallized guanylate cyclase is in an inactive conformation, and the structure provides indications as to how activation might occur. We demonstrate that the two active sites in the dimer exhibit positive cooperativity, with a Hill coefficient of ~1.5. Positive cooperativity has also been observed in the homodimeric mammalian membrane-bound guanylate cyclases. The structure described here provides a reliable model for functional analysis of mammalian guanylate cyclases, which are closely related in sequence.

  1. Adenylate-forming enzymes

    Science.gov (United States)

    Schmelz, Stefan; Naismith, James H.

    2012-01-01

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

  2. Pituitary adenylate cyclase activating polypeptide (PACAP), stress, and sex hormones.

    Science.gov (United States)

    King, S Bradley; Toufexis, Donna J; Hammack, Sayamwong E

    2017-06-14

    Stressor exposure is associated with the onset and severity of many psychopathologies that are more common in women than men. Moreover, the maladaptive expression and function of stress-related hormones have been implicated in these disorders. Evidence suggests that PACAP has a critical role in the stress circuits mediating stress-responding, and PACAP may interact with sex hormones to contribute to sex differences in stress-related disease. In this review, we describe the role of the PACAP/PAC1 system in stress biology, focusing on the role of stress-induced alterations in PACAP expression and signaling in the development of stress-induced behavioral change. Additionally, we present more recent data suggesting potential interactions between stress, PACAP, and circulating estradiol in pathological states, including PTSD. These studies suggest that the level of stress and circulating gonadal hormones may differentially regulate the PACAPergic system in males and females to influence anxiety-like behavior and may be one mechanism underlying the discrepancies in human psychiatric disorders.

  3. In Vitro Assessment of Guanylyl Cyclase Activity of Plant Receptor Kinases

    KAUST Repository

    Raji, Misjudeen

    2017-05-31

    Cyclic nucleotides such as 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP) are increasingly recognized as key signaling molecules in plants, and a growing number of plant mononucleotide cyclases, both adenylate cyclases (ACs) and guanylate cyclases (GCs), have been reported. Catalytically active cytosolic GC domains have been shown to be part of many plant receptor kinases and hence directly linked to plant signaling and downstream cellular responses. Here we detail, firstly, methods to identify and express essential functional GC domains of receptor kinases, and secondly, we describe mass spectrometric methods to quantify cGMP generated by recombinant GCs from receptor kinases in vitro.

  4. Cyclic Nucleotide Monophosphates and Their Cyclases in Plant Signaling

    KAUST Repository

    Gehring, Christoph A.

    2017-10-04

    The cyclic nucleotide monophosphates (cNMPs), and notably 3′,5′-cyclic guanosine monophosphate (cGMP) and 3′,5′-cyclic adenosine monophosphate (cAMP) are now accepted as key signaling molecules in many processes in plants including growth and differentiation, photosynthesis, and biotic and abiotic defense. At the single molecule level, we are now beginning to understand how cNMPs modify specific target molecules such as cyclic nucleotide-gated channels, while at the systems level, a recent study of the Arabidopsis cNMP interactome has identified novel target molecules with specific cNMP-binding domains. A major advance came with the discovery and characterization of a steadily increasing number of guanylate cyclases (GCs) and adenylate cyclases (ACs). Several of the GCs are receptor kinases and include the brassinosteroid receptor, the phytosulfokine receptor, the Pep receptor, the plant natriuretic peptide receptor as well as a nitric oxide sensor. We foresee that in the near future many more molecular mechanisms and biological roles of GCs and ACs and their catalytic products will be discovered and further establish cNMPs as a key component of plant responses to the environment.

  5. Pituitary adenylate cyclase-activating polypeptide promotes eccrine gland sweat secretion

    DEFF Research Database (Denmark)

    Sasaki, S; Watanabe, J; Ohtaki, H;

    2016-01-01

    BACKGROUND: Sweat secretion is the major function of eccrine sweat glands; when this process is disturbed (paridrosis), serious skin problems can arise. To elucidate the causes of paridrosis, an improved understanding of the regulation, mechanisms and factors underlying sweat production is required...... and several exocrine glands, the effects of PACAP on the process of eccrine sweat secretion have not been examined. OBJECTIVES: To investigate the effect of PACAP on eccrine sweat secretion. METHODS: Reverse transcriptase-polymerase chain reaction and immunostaining were used to determine the expression...... and localization of PACAP and its receptors in mouse and human eccrine sweat glands. We injected PACAP subcutaneously into the footpads of mice and used the starch-iodine test to visualize sweat-secreting glands. RESULTS: Immunostaining showed PACAP and PAC1R expression by secretory cells from mouse and human...

  6. Pituitary adenylate cyclase-activating polypeptide stimulates renin secretion via activation of PAC1 receptors

    DEFF Research Database (Denmark)

    Hautmann, Matthias; Friis, Ulla G; Desch, Michael

    2007-01-01

    concentration was significantly lower in PAC1-/- compared with their wild-type littermates under control conditions as well as under a low- or high-salt diet and under treatment with the angiotensin-converting enzyme inhibitor ramipril, whereas no differences in plasma renin concentration between the genotypes......), because PACAP (1-27) applied in concentrations in the physiologic range (10 and 100 pmol/L) did not enhance renin release from isolated kidneys of PAC1 receptor knockout mice (PAC1-/-), whereas it stimulated renin release 1.38- and 2.5-fold in kidneys from wild-type mice. Moreover, plasma renin...... were detectable after water deprivation. These data show that PACAP acting on PAC1 receptors potently stimulates renin release, serving as a tonic enhancer of the renin system in vivo....

  7. Intein-mediated Rapid Purification of Recombinant Human Pituitary Adenylate Cyclase Activating Polypeptide

    Institute of Scientific and Technical Information of China (English)

    Rong-jie YU; An HONG; Yun DAI; Yuan GAO

    2004-01-01

    In order to obtain the recombinant human PACAP efficiently by intein-mediated single column purification, a gene encoding human PACAP was synthesized and cloned into Escherichia coli expression vector pKYB. The recombinant vector pKY-PAC was transferred into E. coli ER2566 cells and the target protein was over-expressed as a fusion to the N-terminus of a self-cleavable affinity tag. After the PACAPintein-CBD fusion protein was purified by chitin-affinity chromatography, the self-cleavage activity of the intein was induced by DTT and the rhPACAP was released from the chitin-bound intein tag. The activity of the rhPACAP to stimulate cyclic AMP accumulation was detected using the human pancreas carcinoma cells SW1990. Twenty-two milligrams of rhPACAP with the purity over 98% was obtained by single column purification from 1 liter of induced culture. The preliminary biological assay indicated that the rhPACAP, which has an extra Met at its N-terminus compared with the native human PACAP, had the similar activity of stimulating cAMP accumulation with the standard PACAP38 in the SW1990 cells. A new efficient production procedure of the active recombinant human PACAP was established.

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

    Science.gov (United States)

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

    2012-01-01

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

  9. Accelerated evolution of the pituitary adenylate cyclase-activating polypeptide precursor gene during human origin

    DEFF Research Database (Denmark)

    Wang, Yin-Qiu; Qian, Ya-Ping; Yang, Su

    2005-01-01

    a strong functional constraint during the course of evolution. However, through comparative sequence analysis, we demonstrated that the PACAP precursor gene underwent an accelerated evolution in the human lineage since the divergence from chimpanzees, and the amino acid substitution rate in humans...... is at least seven times faster than that in other mammal species resulting from strong Darwinian positive selection. Eleven human-specific amino acid changes were identified in the PACAP precursors, which are conserved from murine to African apes. Protein structural analysis suggested that a putative novel...... neuropeptide might have originated during human evolution and functioned in the human brain. Our data suggested that the PACAP precursor gene underwent adaptive changes during human origin and may have contributed to the formation of human cognition. Udgivelsesdato: 2005-Jun...

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

    Science.gov (United States)

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

    2012-01-01

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

  11. Integrative signaling networks of membrane guanylate cyclases: Biochemistry and physiology

    Directory of Open Access Journals (Sweden)

    Rameshwar K Sharma

    2016-09-01

    Full Text Available This monograph presents a historical perspective of cornerstone developments on the biochemistry and physiology of mammalian membrane guanylate cyclases (MGCs, highlighting contributions made by the authors and their collaborators. Upon resolution of early, contentious studies, cyclic GMP emerged, alongside cyclic AMP, as an important intracellular second messenger for hormonal signaling. However, the two signaling pathways differ in significant ways. In the cyclic AMP pathway, hormone binding to a G protein coupled receptor leads to stimulation or inhibition of an adenylate cyclase, whereas the cyclic GMP pathway dispenses with intermediaries; hormone binds to an MGC to affect its activity. Although the cyclic GMP pathway is direct, it is by no means simple. The modular design of the molecule incorporates regulation by ATP binding and phosphorylation. MGCs can form complexes with Ca2+-sensing subunits that either increase or decrease cyclic GMP synthesis, depending on subunit identity. In some systems, co-expression of two Ca2+ sensors, GCAP1 and S100B with ROS-GC1 confers bimodal signaling marked by increases in cyclic GMP synthesis when intracellular Ca2+ concentration rises or falls. Some MGCs monitor or are modulated by carbon dioxide via its conversion to bicarbonate. One MGC even functions as a thermosensor as well as a chemosensor; activity reaches a maximum with a mild drop in temperature. The complexity afforded by these multiple limbs of operation enables MGC networks to perform transductions traditionally reserved for G protein coupled receptors and TRP (Transient Receptor Potential channels and to serve a diverse array of functions, including control over cardiac vasculature, smooth muscle relaxation, blood pressure regulation, cellular growth, sensory transductions, neural plasticity and memory.

  12. Integrative Signaling Networks of Membrane Guanylate Cyclases: Biochemistry and Physiology

    Science.gov (United States)

    Sharma, Rameshwar K.; Duda, Teresa; Makino, Clint L.

    2016-01-01

    This monograph presents a historical perspective of cornerstone developments on the biochemistry and physiology of mammalian membrane guanylate cyclases (MGCs), highlighting contributions made by the authors and their collaborators. Upon resolution of early contentious studies, cyclic GMP emerged alongside cyclic AMP, as an important intracellular second messenger for hormonal signaling. However, the two signaling pathways differ in significant ways. In the cyclic AMP pathway, hormone binding to a G protein coupled receptor leads to stimulation or inhibition of an adenylate cyclase, whereas the cyclic GMP pathway dispenses with intermediaries; hormone binds to an MGC to affect its activity. Although the cyclic GMP pathway is direct, it is by no means simple. The modular design of the molecule incorporates regulation by ATP binding and phosphorylation. MGCs can form complexes with Ca2+-sensing subunits that either increase or decrease cyclic GMP synthesis, depending on subunit identity. In some systems, co-expression of two Ca2+ sensors, GCAP1 and S100B with ROS-GC1 confers bimodal signaling marked by increases in cyclic GMP synthesis when intracellular Ca2+ concentration rises or falls. Some MGCs monitor or are modulated by carbon dioxide via its conversion to bicarbonate. One MGC even functions as a thermosensor as well as a chemosensor; activity reaches a maximum with a mild drop in temperature. The complexity afforded by these multiple limbs of operation enables MGC networks to perform transductions traditionally reserved for G protein coupled receptors and Transient Receptor Potential (TRP) ion channels and to serve a diverse array of functions, including control over cardiac vasculature, smooth muscle relaxation, blood pressure regulation, cellular growth, sensory transductions, neural plasticity and memory.

  13. Cytosolic adenylate changes during exercise in prawn muscle

    Energy Technology Data Exchange (ETDEWEB)

    Thebault, M.T. [College de France, 29 - Concarneau (France); Raffin, J.P.; Pichon, R. [Brest Univ., 29 (France)

    1994-11-01

    {sup 31}P NMR and biochemical analysis were used to assess the effect of heavy exercise on cytosolic adenylate levels in Palaemon serratus abdominal muscle. At rest, the MgATP level corresponded to 85.5% of the total ATP content. The cytosolic adenylate concentrations of the prawn muscle are considerably different from that of vertebrates. The percentage of ADP bound to myofilaments was lower in the prawn muscle. Consequently, the level of free cytosolic AMP was greatly higher (thirty fold higher) than in vertebrate muscle. During vigorous work, the concentration of MgATP dropped and the cytosolic AMP accumulated, while the cytosolic adenine nucleotide pool decreased significantly. The phosphorylation potential value and the ATP/ADP ratio, calculated from the cytosolic adenylate, dropped acutely during the whole period of muscular contractions. On the contrary, the adenylate energy charge calculated from the cytosolic adenylate decreased slightly. Therefore, even in muscle displaying no AMP deamination, the adenylate charge is stabilized during exercise by the dynamic changes between cytosolic and bound adenylate species. (author). 21 refs., 2 tabs.

  14. Structural studies of Schistosoma mansoni adenylate kinases

    Energy Technology Data Exchange (ETDEWEB)

    Marques, I.A. [Universidade Federal de Goias (UFG), Goiania, GO (Brazil); Pereira, H.M.; Garrat, R.C. [Universidade de Sao Paulo (USP-SC), Sao Carlos, SP (Brazil)

    2012-07-01

    Full text: Parasitic diseases are a major cause of death in developing countries, however receive little or no attention from pharmaceutical companies for the development of novel therapies. In this respect, the Center for Structural Molecular Biology (CBME) of the Institute of Physics of Sao Carlos (IFSC / USP) has developed expertise in all stages of the development of active compounds against target enzymes from parasitic diseases. The present work focuses on the adenylate kinase enzymes (ADK's) from Schistosoma mansoni. These enzymes are widely distributed and catalyze the reaction of phosphoryl exchange between nucleotides in the reaction 2ADP to ATP + AMP, which is critical for the cells life cycle. Due to the particular property of the reaction catalyzed, the ADK's are recognized as reporters of the cells energetic state, translating small changes in the balance between ATP and ADP into a large change in concentration of AMP. The genome of S. mansoni was recently sequenced by the Sanger Center in England. On performing searches for genes encoding adenylate kinases we found two such genes. The corresponding gene products were named ADK1 (197 residues) and ADK2 (239 residues), and the two sequences share only 28 percent identity. Both have been cloned into the pET-28a(+)vector, expressed in E. coli and purified. Preliminary tests of activity have been performed only for ADK1 showing it to be catalytically active. Crystallization trials were performed for both proteins and thus far, crystals of ADK1 have been obtained which diffract to 2.05 at the LNLS beamline MX2 and the structure solved by molecular replacement. Understanding, at the atomic level, the function of these enzymes may help in the development of specific inhibitors and may provide tools for developing diagnostic tests for schistosomiasis. (author)

  15. The flagellar adenylate kinases of Trypanosoma cruzi.

    Science.gov (United States)

    Camara, María de los Milagros; Bouvier, León A; Miranda, Mariana R; Pereira, Claudio A

    2015-01-01

    Adenylate kinases (ADK) are key enzymes involved in cell energy management. Trypanosomatids present the highest number of variants in a single cell in comparison with the rest of the living organisms. In this work, we characterized two flagellar ADKs from Trypanosoma cruzi, called TcADK1 and TcADK4, which are also located in the cell cytosol. Interestingly, TcADK1 presents a stage-specific expression. This variant was detected in epimastigotes cells, and was completely absent in trypomastigotes and amastigotes, while TcADK4 is present in the major life cycle stages of T. cruzi. Both variants are also regulated, in opposite ways, along the parasite growth curve suggesting that their expression depends on the intra- and extracellular conditions. Both, TcADK1 and TcADK4 present N-terminal extension that could be responsible for their subcellular localization. The presence of ADK variants in the flagellum would be critical for the provision of energy in a process of high ATP consumption such as cell motility.

  16. Mechanistic investigations on six bacterial terpene cyclases

    Directory of Open Access Journals (Sweden)

    Patrick Rabe

    2016-08-01

    Full Text Available The products obtained by incubation of farnesyl diphosphate (FPP with six purified bacterial terpene cyclases were characterised by one- and two-dimensional NMR spectroscopic methods, allowing for a full structure elucidation. The absolute configurations of four terpenes were determined based on their optical rotary powers. Incubation experiments with 13C-labelled isotopomers of FPP in buffers containing water or deuterium oxide allowed for detailed insights into the cyclisation mechanisms of the bacterial terpene cyclases.

  17. Coupled ATPase-adenylate kinase activity in ABC transporters

    Science.gov (United States)

    Kaur, Hundeep; Lakatos-Karoly, Andrea; Vogel, Ramona; Nöll, Anne; Tampé, Robert; Glaubitz, Clemens

    2016-01-01

    ATP-binding cassette (ABC) transporters, a superfamily of integral membrane proteins, catalyse the translocation of substrates across the cellular membrane by ATP hydrolysis. Here we demonstrate by nucleotide turnover and binding studies based on 31P solid-state NMR spectroscopy that the ABC exporter and lipid A flippase MsbA can couple ATP hydrolysis to an adenylate kinase activity, where ADP is converted into AMP and ATP. Single-point mutations reveal that both ATPase and adenylate kinase mechanisms are associated with the same conserved motifs of the nucleotide-binding domain. Based on these results, we propose a model for the coupled ATPase-adenylate kinase mechanism, involving the canonical and an additional nucleotide-binding site. We extend these findings to other prokaryotic ABC exporters, namely LmrA and TmrAB, suggesting that the coupled activities are a general feature of ABC exporters. PMID:28004795

  18. Recurrent adenylation domain replacement in the microcystin synthetase gene cluster

    Directory of Open Access Journals (Sweden)

    Laakso Kati

    2007-10-01

    Full Text Available Abstract Background Microcystins are small cyclic heptapeptide toxins produced by a range of distantly related cyanobacteria. Microcystins are synthesized on large NRPS-PKS enzyme complexes. Many structural variants of microcystins are produced simulatenously. A recombination event between the first module of mcyB (mcyB1 and mcyC in the microcystin synthetase gene cluster is linked to the simultaneous production of microcystin variants in strains of the genus Microcystis. Results Here we undertook a phylogenetic study to investigate the order and timing of recombination between the mcyB1 and mcyC genes in a diverse selection of microcystin producing cyanobacteria. Our results provide support for complex evolutionary processes taking place at the mcyB1 and mcyC adenylation domains which recognize and activate the amino acids found at X and Z positions. We find evidence for recent recombination between mcyB1 and mcyC in strains of the genera Anabaena, Microcystis, and Hapalosiphon. We also find clear evidence for independent adenylation domain conversion of mcyB1 by unrelated peptide synthetase modules in strains of the genera Nostoc and Microcystis. The recombination events replace only the adenylation domain in each case and the condensation domains of mcyB1 and mcyC are not transferred together with the adenylation domain. Our findings demonstrate that the mcyB1 and mcyC adenylation domains are recombination hotspots in the microcystin synthetase gene cluster. Conclusion Recombination is thought to be one of the main mechanisms driving the diversification of NRPSs. However, there is very little information on how recombination takes place in nature. This study demonstrates that functional peptide synthetases are created in nature through transfer of adenylation domains without the concomitant transfer of condensation domains.

  19. The Cyclase-associated protein Cap1 is important for proper regulation of infection-related morphogenesis in Magnaporthe oryzae.

    Directory of Open Access Journals (Sweden)

    Xiaoying Zhou

    2012-09-01

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

  20. The Cyclase-associated protein Cap1 is important for proper regulation of infection-related morphogenesis in Magnaporthe oryzae.

    Science.gov (United States)

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

    2012-09-01

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

  1. Nucleoside triphosphate synthesis catalysed by adenylate kinase is ADP dependent

    DEFF Research Database (Denmark)

    Willemoës, Martin; Kilstrup, Mogens

    2005-01-01

    Adenylate kinase (Adk) that catalyses the synthesis of ADP from ATP and AMP has also been shown to perform an ATP dependent phosphorylation of ribo- and deoxynucleoside diphosphates to their corresponding nucleoside triphosphate; ATP+(d)NDPADP+(d)NTP. This reaction, suggested to occur by the tran......Adenylate kinase (Adk) that catalyses the synthesis of ADP from ATP and AMP has also been shown to perform an ATP dependent phosphorylation of ribo- and deoxynucleoside diphosphates to their corresponding nucleoside triphosphate; ATP+(d)NDPADP+(d)NTP. This reaction, suggested to occur...

  2. Characterization of two unusual guanylyl cyclases from Dictyostelium

    NARCIS (Netherlands)

    Roelofs, Jeroen; Haastert, Peter J.M. van

    2002-01-01

    Guanylyl cyclase A (GCA) and soluble guanylyl cyclase (sGC) encode GCs in Dictyostelium and have a topology similar to 12-transmembrane and soluble adenylyl cyclase, respectively. We demonstrate that all detectable GC activity is lost in a cell line in which both genes have been inactivated. Cell li

  3. Nucleoside triphosphate synthesis catalysed by adenylate kinase is ADP dependent

    DEFF Research Database (Denmark)

    Willemoes, Martin; Kilstrup, M.

    2005-01-01

    Adenylate kinase (Adk) that catalyses the synthesis of ADP from ATP and AMP has also been shown to perform an ATP dependent phosphorylation of ribo- and deoxynucleoside diphosphates to their corresponding nucleoside triphosphate; ATP + (d)NDP ¿ ADP + (d)NTP. This reaction, suggested to occur...

  4. Y1 receptors for neuropeptide Y are coupled to mobilization of intracellular calcium and inhibition of adenylate cyclase

    DEFF Research Database (Denmark)

    Aakerlund, L; Gether, U; Fuhlendorff, J;

    1990-01-01

    Two types of binding sites have previously been described for neuropeptide Y (NPY), called Y1 and Y2 receptors. The intracellular events following Y1 receptor activation was studied in the human neuroblastoma cell line SK-N-MC. Both NPY and the specific Y1 receptor ligand, [Leu31,Pro34]-NPY, caused...

  5. Alternative Splicing of the Pituitary Adenylate Cyclase-Activating Polypeptide Receptor PAC1: Mechanisms of Fine Tuning of Brain Activity

    Directory of Open Access Journals (Sweden)

    Janna eBlechman

    2013-05-01

    Full Text Available Alternative splicing of the precursor mRNA encoding for the neuropeptide receptor PAC1/ADCYAP1R1 generates multiple protein products that exhibit pleiotropic activities. Recent studies in mammals and zebrafish have implicated some of these splice isoforms in control of both cellular and body homeostasis. Here, we review the regulation of PAC1 splice variants and their underlying signal transduction and physiological processes in the nervous system.

  6. Innervation of the rat pineal gland by pituitary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive nerve fibres

    DEFF Research Database (Denmark)

    Møller, Morten; Fahrenkrug, Jan; Hannibal, J.

    1999-01-01

    Calcitonin gene-related peptide, vasoactive intestinal peptide, neuropeptide Y, colocalization, trigeminal ganglion, rat (Wistar)......Calcitonin gene-related peptide, vasoactive intestinal peptide, neuropeptide Y, colocalization, trigeminal ganglion, rat (Wistar)...

  7. Plant-activated bacterial receptor adenylate cyclases modulate epidermal infection in the Sinorhizobium meliloti–Medicago symbiosis

    OpenAIRE

    Tian, Chang Fu; Garnerone, Anne-Marie; Mathieu-Demazière, Céline; Masson-Boivin, Catherine; Batut, Jacques

    2012-01-01

    Legumes and soil bacteria called rhizobia have coevolved a facultative nitrogen-fixing symbiosis. Establishment of the symbiosis requires bacterial entry via root hair infection threads and, in parallel, organogenesis of nodules that subsequently are invaded by bacteria. Tight control of nodulation and infection is required to maintain the mutualistic character of the interaction. Available evidence supports a passive bacterial role in nodulation and infection after the microsymbiont has trig...

  8. Investigation of the pathophysiological mechanisms of migraine attacks induced by pituitary adenylate cyclase-activating polypeptide-38

    DEFF Research Database (Denmark)

    Amin, Faisal Mohammad; Hougaard, Anders; Schytz, Henrik W

    2014-01-01

    aura were randomly allocated to intravenous infusion of PACAP38 (10 pmol/kg/min) or vasoactive intestinal polypeptide (8 pmol/kg/min) over 20 min. We recorded incidence of migraine during and after infusion (0-24 h). Magnetic resonance angiography of selected extra- and intracranial arteries, blood...... peptides induced marked dilatation of the extracranial (P 0.05). PACAP38-induced vasodilatation was longer lasting (>2 h), whereas vasoactive intestinal polypeptide-induced dilatation was normalized after 2 h. We recorded elevated plasma PACAP38 at 1 h after...... and elevated plasma PACAP38 before onset of migraine-like attacks. PACAP38 has a much higher affinity for the PAC1 receptor and we therefore suggest that migraine induction by PACAP38 may be because of activation of the PAC1 receptor, which may be a future anti-migraine drug target....

  9. Bicarbonate-Regulated Soluble Adenylyl Cyclase

    Directory of Open Access Journals (Sweden)

    Wuttke MS

    2001-07-01

    Full Text Available Soluble adenylyl cyclase (sAC represents a novel form of mammalian adenylyl cyclase structurally, molecularly, and biochemically distinct from the G protein-regulated, transmembrane adenylyl cyclases (tmACs. sAC possesses no transmembrane domains and is insensitive to classic modulators of tmACs, such as heterotrimeric G proteins and P site ligands. Thus, sAC defines an independently regulated cAMP signaling system within mammalian cells. sAC is directly stimulated by bicarbonate ion both in vivo in heterologously expressing cells and in vitro using purified protein. sAC appears to be the predominant form of adenylyl cyclase (AC in mammalian sperm, and its direct activation by bicarbonate provides a mechanism for generating the cAMP required to complete the bicarbonate-induced processes necessary for fertilization, including hyperactivated motility, capacitation, and the acrosome reaction. Immunolocalization studies reveal sAC is also abundantly expressed in other tissues which respond to bicarbonate or carbon dioxide levels suggesting it may function as a general bicarbonate/CO(2 sensor throughout the body.

  10. Allosteric activation of Bordetella pertussis adenylyl cyclase by calmodulin: molecular dynamics and mutagenesis studies.

    Science.gov (United States)

    Selwa, Edithe; Davi, Marilyne; Chenal, Alexandre; Sotomayor-Pérez, Ana-Cristina; Ladant, Daniel; Malliavin, Thérèse E

    2014-07-25

    Adenylyl cyclase (AC) toxin is an essential toxin that allows Bordetella pertussis to invade eukaryotic cells, where it is activated after binding to calmodulin (CaM). Based on the crystal structure of the AC catalytic domain in complex with the C-terminal half of CaM (C-CaM), our previous molecular dynamics simulations (Selwa, E., Laine, E., and Malliavin, T. (2012) Differential role of calmodulin and calcium ions in the stabilization of the catalytic domain of adenyl cyclase CyaA from Bordetella pertussis. Proteins 80, 1028–1040) suggested that three residues (i.e. Arg(338), Asn(347), and Asp(360)) might be important for stabilizing the AC/CaM interaction. These residues belong to a loop-helix-loop motif at the C-terminal end of AC, which is located at the interface between CaM and the AC catalytic loop. In the present study, we conducted the in silico and in vitro characterization of three AC variants, where one (Asn(347); ACm1A), two (Arg(338) and Asp(360); ACm2A), or three residues (Arg(338), Asn(347), and Asp(360); ACm3A) were substituted with Ala. Biochemical studies showed that the affinities of ACm1A and ACm2A for CaM were not affected significantly, whereas that of ACm3A was reduced dramatically. To understand the effects of these modifications, molecular dynamics simulations were performed based on the modified proteins. The molecular dynamics trajectories recorded for the ACm3AC-CaM complex showed that the calcium-binding loops of C-CaM exhibited large fluctuations, which could be related to the weakened interaction between ACm3A and its activator. Overall, our results suggest that the loop-helix-loop motif at the C-terminal end of AC is crucial during CaM binding for stabilizing the AC catalytic loop in an active configuration.

  11. Diterpene Cyclases and the Nature of the Isoprene Fold

    Science.gov (United States)

    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

  12. An aberrant adenylate kinase isoenzyme from the serum of patients with Duchenne muscular dystrophy.

    Science.gov (United States)

    Hamada, M; Okuda, H; Oka, K; Watanabe, T; Ueda, K; Nojima, M; Kuby, S A; Manship, M; Tyler, F H; Ziter, F A

    1981-08-13

    The sera from patients with human Duchenne (X-linked) progressive muscular dystrophy contain elevated adenylate kinase (ATP: AMP phosphotransferase, EC 2.7.4.3) activities, in addition to their characteristically high creatine kinase (ATP; creatine N-phosphotransferase, EC 2.7.3.2) activities. By agarose gel electrophoresis of human Duchenne dystrophic serum, the presence of an apparently normal human serum adenylate kinase together with a variant species of adenylate kinase was detected. The latter enzyme species appeared, in its mobility, to be similar to that of the normal human liver-type adenylate kinase. The presence of this aberrant liver-type adenylate kinase could also be demonstrated by characteristic (for the liver type) inhibition patterns with P1,P5-di-(adenosine-5')pentaphosphate, 5,5'-dithiobis(2-nitrobenzoate) and phosphoenolpyruvate. On the other hand, by inhibition titrations with an anti-muscle-type adenylate kinase, hemolysates from the erythrocytes of several Duchenne and Becker's dystrophics were found to contain approx. 96% muscle-type adenylate kinase and their serum approx. 97% muscle-type adenylate kinase. These same patients contained approx. 89% M-M type creatine kinase in their serum (by inhibition against anti-human muscle-type creatine kinase) indicative of the presence also of M-B plus B-B type active isoenzymes. All of these data can best be explained by the presence of a variant or mutant adenylate kinase isoenzyme in the dystrophic serum. This isoenzyme appears to resemble the liver type in its inhibition patterns with P1,P5-di(adenosine-5')pentaphosphate, 5,5'-dithiobis(2-nitrobenzoate) and phosphoenolpyruvate, and in its heat stability (compare also the agarose gel electrophoresis pattern); but structurally, it is a muscle type, or derived from a muscle type, as shown immunologically by inhibition reactions with anti-muscle-type adenylate kinase. Whether this is a fetal-type isoenzyme of adenylate kinase will require further

  13. Effect of association with adenylyl cyclase-associated protein on the interaction of yeast adenylyl cyclase with Ras protein.

    Science.gov (United States)

    Shima, F; Yamawaki-Kataoka, Y; Yanagihara, C; Tamada, M; Okada, T; Kariya, K; Kataoka, T

    1997-03-01

    Posttranslational modification of Ras protein has been shown to be critical for interaction with its effector molecules, including Saccharomyces cerevisiae adenylyl cyclase. However, the mechanism of its action was unknown. In this study, we used a reconstituted system with purified adenylyl cyclase and Ras proteins carrying various degrees of the modification to show that the posttranslational modification, especially the farnesylation step, is responsible for 5- to 10-fold increase in Ras-dependent activation of adenylyl cyclase activity even though it has no significant effect on their binding affinity. The stimulatory effect of farnesylation is found to depend on the association of adenylyl cyclase with 70-kDa adenylyl cyclase-associated protein (CAP), which was known to be required for proper in vivo response of adenylyl cyclase to Ras protein, by comparing the levels of Ras-dependent activation of purified adenylyl cyclase with and without bound CAP. The region of CAP required for this effect is mapped to its N-terminal segment of 168 amino acid residues, which coincides with the region required for the in vivo effect. Furthermore, the stimulatory effect is successfully reconstituted by in vitro association of CAP with the purified adenylyl cyclase molecule lacking the bound CAP. These results indicate that the association of adenylyl cyclase with CAP is responsible for the stimulatory effect of posttranslational modification of Ras on its activity and that this may be the mechanism underlying its requirement for the proper in vivo cyclic AMP response.

  14. G-protein-mediated interconversions of cell-surface cAMP receptors and their involvement in excitation and desensitization of guanylate cyclase in Dictyostelium discoideum

    Energy Technology Data Exchange (ETDEWEB)

    van Haastert, P.J.; de Wit, R.J.; Janssens, P.M.; Kesbeke, F.; DeGoede, J.

    1986-05-25

    In Dictyostelium discoideum cells, extracellular cAMP induces the rapid (within 2 s) activation of guanylate cyclase, which is followed by complete desensitization after about 10 s. cAMP binding to these cells is heterogeneous, showing a subclass of fast dissociating sites coupled to adenylate cyclase (A-sites) and a subclass of slowly dissociating sites coupled to guanylate cyclase (B-sites). The kinetics of the B-sites were further investigated on a seconds time scale. Statistical analysis of the association of (/sup 3/H)cAMP to the B-sites and dissociation of the complex revealed that the receptor can exist in three states which interconvert according to the following scheme. cAMP binds to the BF-state (off-rate 2.5 s) which rapidly (t1/2 = 3 s) converts to the BS-state (off-rate 15 s) and subsequently (without a detectable delay) into the BSS-state (off-rate 150 s). In membranes, both the BS- and BSS-states are converted to the BF-state by GTP and GDP, suggesting the involvement of a G-protein. Densensitized cells show a 80% reduction of the formation of the BSS-state, but no reduction of the BF- or BS-state. These data are combined into a model in which the transitions of the B-sites are mediated by a G-protein; activation of the G-protein and guanylate cyclase is associated with the transition of the BS- to the BSS-state of the receptor, whereas desensitization is associated with the inhibition of this transition.

  15. Isoform-targeted regulation of cardiac adenylyl cyclase.

    Science.gov (United States)

    Ishikawa, Yoshihiro

    2003-01-01

    Numerous attempts have been made to develop strategies for regulating the intracellular cyclic AMP signal pharmacologically, with an intention to establish either new medical therapeutic methods or experimental tools. In the past decades, many pharmacological reagents have been identified that regulate this pathway at the level of the receptor. G protein, adenylyl cyclase, cyclic AMP, protein kinase A and phosphodiesterase. Since the cloning of adenylyl cyclase isoforms during the 1990s, investigators including ourselves have tried to find reagents that regulate the activity of this enzyme directly in an isoform-dependent manner. The ultimate goal of developing such reagents would be to regulate the cyclic AMP signal in an organ-dependent manner. Ourselves and other workers have reported that such reagents may vary from a simple cation to kinases. In a more recent study, using the results from crystallographic studies and computer-assisted drug design programs, we have identified subtype-selective regulators of adenylyl cyclase. Such regulators are mostly based upon forskolin, a diterpene compound obtained from Coleus forskolii, that acts directly on adenylyl cyclase to increase the intracellular levels of cyclic AMP. Similarly, novel reagents have been identified that inhibit a specific adenylyl cyclase isoform (e.g. type 5 adenylyl cyclase). Such reagents would potentially provide a new therapeutic strategy to treat hypertension, for example, as well as methods to selectively stimulate or inhibit this adenylyl cyclase isoform, which may be reminiscent of overexpression or knocking out of the cardiac adenylyl cyclase isoform by the use of a pharmacological method.

  16. Crystallization of cyclase-associated protein from Dictyostelium discoideum.

    Science.gov (United States)

    Hofmann, Andreas; Hess, Sonja; Noegel, Angelika A; Schleicher, Michael; Wlodawer, Alexander

    2002-10-01

    Cyclase-associated protein (CAP) is a conserved two-domain protein that helps to activate the catalytic activity of adenylyl cyclase in the cyclase-bound state through interaction with Ras, which binds to the cyclase in a different region. With its other domain, CAP can bind monomeric actin and therefore also carries a cytoskeletal function. The protein is thus involved in Ras/cAMP-dependent signal transduction and most likely serves as an adapter protein translocating the adenylyl cyclase complex to the actin cytoskeleton. Crystals belonging to the orthorhombic space group C222, with unit-cell parameters a = 71.2, b = 75.1, c = 162.9 A, have been obtained from Dictyostelium discoideum CAP carrying a C-terminal His tag. A complete native data set extending to 2.2 A resolution was collected from a single crystal using an in-house X-ray system. The asymmetric unit contains one molecule of CAP.

  17. Adenylate kinase 1 knockout mice have normal thiamine triphosphate levels.

    Science.gov (United States)

    Makarchikov, Alexander F; Wins, Pierre; Janssen, Edwin; Wieringa, Bé; Grisar, Thierry; Bettendorff, Lucien

    2002-10-21

    Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues and it may act as a phosphate donor for the phosphorylation of proteins, suggesting a potential role in cell signaling. Two mechanisms have been proposed for the enzymatic synthesis of ThTP. A thiamine diphosphate (ThDP) kinase (ThDP+ATP if ThTP+ADP) has been purified from brewer's yeast and shown to exist in rat liver. However, other data suggest that, at least in skeletal muscle, adenylate kinase 1 (AK1) is responsible for ThTP synthesis. In this study, we show that AK1 knockout mice have normal ThTP levels in skeletal muscle, heart, brain, liver and kidney, demonstrating that AK1 is not responsible for ThTP synthesis in those tissues. We predict that the high ThTP content of particular tissues like the Electrophorus electricus electric organ, or pig and chicken skeletal muscle is more tightly correlated with high ThDP kinase activity or low soluble ThTPase activity than with non-stringent substrate specificity and high activity of adenylate kinase.

  18. Calcium regulation of adenylyl cyclase relevance for endocrine control.

    Science.gov (United States)

    Antoni, F A

    1997-01-01

    A fundamental process in the hormonal regulation of body functions is the conversion of the intercellular signal into an intracellular signal. The first recognized intracellular messengers mediating the actions of hormones were calcium ions (Ca(2+)) and adenosine 3':5' monophosphate (cAMP), which is synthesized from ATP by adenylyl cyclase. Recent work on the structure of adenylyl cyclases has shown that these enzymes are individually tailored molecular machines controlled by diverse Ca(2+)-dependent mechanisms. These include allosteric regulation of enzyme activity through the Ca(2+)-receptor protein calmodulin, apparently direct actions of Ca(2+)on the cyclase catalytic moiety and phosphorylation/dephosphorylation by Ca(2+)-regulated protein kinases and protein phosphatases. This article is a brief review of the recent developments in the area of cyclase control that forecast a major revival of the interest in cAMP-Ca(2+)interactions. (c) 1997, Elsevier Science Inc. (Trends Endocrinol Metab 1997;8:7-14).

  19. Control of guanylate cyclase activity in the rod outer segment.

    Science.gov (United States)

    Pannbacker, R G

    1973-12-14

    Mammalian photoreceptors contain a guanylate cyclase which has a high specific activity and is inhibited by exposure of the rod outer segment to light. Several minutes are required for this inhibition to take effect, indicating that it is not a step in visual excitation. The activity of the enzyme is sensitive to the concentration of calcium ion in the medium, suggesting that light-induced changes in calcium distribution in the photoreceptor could control guanylate cyclase activity.

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

    Directory of Open Access Journals (Sweden)

    Shiva Mansouri

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

  1. Molecular and Functional Characterization of a Trypanosoma cruzi Nuclear Adenylate Kinase Isoform

    OpenAIRE

    María de los Milagros Cámara; Bouvier, León A.; Canepa, Gaspar E.; Mariana R Miranda; Pereira, Claudio A.

    2013-01-01

    Trypanosoma cruzi, the etiological agent of Chagas’ disease, is an early divergent eukaryote in which control of gene expression relies mainly in post-transcriptional mechanisms. Transcription levels are globally up and down regulated during the transition between proliferating and non-proliferating life-cycle stages. In this work we characterized a nuclear adenylate kinase isoform (TcADKn) that is involved in ribosome biogenesis. Nuclear adenylate kinases have been recently described in a fe...

  2. Optimization of ATP synthase function in mitochondria and chloroplasts via the adenylate kinase equilibrium

    Directory of Open Access Journals (Sweden)

    Abir U Igamberdiev

    2015-01-01

    Full Text Available The bulk of ATP synthesis in plants is performed by ATP synthase, the main bioenergetics engine of cells, operating both in mitochondria and in chloroplasts. The reaction mechanism of ATP synthase has been studied in detail for over half a century; however, its optimal performance depends also on the steady delivery of ATP synthase substrates and the removal of its products. For mitochondrial ATP synthase, we analyze here the provision of stable conditions for (i the supply of ADP and Mg2+, supported by adenylate kinase (AK equilibrium in the intermembrane space, (ii the supply of phosphate via membrane transporter in symport with H+, and (iii the conditions of outflow of ATP by adenylate transporter carrying out the exchange of free adenylates. We also show that, in chloroplasts, AK equilibrates adenylates and governs Mg2+ contents in the stroma, optimizing ATP synthase and Calvin cycle operation, and affecting the import of inorganic phosphate in exchange with triose phosphates. It is argued that chemiosmosis is not the sole component of ATP synthase performance, which also depends on AK-mediated equilibrium of adenylates and Mg2+, adenylate transport and phosphate release and supply.

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

    Energy Technology Data Exchange (ETDEWEB)

    Tumbale, Percy [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States). Lab. of Structural Biology; Williams, Jessica S. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States). Lab. of Structural Biology; Schellenberg, Matthew J. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States). Lab. of Structural Biology; Kunkel, Thomas A. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States). Lab. of Structural Biology and Lab. of Molecular Genetics; Williams, R. Scott [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States). Lab. of Structural Biology and Lab. Molecular Genetics

    2013-12-22

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

  4. Adenylate kinase locus 1 polymorphism and feto-placental development.

    Science.gov (United States)

    Fulvia, Gloria-Bottini; Antonio, Pietroiusti; Anna, Neri; Patrizia, Saccucci; Ada, Amante; Egidio, Bottini; Andrea, Magrini

    2011-12-01

    Recently our group has found that the correlation between birth weight and placental weight - an index of a balanced feto-placental unit development - is influenced by genetic factors. Since adenylate kinase locus 1 (AK₁) is a polymorphic enzyme that plays an important role in the synthesis of nucleotides required for many metabolic functions, we have investigated the possible role of its genetic variability in the correlation between birth weight and placental weight. 342 consecutive healthy newborn infants from the population of Rome (Italy) and 286 puerperae from another population from Central Italy were studied. The correlation coefficient between birth weight and placental weight is much higher in infants with low activity AK₁2-1 phenotype than in those with high activity AK₁1 phenotype. The difference between AK₁ and AK₁2-1 is well marked only in newborns with a gestational age greater than 38 weeks and it is not influenced by sex, maternal age and maternal smoking. A similar pattern is observed with maternal AK₁ phenotype. These results suggest that the difference in enzymatic activity between AK₁ phenotypes influencing the equilibrium among ATP, ADP, AMP and adenosine could have an important role in a balanced development of feto-placental unit. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  5. Conformational dynamics of a ligand-free adenylate kinase.

    Directory of Open Access Journals (Sweden)

    Hyun Deok Song

    Full Text Available Adenylate kinase (AdK is a phosphoryl-transfer enzyme with important physiological functions. Based on a ligand-free open structure and a ligand-bound closed structure solved by crystallography, here we use molecular dynamics simulations to examine the stability and dynamics of AdK conformations in the absence of ligands. We first perform multiple simulations starting from the open or the closed structure, and observe their free evolutions during a simulation time of 100 or 200 nanoseconds. In all seven simulations starting from the open structure, AdK remained stable near the initial conformation. The eight simulations initiated from the closed structure, in contrast, exhibited large variation in the subsequent evolutions, with most (seven undergoing large-scale spontaneous conformational changes and approaching or reaching the open state. To characterize the thermodynamics of the transition, we propose and apply a new sampling method that employs a series of restrained simulations to calculate a one-dimensional free energy along a curved pathway in the high-dimensional conformational space. Our calculated free energy profile features a single minimum at the open conformation, and indicates that the closed state, with a high (∼13 kcal/mol free energy, is not metastable, consistent with the observed behaviors of the unrestrained simulations. Collectively, our simulations suggest that it is energetically unfavorable for the ligand-free AdK to access the closed conformation, and imply that ligand binding may precede the closure of the enzyme.

  6. Molecular Physiology of Membrane Guanylyl Cyclase Receptors.

    Science.gov (United States)

    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.

  7. Role of glutaminyl cyclases in thyroid carcinomas.

    Science.gov (United States)

    Kehlen, Astrid; Haegele, Monique; Menge, Katja; Gans, Kathrin; Immel, Uta-Dorothee; Hoang-Vu, Cuong; Klonisch, Thomas; Demuth, Hans-Ulrich

    2013-02-01

    CCL2 is a chemokine known to recruit monocytes/macrophages to sites of inflammation. CCL2 is also associated with tumor progression in several cancer types. Recently, we showed that the N-terminus of CCL2 is modified to a pyroglutamate (pE)-residue by both glutaminyl cyclases (QC (QPCT)) and its isoenzyme (isoQC (QPCTL)). The pE-residue increases stability against N-terminal degradation by aminopeptidases. Here, we report an upregulation of QPCT expression in tissues of patients with thyroid carcinomas compared with goiter tissues, whereas QPCTL was not regulated. In thyroid carcinoma cell lines, QPCT gene expression correlates with the mRNA levels of its substrate CCL2. Both QPCT and CCL2 are regulated in a NF-κB-dependent pathway shown by stimulation with TNFa and IL1b as well as by inhibition with the IKK2 inhibitor and RNAi of p50. In the culture supernatant of thyroid carcinoma cells, equal amounts of pECCL2 and total CCL2 were detected by two ELISAs discriminating between total CCL2 and pECCL2, concluding that all CCL2 is secreted as pECCL2. Activation of the CCL2/CCR2 pathway by recombinant CCL2 increased tumor cell migration of FTC238 cells in scratch assays as well as thyroid carcinoma cell-derived CCL2-induced migration of monocytic THP1 cells. Suppression of CCL2 signaling by CCR2 antagonist, IKK2 inhibitor, and QPCT RNAi reduced FTC238 cell growth measured by WST8 proliferation assays. Our results reveal new evidence for a novel role of QC in thyroid carcinomas and provide an intriguing rationale for the use of QC inhibitors as a means of blocking pECCL2 formation and preventing thyroid cancer metastasis.

  8. Nucleotidyl cyclase activity of particulate guanylyl cyclase A: comparison with particulate guanylyl cyclases E and F, soluble guanylyl cyclase and bacterial adenylyl cyclases CyaA and edema factor.

    Directory of Open Access Journals (Sweden)

    Kerstin Y Beste

    Full Text Available Guanylyl cyclases (GCs regulate many physiological processes by catalyzing the synthesis of the second messenger cGMP. The GC family consists of seven particulate GCs (pGCs and a nitric oxide-activated soluble GC (sGC. Rat sGC α1β1 possesses much broader substrate specificity than previously assumed. Moreover, the exotoxins CyaA from Bordetella pertussis and edema factor (EF from Bacillus anthracis possess nucleotidyl cyclase (NC activity. pGC-A is a natriuretic peptide-activated homodimer with two catalytic sites that act cooperatively. Here, we studied the NC activity of rat pGC-A in membranes of stably transfected HEK293 cells using a highly sensitive and specific HPLC-MS/MS technique. GTP and ITP were effective, and ATP and XTP were only poor, pGC-A substrates. In contrast to sGC, pGC-A did not use CTP and UTP as substrates. pGC-E and pGC-F expressed in bovine rod outer segment membranes used only GTP as substrate. In intact HEK293 cells, pGC-A generated only cGMP. In contrast to pGCs, EF and CyaA showed very broad substrate-specificity. In conclusion, NCs exhibit different substrate-specificities, arguing against substrate-leakiness of enzymes and pointing to distinct physiological functions of cyclic purine and pyrimidine nucleotides.

  9. Computational identification of candidate nucleotide cyclases in higher plants

    KAUST Repository

    Wong, Aloysius Tze

    2013-09-03

    In higher plants guanylyl cyclases (GCs) and adenylyl cyclases (ACs) cannot be identified using BLAST homology searches based on annotated cyclic nucleotide cyclases (CNCs) of prokaryotes, lower eukaryotes, or animals. The reason is that CNCs are often part of complex multifunctional proteins with different domain organizations and biological functions that are not conserved in higher plants. For this reason, we have developed CNC search strategies based on functionally conserved amino acids in the catalytic center of annotated and/or experimentally confirmed CNCs. Here we detail this method which has led to the identification of >25 novel candidate CNCs in Arabidopsis thaliana, several of which have been experimentally confirmed in vitro and in vivo. We foresee that the application of this method can be used to identify many more members of the growing family of CNCs in higher plants. © Springer Science+Business Media New York 2013.

  10. Computational identification of candidate nucleotide cyclases in higher plants.

    Science.gov (United States)

    Wong, Aloysius; Gehring, Chris

    2013-01-01

    In higher plants guanylyl cyclases (GCs) and adenylyl cyclases (ACs) cannot be identified using BLAST homology searches based on annotated cyclic nucleotide cyclases (CNCs) of prokaryotes, lower eukaryotes, or animals. The reason is that CNCs are often part of complex multifunctional proteins with different domain organizations and biological functions that are not conserved in higher plants. For this reason, we have developed CNC search strategies based on functionally conserved amino acids in the catalytic center of annotated and/or experimentally confirmed CNCs. Here we detail this method which has led to the identification of >25 novel candidate CNCs in Arabidopsis thaliana, several of which have been experimentally confirmed in vitro and in vivo. We foresee that the application of this method can be used to identify many more members of the growing family of CNCs in higher plants.

  11. Deducing the origin of soluble adenylyl cyclase, a gene lost in multiple lineages

    NARCIS (Netherlands)

    Roelofs, Jeroen; Haastert, Peter J.M. van

    2002-01-01

    The family of eukaryotic adenylyl cyclases consists of a very large group of 12 transmembrane adenylyl cyclases and a very small group of soluble adenylyl cyclase (sAC). Orthologs of human sAC are present in rat Diclyostelium and bacteria but absent from the completely sequenced genomes of Drosophil

  12. Tyrosine phosphorylation of the human guanylyl cyclase C receptor

    Indian Academy of Sciences (India)

    Rashna Bhandari; Roy Mathew; K Vijayachandra; Sandhya S Visweswariah

    2000-12-01

    Tyrosine phosphorylation events are key components of several cellular signal transduction pathways. This study describes a novel method for identification of substrates for tyrosine kinases. Co-expression of the tyrosine kinase EphB1 with the intracellular domain of guanylyl cyclase C (GCC) in Escherichia coli cells resulted in tyrosine phosphorylation of GCC, indicating that GCC is a potential substrate for tyrosine kinases. Indeed, GCC expressed in mammalian cells is tyrosine phosphorylated, suggesting that tyrosine phosphorylation may play a role in regulation of GCC signalling. This is the first demonstration of tyrosine phosphorylation of any member of the family of membrane-associated guanylyl cyclases.

  13. AKAPs and Adenylyl Cyclase in Cardiovascular Physiology and Pathology

    Science.gov (United States)

    Efendiev, Riad; Dessauer, Carmen W.

    2011-01-01

    Cyclic AMP, generated by adenylyl cyclase (AC), serves as a second messenger in signaling pathways regulating many aspects of cardiac physiology including contraction rate and action potential duration, and in the pathophysiology of hypertrophy and heart failure. A kinase-anchoring proteins (AKAPs) localize the effect of cAMP in space and time by organizing receptors, adenylyl cyclase, protein kinase A and other components of the cAMP cascade into multiprotein complexes. In this review we discuss how interaction of AKAPs with distinct AC isoforms affects cardiovascular physiology. PMID:21978991

  14. Synthetic inhibitors of adenylate kinases in the assays for ATPases and phosphokinases.

    Science.gov (United States)

    Feldhau, P; Fröhlich, T; Goody, R S; Isakov, M; Schirmer, R H

    1975-09-01

    1. Procedures are given for the syntheses of alpha,omega-dinucleoside 5'-polyphosphates as inhibitors of adenylate kinases. The following order for the ability of inhibiting pig muscle adenylate kinase was observed: Ap5A greater than 1:N6-etheno-Ap5A greater than Ap6A greater than Gp5A greater than Ap4A greater than Up5A. The synthesis of adenosine tetraphosphate, the starting material for Ap5A, is also described. 2. One molecule of pig muscle adenylate kinase binds one molecule of Ap5A. The difference spectrum of Ap5A-adenylate kinase with its maximum of 5050 M-1 - cm-1 at 271 nm, as well as the fluorescence properties of 1:N6-etheno-Ap5A can be used for kinetic and binding studies. 3. The specific binding of the negatively charged Ap5A was exploited in the preparation of human muscle adenylate kinase. The enzyme was purified to homogeneity with an overall yield of 65%, the absolute value being 70 mg per kg of muscle. 4. The effect of Ap5A on adenylate kinase in extracts of various cells and cell organelles was tested. A ratio of 1:50 (mol/mol) for Ap5A to other nucleotides was used for suppressing the adenylate kinase activity in extracts of mammalian and insect skeletal muscel, of human erythrocytes and of Staphylococcus aureus. A ratio of 1:5 was found to be necessary for the adenylate kinase from tobacco leaves and spinach chloroplasts, and a ratio of 2:1 was needed for suppressing the adenylate kinase from bovine liver mitochondria, human kidney homogenate and from Escherichia coli. Ap5A appears not to be metabolized in any of the above extracts. These results indicate that Ap5A can be used for evaluating the contribution of adenylate kinase to the production of ATP fro ADP in energy-transducing systems. 5. Contaminating adenylate kinase can be inhibited by a concentration of Ap5A which does not interfere in the study of many (phospho)kinases and ATPases. The applications of Ap5A in the assay for nucleoside diphosphokinase and in the study of mechanical and

  15. Ectopic expression of cyclase associated protein CAP restores the streaming and aggregation defects of adenylyl cyclase a deficient Dictyostelium discoideum cells

    OpenAIRE

    Sultana Hameeda; Neelakanta Girish; Rivero Francisco; Blau-Wasser Rosemarie; Schleicher Michael; Noegel Angelika A

    2012-01-01

    Abstract Background Cell adhesion, an integral part of D. discoideum development, is important for morphogenesis and regulated gene expression in the multicellular context and is required to trigger cell-differentiation. G-protein linked adenylyl cyclase pathways are crucially involved and a mutant lacking the aggregation specific adenylyl cyclase ACA does not undergo multicellular development. Results Here, we have investigated the role of cyclase-associated protein (CAP), an important regul...

  16. [Role of the adenyl cyclase system in achieving the immunogenesis-stimulating action of bacterial lipopolysaccharides--pyrogenal and endogenous serum pyrogen].

    Science.gov (United States)

    Dzheksenbaev, O Sh; Selezneva, V P; Loginova, V T

    1976-05-01

    Experiments were conducted on rabbits immunized intraperitoneally with corpuscular typhoid vaccine; the number of antibody-forming cells in the spleen proved to increase after tha administration of bacterial lipopolysaccharide (LPS)--pyropeneal, and endogenous serum pyrogen (EPS) together with theopylline. The data obtained indicated that the adenylcyclase system played a certain role in the mechanism of the stimulating action of LPS and EPS.

  17. Stimulation of adenylate cyclase in relation to dopamine-induced long-term enhancement (LTE) of muscarinic depolarization in the rabbit superior cervical ganglion.

    Science.gov (United States)

    Mochida, S; Kobayashi, H; Libet, B

    1987-02-01

    Dopamine (DA) induction of the long-term enhancement (LTE) of the slow muscarinic depolarizing response to methacholine (MCh), equivalent to the slow EPSP (S-EPSP), was previously found to be mimicked by exogenous cyclic AMP (cAMP) in the rabbit superior cervical ganglion (SCG). DA-induced LTE of the S-EPSP was shown to be depressed by some DA antagonists. We now show that DA (15 microM), its analog, 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (ADTN), and a D2 receptor antagonist, metoclopramide, each can induce both LTE of MCh depolarization and an increase in ganglionic cAMP. Conversely, antagonists of DA-induced LTE also depress DA-induced rises in cAMP; these antagonists include haloperidol (1 microM), both (+) and (-) enantiomers of butaclamol (0.7-7 microM), flupenthixol (1 microM), and (+)-R-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7-o l (SCH-23390) (7 microM). The selective D2 antagonists sulpiride (10 microM) and domperidone (10 microM) affect neither DA action. Alpha-2 adrenergic agonists (alpha-methyl-norepinephrine and clonidine) produce no LTE; alpha-antagonist dihydroergotamine (35 microM) does not affect either DA action, although it can completely block the hyperpolarizing response to DA or other catecholamines. Beta-antagonist propranolol (5 microM) partially depresses DA-induced rises in cAMP but has no effect on the DA-induced LTE. (Butaclamol and propranolol in combination can completely block the cAMP rise induced by DA.) Beta-agonist isoproterenol can induce appreciable LTE of MCh depolarization, but this LTE is not depressed by propranolol (10 microM). Isoproterenol can elicit a substantial rise in cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. GABAB受体与腺苷酸环化酶偶联环节的脱敏研究%STUDIES ON DESENSITIZATION OF GABAB RECEPTOR COUPLED ADENYLATE CYCLASE

    Institute of Scientific and Technical Information of China (English)

    俞在芳; 程冠军; 胡本荣

    1997-01-01

    将突触体膜与佛波酯(PMA),GABAB受体激动剂巴氯芬(Baclofen,BAL)预孵育一定时间后,BAL对腺苷酸环化酶(AC)基础活性及forskolin刺激的AC活性的抑制率显著降低(脱敏);而forskolin预孵育时,BAL对基础及forskolin刺激的AC活性的抑制率不变,表明GABAB受体与AC偶联环节的脱敏机制涉及蛋白激酶C(PKC)激活,而与蛋白激酶A无关,脱敏时GABAB受体的Kd值增加.本实验提示,可能由于PKC激活导致GABAB受体结构或构象改变,使受体-G蛋白脱偶联而出现脱敏现象.

  19. H3 receptor-mediated inhibition of noradrenaline release: an investigation into the involvement of Ca2+ and K+ ions, G protein and adenylate cyclase.

    Science.gov (United States)

    Schlicker, E; Kathmann, M; Detzner, M; Exner, H J; Göthert, M

    1994-07-01

    The present study was aimed at the identification of mechanisms following the activation of histamine H3 receptors. Mouse brain cortex slices preincubated with 3H-noradrenaline were superfused and the (H3 receptor-mediated) effect of histamine on the electrically evoked tritium overflow was studied under a variety of conditions. The extent of inhibition produced by histamine was inversely related to the frequency of stimulation used to evoke tritium overflow and to the Ca2+ concentration in the superfusion medium. An activator (levcromakalim) and blocker (glibenclamide) of ATP-dependent K+ channels did not affect the electrically evoked tritium overflow and its inhibition by histamine. A blocker of voltage-sensitive K+ channels, tetraethylammonium (TEA), increased the evoked overflow and attenuated the inhibitory effect of histamine. TEA also reduced the inhibitory effect of noradrenaline and prostaglandin E2 on the evoked overflow. When the facilitatory effect of TEA on the evoked overflow was compensated for by reducing the Ca2+ concentration in the superfusion medium, TEA did no longer attenuate the effect of histamine. Exposure of the slices to the SH group-alkylating agent N-ethylmaleimide increased the evoked overflow and attenuated the inhibitory effect of histamine; both effects were counteracted by the SH group-protecting agent dithiothreitol, which, by itself, did not affect the evoked overflow and its inhibition by histamine. Mouse brain cortex membranes were used to study the effect of the H3 receptor agonist R-(-)-alpha-methylhistamine on the basal cAMP accumulation and on the accumulation stimulated by forskolin or noradrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Neuronal localization of pituitary adenylate cyclase-activating polypeptide 38 in the adrenal medulla and growth-inhibitory effect on chromaffin cells

    DEFF Research Database (Denmark)

    Frödin, M; Hannibal, J; Wulff, B S

    1995-01-01

    medulla showed PACAP38 immunoreactivity in a widely distributed network of delicate nerve fibers surrounding the chromaffin cells. In a primary culture system, PACAP38 inhibited growth factor-stimulated DNA synthesis by 90% in neonatal and adult rat chromaffin cells with half-maximal inhibition at 4 and 0...

  1. A subnanomolar concentration of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) pre-synaptically modulates glutamatergic transmission in the rat hippocampus acting through acetylcholine.

    Science.gov (United States)

    Pecoraro, Valeria; Sardone, Lara Maria; Chisari, Mariangela; Licata, Flora; Li Volsi, Guido; Perciavalle, Vincenzo; Ciranna, Lucia; Costa, Lara

    2017-01-06

    The neuropeptide PACAP modulates synaptic transmission in the hippocampus exerting multiple effects through different receptor subtypes: the underlying mechanisms have not yet been completely elucidated. The neurotransmitter acetylcholine (ACh) also exerts a well-documented modulation of hippocampal synaptic transmission and plasticity. Since PACAP was shown to stimulate ACh release in the hippocampus, we tested whether PACAP acting through ACh might indirectly modulate glutamate-mediated synaptic transmission at a pre- and/or at a post-synaptic level. Using patch clamp on rat hippocampal slices, we tested PACAP effects on stimulation-evoked AMPA receptor-mediated excitatory post-synaptic currents (EPSCsAMPA) in the CA3-CA1 synapse and on spontaneous miniature EPSCs (mEPSCs) in CA1 pyramidal neurons. A subnanomolar dose of PACAP (0.5nM) decreased EPSCsAMPA amplitude, enhanced EPSC paired-pulse facilitation (PPF) and reduced mEPSC frequency, indicating a pre-synaptic decrease of glutamate release probability: these effects were abolished by simultaneous blockade of muscarinic and nicotinic ACh receptors, indicating the involvement of endogenous ACh. The effect of subnanomolar PACAP was abolished by a PAC1 receptor antagonist but not by a VPAC receptor blocker. At a higher concentration (10nM), PACAP inhibited EPSCsAMPA: this effect persisted in the presence of ACh receptor antagonists and did not involve any change in PPF or in mEPSC frequency, thus was not mediated by ACh and was exerted post- synaptically on CA1 pyramidal neurons. We suggest that a high-affinity PAC1 receptor pre-synaptically modulates hippocampal glutamatergic transmission acting through ACh. Therefore, administration of PACAP at very low doses might be envisaged in cognitive diseases with reduced cholinergic transmission.

  2. Distribution of vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, nitric oxide synthase, and their receptors in human and rat sphenopalatine ganglion

    DEFF Research Database (Denmark)

    Csati, A; Tajti, J; Kuris, A

    2012-01-01

    Cranial parasympathetic outflow is mediated through the sphenopalatine ganglion (SPG). The present study was performed to examine the expression of the parasympathetic signaling transmitters and their receptors in human and rat SPG. Indirect immunofluorescence technique was used for the demonstra...

  3. A FRET-Based Method for Probing the Conformational Behavior of an Intrinsically Disordered Repeat Domain from Bordetella pertussis Adenylate Cyclase

    Science.gov (United States)

    2009-10-22

    ACKNOWLEDGMENT We thank Carol Li for technical help and Elliot Campbell for help with the stopped-flow fluorescence measurements. Dr. Ladant (Institute...387–401. 21. Ringler, P., and Schulz, G. E. (2003) Self-assembly of proteins into designed networks. Science 302, 106–109. 22. Reiersen, H., and Rees

  4. Soluble guanylate cyclase : a potential therapeutic target for heart failure

    NARCIS (Netherlands)

    Gheorghiade, Mihai; Marti, Catherine N.; Sabbah, Hani N.; Roessig, Lothar; Greene, Stephen J.; Boehm, Michael; Burnett, John C.; Campia, Umberto; Cleland, John G. F.; Collins, Sean P.; Fonarow, Gregg C.; Levy, Phillip D.; Metra, Marco; Pitt, Bertram; Ponikowski, Piotr; Sato, Naoki; Voors, Adriaan A.; Stasch, Johannes-Peter; Butler, Javed

    2013-01-01

    The number of annual hospitalizations for heart failure (HF) and the mortality rates among patients hospitalized for HF remains unacceptably high. The search continues for safe and effective agents that improve outcomes when added to standard therapy. The nitric oxide (NO)-soluble guanylate cyclase

  5. Multilevel control of glucose homeostasis by adenylyl cyclase 8

    NARCIS (Netherlands)

    Raoux, Matthieu; Vacher, Pierre; Papin, Julien; Picard, Alexandre; Kostrzewa, Elzbieta; Devin, Anne; Gaitan, Julien; Limon, Isabelle; Kas, Martien J.; Magnan, Christophe; Lang, Jochen

    2015-01-01

    Aims/hypothesis: Nutrient homeostasis requires integration of signals generated by glucose metabolism and hormones. Expression of the calcium-stimulated adenylyl cyclase ADCY8 is regulated by glucose and the enzyme is capable of integrating signals from multiple pathways. It may thus have an

  6. General base-general acid catalysis by terpenoid cyclases.

    Science.gov (United States)

    Pemberton, Travis A; Christianson, David W

    2016-07-01

    Terpenoid cyclases catalyze the most complex reactions in biology, in that more than half of the substrate carbon atoms often undergo changes in bonding during the course of a multistep cyclization cascade that proceeds through multiple carbocation intermediates. Many cyclization mechanisms require stereospecific deprotonation and reprotonation steps, and most cyclization cascades are terminated by deprotonation to yield an olefin product. The first bacterial terpenoid cyclase to yield a crystal structure was pentalenene synthase from Streptomyces exfoliatus UC5319. This cyclase generates the hydrocarbon precursor of the pentalenolactone family of antibiotics. The structures of pentalenene synthase and other terpenoid cyclases reveal predominantly nonpolar active sites typically lacking amino acid side chains capable of serving general base-general acid functions. What chemical species, then, enables the Brønsted acid-base chemistry required in the catalytic mechanisms of these enzymes? The most likely candidate for such general base-general acid chemistry is the co-product inorganic pyrophosphate. Here, we briefly review biological and nonbiological systems in which phosphate and its derivatives serve general base and general acid functions in catalysis. These examples highlight the fact that the Brønsted acid-base activities of phosphate derivatives are comparable to the Brønsted acid-base activities of amino acid side chains.

  7. Multilevel control of glucose homeostasis by adenylyl cyclase 8

    NARCIS (Netherlands)

    Raoux, Matthieu; Vacher, Pierre; Papin, Julien; Picard, Alexandre; Kostrzewa, Elzbieta; Devin, Anne; Gaitan, Julien; Limon, Isabelle; Kas, Martien J.; Magnan, Christophe; Lang, Jochen

    2015-01-01

    Aims/hypothesis: Nutrient homeostasis requires integration of signals generated by glucose metabolism and hormones. Expression of the calcium-stimulated adenylyl cyclase ADCY8 is regulated by glucose and the enzyme is capable of integrating signals from multiple pathways. It may thus have an importa

  8. The 70-kilodalton adenylyl cyclase-associated protein is not essential for interaction of Saccharomyces cerevisiae adenylyl cyclase with RAS proteins.

    OpenAIRE

    Wang, J; Suzuki, N.; Kataoka, T

    1992-01-01

    In the yeast Saccharomyces cerevisiae, adenylyl cyclase is regulated by RAS proteins. We show here that the yeast adenylyl cyclase forms at least two high-molecular-weight complexes, one with the RAS protein-dependent adenylyl cyclase activity and the other with the Mn(2+)-dependent activity, which are separable by their size difference. The 70-kDa adenylyl cyclase-associated protein (CAP) existed in the former complex but not in the latter. Missense mutations in conserved motifs of the leuci...

  9. Adenylate kinase-independent thiamine triphosphate accumulation under severe energy stress in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Wins Pierre

    2008-01-01

    Full Text Available Abstract Background Thiamine triphosphate (ThTP exists in most organisms and might play a role in cellular stress responses. In E. coli, ThTP is accumulated in response to amino acid starvation but the mechanism of its synthesis is still a matter of controversy. It has been suggested that ThTP is synthesized by an ATP-dependent specific thiamine diphosphate kinase. However, it is also known that vertebrate adenylate kinase 1 catalyzes ThTP synthesis at a very low rate and it has been postulated that this enzyme is responsible for ThTP synthesis in vivo. Results Here we show that bacterial, as vertebrate adenylate kinases are able to catalyze ThTP synthesis, but at a rate more than 106-fold lower than ATP synthesis. This activity is too low to explain the high rate of ThTP accumulation observed in E. coli during amino acid starvation. Moreover, bacteria from the heat-sensitive CV2 strain accumulate high amounts of ThTP (>50% of total thiamine at 37°C despite complete inactivation of adenylate kinase and a subsequent drop in cellular ATP. Conclusion These results clearly demonstrate that adenylate kinase is not responsible for ThTP synthesis in vivo. Furthermore, they show that E. coli accumulate large amounts of ThTP under severe energy stress when ATP levels are very low, an observation not in favor of an ATP-dependent mechanisms for ThTP synthesis.

  10. Skeletal muscle contractile performance and ADP accumulation in adenylate kinase-deficient mice

    NARCIS (Netherlands)

    Hancock, C.R.; Janssen, E.E.W.; Terjung, R.L.

    2005-01-01

    The production of AMP by adenylate kinase (AK) and subsequent deamination by AMP deaminase limits ADP accumulation during conditions of high-energy demand in skeletal muscle. The goal of this study was to investigate the consequences of AK deficiency (-/-) on adenine nucleotide management and whole

  11. Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

    Energy Technology Data Exchange (ETDEWEB)

    Z Zhang; R Zhou; J Sauder; P Tonge; S Burley; S Swaminathan

    2011-12-31

    Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.

  12. Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Z.; Swaminathan, S.; Zhou, R.; Sauder, J. M.; Tonge, P. J.; Burley, S. K.

    2011-02-18

    Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.

  13. Coiled-coil interaction of N-terminal 36 residues of cyclase-associated protein with adenylyl cyclase is sufficient for its function in Saccharomyces cerevisiae ras pathway.

    Science.gov (United States)

    Nishida, Y; Shima, F; Sen, H; Tanaka, Y; Yanagihara, C; Yamawaki-Kataoka, Y; Kariya, K; Kataoka, T

    1998-10-23

    In the budding yeast Saccharomyces cerevisiae, association with the 70-kDa cyclase-associated protein (CAP) is required for proper response of adenylyl cyclase to Ras proteins. We show here that a small segment comprising the N-terminal 36 amino acid residues of CAP is sufficient for association with adenylyl cyclase as well as for its function in the Ras-adenylyl cyclase pathway as assayed by the ability to confer RAS2(Val-19)-dependent heat shock sensitivity to yeast cells. The CAP-binding site of adenylyl cyclase was mapped to a segment of 119 amino acid residues near its C terminus. Both of these regions contained tandem repetitions of a heptad motif alphaXXalphaXXX (where alpha represents a hydrophobic amino acid and X represents any amino acid), suggesting a coiled-coil interaction. When mutants of CAP defective in associating with adenylyl cyclase were isolated by screening of a pool of randomly mutagenized CAP, they were found to carry substitution mutations in one of the key hydrophobic residues in the heptad repeats. Furthermore, mutations of the key hydrophobic residues in the heptad repeats of adenylyl cyclase also resulted in loss of association with CAP. These results indicate the coiled-coil mechanism as a basis of the CAP-adenylyl cyclase interaction.

  14. The 70-kilodalton adenylyl cyclase-associated protein is not essential for interaction of Saccharomyces cerevisiae adenylyl cyclase with RAS proteins.

    Science.gov (United States)

    Wang, J; Suzuki, N; Kataoka, T

    1992-11-01

    In the yeast Saccharomyces cerevisiae, adenylyl cyclase is regulated by RAS proteins. We show here that the yeast adenylyl cyclase forms at least two high-molecular-weight complexes, one with the RAS protein-dependent adenylyl cyclase activity and the other with the Mn(2+)-dependent activity, which are separable by their size difference. The 70-kDa adenylyl cyclase-associated protein (CAP) existed in the former complex but not in the latter. Missense mutations in conserved motifs of the leucine-rich repeats of the catalytic subunit of adenylyl cyclase abolished the RAS-dependent activity, which was accompanied by formation of a very high molecular weight complex having the Mn(2+)-dependent activity. Contrary to previous results, disruption of the gene encoding CAP did not alter the extent of RAS protein-dependent activation of adenylyl cyclase, while a concomitant decrease in the size of the RAS-responsive complex was observed. These results indicate that CAP is not essential for interaction of the yeast adenylyl cyclase with RAS proteins even though it is an inherent component of the RAS-responsive adenylyl cyclase complex.

  15. Intracellular cAMP signaling by soluble adenylyl cyclase.

    Science.gov (United States)

    Tresguerres, Martin; Levin, Lonny R; Buck, Jochen

    2011-06-01

    Soluble adenylyl cyclase (sAC) is a recently identified source of the ubiquitous second messenger cyclic adenosine 3',5' monophosphate (cAMP). sAC is distinct from the more widely studied source of cAMP, the transmembrane adenylyl cyclases (tmACs); its activity is uniquely regulated by bicarbonate anions, and it is distributed throughout the cytoplasm and in cellular organelles. Due to its unique localization and regulation, sAC has various functions in a variety of physiological systems that are distinct from tmACs. In this review, we detail the known functions of sAC, and we reassess commonly held views of cAMP signaling inside cells.

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

    Science.gov (United States)

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

    2015-05-29

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

  17. NCBI nr-aa BLAST: CBRC-ACAR-01-0303 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ACAR-01-0303 ref|NP_066939.1| brain adenylate cyclase 1 [Homo sapiens] sp|Q088...28|ADCY1_HUMAN Adenylate cyclase type 1 (Adenylate cyclase type I) (ATP pyrophosphate-lyase 1) (Adenylyl cyclase... 1) (Ca(2+)/calmodulin-activated adenylyl cyclase) gb|EAL23741.1| adenylate cyclase 1 (brain) [Homo sapiens] NP_066939.1 6e-54 57% ...

  18. Overexpression of functional human oxidosqualene cyclase in Escherichia coli

    DEFF Research Database (Denmark)

    Kürten, Charlotte; Uhlén, Mathias; Syrén, Per-Olof

    2015-01-01

    The generation of multicyclic scaffolds from linear oxidosqualene by enzymatic polycyclization catalysis constitutes a cornerstone in biology for the generation of bioactive compounds. Human oxidosqualene cyclase (hOSC) is a membrane-bound triterpene cyclase that catalyzes the formation of the te......The generation of multicyclic scaffolds from linear oxidosqualene by enzymatic polycyclization catalysis constitutes a cornerstone in biology for the generation of bioactive compounds. Human oxidosqualene cyclase (hOSC) is a membrane-bound triterpene cyclase that catalyzes the formation...

  19. Assessment of industrial sewage impacts by adenylate energy charge measurements in the bivalve Cerastoderma edule.

    Science.gov (United States)

    Picado, A M; Le Gal, Y

    1990-02-01

    Laboratory toxicity tests performed on the bivalve Cerastoderma edule submitted to sublethal concentrations of paper mill effluent revealed significant decreases of adenylate energy charge (AEC), and changes in the total adenylate pool were observed in a 24-hr period even for the lowest concentration of pollutant tested. Field transfer experiments of C. edule from a safe zone to polluted areas of the Sado estuary were carried out at two different times of the year. Close proximity to sewage outfall was shown to result in significant decreases in AEC values within 24 hr. One week after transfer, either normal AEC values were found or the organisms died, depending on the location of the sampling station.

  20. Analysis of the function of the 70-kilodalton cyclase-associated protein (CAP) by using mutants of yeast adenylyl cyclase defective in CAP binding.

    Science.gov (United States)

    Wang, J; Suzuki, N; Nishida, Y; Kataoka, T

    1993-07-01

    In Saccharomyces cerevisiae, adenylyl cyclase forms a complex with the 70-kDa cyclase-associated protein (CAP). By in vitro mutagenesis, we assigned a CAP-binding site of adenylyl cyclase to a small segment near its C terminus and created mutants which lost the ability to bind CAP. CAP binding was assessed first by observing the ability of the overproduced C-terminal 150 residues of adenylyl cyclase to sequester CAP, thereby suppressing the heat shock sensitivity of yeast cells bearing the activated RAS2 gene (RAS2Val-19), and then by immunoprecipitability of adenylyl cyclase activity with anti-CAP antibody and by direct measurement of the amount of CAP bound. Yeast cells whose chromosomal adenylyl cyclase genes were replaced by the CAP-nonbinding mutants possessed adenylyl cyclase activity fully responsive to RAS2 protein in vitro. However, they did not exhibit sensitivity to heat shock in the RAS2Val-19 background. When glucose-induced accumulation of cyclic AMP (cAMP) was measured in these mutants carrying RAS2Val-19, a rapid transient rise indistinguishable from that of wild-type cells was observed and a high peak level and following persistent elevation of the cAMP concentration characteristic of RAS2Val-19 were abolished. In contrast, in the wild-type RAS2 background, similar cyclase gene replacement did not affect the glucose-induced cAMP response. These results suggest that the association with CAP, although not involved in the in vivo response to the wild-type RAS2 protein, is somehow required for the exaggerated response of adenylyl cyclase to activated RAS2.

  1. Dysregulation of Alternative Poly-adenylation as a Potential Player in Autism Spectrum Disorder

    Science.gov (United States)

    Szkop, Krzysztof J.; Cooke, Peter I. C.; Humphries, Joanne A.; Kalna, Viktoria; Moss, David S.; Schuster, Eugene F.; Nobeli, Irene

    2017-01-01

    We present here the hypothesis that alternative poly-adenylation (APA) is dysregulated in the brains of individuals affected by Autism Spectrum Disorder (ASD), due to disruptions in the calcium signaling networks. APA, the process of selecting different poly-adenylation sites on the same gene, yielding transcripts with different-length 3′ untranslated regions (UTRs), has been documented in different tissues, stages of development and pathologic conditions. Differential use of poly-adenylation sites has been shown to regulate the function, stability, localization and translation efficiency of target RNAs. However, the role of APA remains rather unexplored in neurodevelopmental conditions. In the human brain, where transcripts have the longest 3′ UTRs and are thus likely to be under more complex post-transcriptional regulation, erratic APA could be particularly detrimental. In the context of ASD, a condition that affects individuals in markedly different ways and whose symptoms exhibit a spectrum of severity, APA dysregulation could be amplified or dampened depending on the individual and the extent of the effect on specific genes would likely vary with genetic and environmental factors. If this hypothesis is correct, dysregulated APA events might be responsible for certain aspects of the phenotypes associated with ASD. Evidence supporting our hypothesis is derived from standard RNA-seq transcriptomic data but we suggest that future experiments should focus on techniques that probe the actual poly-adenylation site (3′ sequencing). To address issues arising from the use of post-mortem tissue and low numbers of heterogeneous samples affected by confounding factors (such as the age, gender and health of the individuals), carefully controlled in vitro systems will be required to model the effect of calcium signaling dysregulation in the ASD brain. PMID:28955198

  2. NMR studies of the AMP-binding site and mechanism of adenylate kinase

    Energy Technology Data Exchange (ETDEWEB)

    Fry, D.C.; Kuby, S.A.; Mildvan, A.S.

    1987-03-24

    NMR has previously been used to determine the conformation of enzyme-bound MgATP and to locate the MgATP-binding site on adenylate kinase. To determine the conformation and location of the other substrate, AMP, distances have been measured from Cr/sup 3 +/AMPPCP, a linear competitive inhibitor with respect to MgATP, to six protons and to the phosphorus atom of AMP on adenylate kinase, with the paramagnetic probe-T/sub 1/ method. Time-dependent nuclear Overhauser effects (NOEs) have been used to measure five interproton distances on enzyme-bound AMP. These distances were used to determine the conformation of bound AMP in addition to its position with respect to metal-ATP. Ten intermolecular NOEs, from protons of the enzyme to those of AMP, were detected, indicating the proximity of at least three hydrophobic amino acids to bound AMP. These constraints, together with the conformation of AMP and the intersubstrate distances, were used to position AMP into the X-ray structure of adenylate kinase. The AMP binding site is found to be near Leu-116, Arg-171, Val-173, Val-182, and Leu-190; all of these residues have been found to be invariant in muscle-type rabbit, calf, human, porcine.

  3. Multiforms of mammalian adenylate kinase and its monoclonal antibody against AK1.

    Science.gov (United States)

    Kurokawa, Y; Takenaka, H; Sumida, M; Oka, K; Hamada, M; Kuby, S A

    1990-01-01

    An attempt has been made to determine the intracellular distribution of the multiforms of the adenylate kinase (AK) isoenzymes in mammalian tissues, to shed some light on their physiological roles, especially in energy metabolism. The adenylate kinase zymograms obtained from isoelectric focusing yielded two typical isoform patterns: (1) with a pI greater than or equal to 9 and 8.6, specific for bovine skeletal muscle, heart, aorta and brain, and (2) with a pI = 7.9 and 7.1, specific for liver and kidney. Pattern (1) was attributed to the cytosolic isoenzyme (AK1) as demonstrated by immunostaining with anti-AK1. Pattern (2) was attributed to the mitochondrial isoenzyme (AK2). These results were largely confirmed by chromatofocusing experiments. The AK1 isoenzyme was partially purified from the cytosol fraction of bovine aortic smooth muscle and had an apparent Mr of 23.5 kilodaltons. Its kinetic features are discussed from a comparative standpoint. Finally, the human serum AK1 isoform was also detected by Western blotting with a monoclonal antibody directed against crystalline porcine muscle AK1. These results are to form the basis of further studies on the 'aberrant' adenylate kinase isoenzyme from the serum of Duchenne muscular dystrophics.

  4. Adenylate kinase from Streptococcus pneumoniae is essential for growth through its catalytic activity

    Directory of Open Access Journals (Sweden)

    Trung Thanh Thach

    2014-01-01

    Full Text Available Streptococcus pneumoniae (pneumococcus infection causes more than 1.6 million deaths worldwide. Pneumococcal growth is a prerequisite for its virulence and requires an appropriate supply of cellular energy. Adenylate kinases constitute a major family of enzymes that regulate cellular ATP levels. Some bacterial adenylate kinases (AdKs are known to be critical for growth, but the physiological effects of AdKs in pneumococci have been poorly understood at the molecular level. Here, by crystallographic and functional studies, we report that the catalytic activity of adenylate kinase from S. pneumoniae (SpAdK serotype 2 D39 is essential for growth. We determined the crystal structure of SpAdK in two conformations: ligand-free open form and closed in complex with a two-substrate mimic inhibitor adenosine pentaphosphate (Ap5A. Crystallographic analysis of SpAdK reveals Arg-89 as a key active site residue. We generated a conditional expression mutant of pneumococcus in which the expression of the adk gene is tightly regulated by fucose. The expression level of adk correlates with growth rate. Expression of the wild-type adk gene in fucose-inducible strains rescued a growth defect, but expression of the Arg-89 mutation did not. SpAdK increased total cellular ATP levels. Furthermore, lack of functional SpAdK caused a growth defect in vivo. Taken together, our results demonstrate that SpAdK is essential for pneumococcal growth in vitro and in vivo.

  5. The intact CFTR protein mediates ATPase rather than adenylate kinase activity.

    Science.gov (United States)

    Ramjeesingh, Mohabir; Ugwu, Francisca; Stratford, Fiona L L; Huan, Ling-Jun; Li, Canhui; Bear, Christine E

    2008-06-01

    The two NBDs (nucleotide-binding domains) of ABC (ATP-binding-cassette) proteins function in a complex to mediate ATPase activity and this activity has been linked to their regulated transport activity. A similar model has been proposed for CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel defective in cystic fibrosis, wherein ATP binding and hydrolysis regulate the channel gate. Recently, it was shown that the individual NBDs isolated from CFTR primarily mediate adenylate kinase activity, raising the possibility that this activity may also contribute to gating of the CFTR channel. However, this present study shows that whereas the isolated NBDs exhibit adenylate kinase activity, the full-length purified and reconstituted CFTR protein functions as an ATPase, arguing that the enzymatic activity of the NBDs is dependent on their molecular context and appropriate domain-domain assembly. As expected, the disease-causing mutant bearing a mutation in the ABC signature motif, CFTR-G551D, exhibited a markedly reduced ATPase activity. Furthermore, mutation of the putative catalytic base in CFTR caused a reduction in ATPase activity, with the CFTR-E1371Q mutant supporting a low level of residual activity. Neither of these mutants exhibited detectable adenylate kinase activity. Together, these findings support the concept that the molecular mechanism of action of CFTR is dependent on ATP binding and hydrolysis, and that the structure of prokaryotic ABC ATPases provide a useful template for understanding their mechanism of action.

  6. A non-canonical peptide synthetase adenylates 3-methyl-2-oxovaleric acid for auriculamide biosynthesis

    Directory of Open Access Journals (Sweden)

    Daniel Braga

    2016-12-01

    Full Text Available Auriculamide is the first natural product known from the predatory bacterium Herpetosiphon aurantiacus. It is composed of three unusual building blocks, including the non-proteinogenic amino acid 3-chloro-L-tyrosine, the α-hydroxy acid L-isoleucic acid, and a methylmalonyl-CoA-derived ethane unit. A candidate genetic locus for auriculamide biosynthesis was identified and encodes four enzymes. Among them, the non-canonical 199 kDa four-domain nonribosomal peptide synthetase, AulA, is extraordinary in that it features two consecutive adenylation domains. Here, we describe the functional characterization of the recombinantly produced AulA. The observed activation of 3-methyl-2-oxovaleric acid by the enzyme supports the hypothesis that it participates in the biosynthesis of auriculamide. An artificially truncated version of AulA that lacks the first adenylation domain activated this substrate like the full-length enzyme which shows that the first adenylation domain is dispensable. Additionally, we provide evidence that the enzyme tolerates structural variation of the substrate. α-Carbon substituents significantly affected the substrate turnover. While all tested aliphatic α-keto acids were accepted by the enzyme and minor differences in chain size and branches did not interfere with the enzymatic activity, molecules with methylene α-carbons led to low turnover. Such enzymatic plasticity is an important attribute to help in the perpetual search for novel molecules and to access a greater structural diversity by mutasynthesis.

  7. Discovery of LRE1 as a specific and allosteric inhibitor of soluble adenylyl cyclase.

    Science.gov (United States)

    Ramos-Espiritu, Lavoisier; Kleinboelting, Silke; Navarrete, Felipe A; Alvau, Antonio; Visconti, Pablo E; Valsecchi, Federica; Starkov, Anatoly; Manfredi, Giovanni; Buck, Hannes; Adura, Carolina; Zippin, Jonathan H; van den Heuvel, Joop; Glickman, J Fraser; Steegborn, Clemens; Levin, Lonny R; Buck, Jochen

    2016-10-01

    The prototypical second messenger cAMP regulates a wide variety of physiological processes. It can simultaneously mediate diverse functions by acting locally in independently regulated microdomains. In mammalian cells, two types of adenylyl cyclase generate cAMP: G-protein-regulated transmembrane adenylyl cyclases and bicarbonate-, calcium- and ATP-regulated soluble adenylyl cyclase (sAC). Because each type of cyclase regulates distinct microdomains, methods to distinguish between them are needed to understand cAMP signaling. We developed a mass-spectrometry-based adenylyl cyclase assay, which we used to identify a new sAC-specific inhibitor, LRE1. LRE1 bound to the bicarbonate activator binding site and inhibited sAC via a unique allosteric mechanism. LRE1 prevented sAC-dependent processes in cellular and physiological systems, and it will facilitate exploration of the therapeutic potential of sAC inhibition.

  8. Expression of the Intestinal Biomarkers Guanylyl Cyclase C and CDX2 in Poorly Differentiated Colorectal Carcinomas

    Science.gov (United States)

    Winn, Brody; Tavares, Rosemarie; Matoso, Andres; Noble, Lelia; Fanion, Jacqueline; Waldman, Scott A.; Resnick, Murray B.

    2009-01-01

    Guanylyl cyclase C a receptor for bacterial diarrheagenic enterotoxins is expressed selectively by intestinal epithelium and is an endogenous downstream target of CDX2. The expression of Guanylyl cyclase C is preserved throughout the adenoma/carcinoma sequence in the colorectum. Detection of Guanylyl cyclase C expression by RT-PCR is currently being validated as a technique to identify occult lymph node metastases in patients with colorectal cancer and for circulating cells in the blood for postoperative surveillance. Although Guanylyl cyclase C is widely expressed by well differentiated colorectal cancer, its expression in poorly differentiated colorectal cancer has not been evaluated. A tissue microarray was created from 69 archival specimens including 44 poorly differentiated, 15 undifferentiated or medullary and 10 signet ring cell colorectal carcinomas. Matched normal colonic mucosa was used as a positive control. Immunohistochemical staining for Guanylyl cyclase C and CDX2 was evaluated as positive or negative based on at least a 10% extent of staining. Out of the 69 tumor samples 75%, 47%, and 90% of the poorly differentiated, medullary and signet ring cell tumors were positive for Guanylyl cyclase C and 75%, 40% and 90% of these subsets were positive for CDX2 respectively. There was excellent correlation between Guanylyl cyclase C and CDX2 expression on a case per case basis (p<0.0001). There was also a statistically significant difference in the GCC staining pattern between MC and PDC (p=0.05). Immunopositivity for Guanylyl cyclase C was greater than 95% in a separately stained microarray series of well/moderately differentiated colorectal carcinomas. In conclusion, Guanylyl cyclase C expression is lost in a quarter of poorly differentiated and half of undifferentiated colorectal carcinomas. Therefore the utility of Guanylyl cyclase C expression as a diagnostic marker for colorectal carcinoma may be questionable in poorly differentiated colorectal

  9. Inhibition of a plant sesquiterpene cyclase by mevinolin.

    Science.gov (United States)

    Vögeli, U; Chappell, J

    1991-07-01

    The specificity of mevinolin as an inhibitor of sterol and sesquiterpene metabolism in tobacco cell suspension cultures was examined. Exogenous mevinolin inhibited [14C]acetate, but not [3H]mevalonate incorporation into free sterols. In contrast, mevinolin inhibited the incorporation of both [14C]acetate and [3H]mevalonate into capsidiol, an extracellular sesquiterpene. Microsomal 3-hydroxy-3-methylglutaryl Coenzyme A reductase was inhibited greater than 90% by microM mevinolin, while squalene synthetase was insensitive to even 600 microM mevinolin. Sesquiterpene cyclase, the first branch point enzyme specific for sesquiterpene biosynthesis, was inhibited in a dose-dependent manner by mevinolin with a 50% reduction in activity at 100 microM. Kinetic analysis indicated that the mechanism for inhibition was complex with mevinolin acting as both a competitive and noncompetitive inhibitor. The results suggest that the mevinolin inhibition of [3H]mevalonate incorporation into extracellular sesquiterpenes can, in part, be attributed to a secondary, but specific, site of inhibition, the sesquiterpene cyclase.

  10. NCBI nr-aa BLAST: CBRC-ACAR-01-1123 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ACAR-01-1123 ref|NP_899200.1| adenylate cyclase 5 [Homo sapiens] sp|O95622|ADCY5_HUMAN Adenylate cyclas...e type 5 (Adenylate cyclase type V) (ATP pyrophosphate-lyase 5) (Adenylyl cyclase 5) NP_899200.1 2e-91 52% ...

  11. NMR studies of the AMP-binding site and mechanism of adenylate kinase.

    Science.gov (United States)

    Fry, D C; Kuby, S A; Mildvan, A S

    1987-03-24

    NMR has previously been used to determine the conformation of enzyme-bound MgATP and to locate the MgATP-binding site on adenylate kinase [Fry, D. C., Kuby, S. A., & Mildvan, A. S. (1985) Biochemistry 24, 4680-4694]. To determine the conformation and location of the other substrate, AMP, distances have been measured from Cr3+AMPPCP, a linear competitive inhibitor with respect to MgATP, to six protons and to the phosphorus atom of AMP on adenylate kinase, with the paramagnetic probe-T1 method. Time-dependent nuclear Overhauser effects (NOEs) have been used to measure five interproton distances on enzyme-bound AMP. These distances were used to determine the conformation of bound AMP in addition to its position with respect to metal-ATP. Enzyme-bound AMP exhibits a high anti-glycosyl torsional angle (chi = 110 +/- 10 degrees), a 3'-endo,2'-exo ribose pucker (delta = 105 +/- 10 degrees), and gauche-trans orientations about the C4'-C5' bond (gamma = 180 +/- 10 degrees) and the C5'-O5' bond (beta = 170 +/- 20 degrees). The distance from Cr3+ to the phosphorus of AMP is 5.9 +/- 0.3 A, indicating a reaction coordinate distance of approximately 3 A, which is consistent with an associative SN2 mechanism for the phosphoryl transfer. Ten intermolecular NOEs, from protons of the enzyme to those of AMP, were detected, indicating the proximity of at least three hydrophobic amino acids to bound AMP. These constraints, together with the conformation of AMP and the intersubstrate distances, were used to position AMP into the X-ray structure of adenylate kinase. The AMP binding site is found to be near (less than or equal to 4 A from) Leu-116, Arg-171, Val-173, Val-182, and Leu-190; all of these residues have been found to be invariant in muscle-type rabbit, calf, human, porcine [Kuby, S. A., Palmieri, R. H., Frischat, A., Fischer, A. H., Wu, L. H., Maland, L., & Manship, M. (1984) Biochemistry 23, 2393-2399], and chicken adenylate kinase [Kishi, F., Maruyama, M., Tanizawa, Y

  12. Ibogaine and noribogaine potentiate the inhibition of adenylyl cyclase activity by opioid and 5-HT receptors.

    Science.gov (United States)

    Rabin, R A; Winter, J C

    1996-12-05

    The effects of the putative anti-addictive compound ibogaine and its principal metabolite, noribogaine, on adenylyl cyclase activity were determined in various areas of the rat brain. Neither compound altered either basal or forskolin-stimulated adenylyl cyclase activities in the frontal cortex, midbrain or striatum. However, in all three brain areas the addition of ibogaine and noribogaine significantly enhanced inhibition of adenylyl cyclase activity by a maximally effective concentration of morphine. Similarly, both compounds also potentiated the inhibition of hippocampal adenylyl cyclase activity by a maximally effective concentration of 5-hydroxytryptamine (5-HT). Although ibogaine appears to be more potent than noribogaine in augmenting opioid- and 5-HT-mediated inhibition of adenylyl cyclase activity, both compounds appear to be of comparable efficacy. Neither compound, however, modified the inhibitory action of the muscarinic acetylcholine agonist, carbachol, on adenylyl cyclase activity. The present data indicate that ibogaine and noribogaine cause a selective increase in receptor-mediated inhibition of adenylyl cyclase activity. This potentiation may be involved in the pharmacological actions of these compounds.

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

    Science.gov (United States)

    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.

  14. Identification of a fourth family of lycopene cyclases in photosynthetic bacteria.

    Science.gov (United States)

    Maresca, Julia A; Graham, Joel E; Wu, Martin; Eisen, Jonathan A; Bryant, Donald A

    2007-07-10

    A fourth and large family of lycopene cyclases was identified in photosynthetic prokaryotes. The first member of this family, encoded by the cruA gene of the green sulfur bacterium Chlorobium tepidum, was identified in a complementation assay with a lycopene-producing strain of Escherichia coli. Orthologs of cruA are found in all available green sulfur bacterial genomes and in all cyanobacterial genomes that lack genes encoding CrtL- or CrtY-type lycopene cyclases. The cyanobacterium Synechococcus sp. PCC 7002 has two homologs of CruA, denoted CruA and CruP, and both were shown to have lycopene cyclase activity. Although all characterized lycopene cyclases in plants are CrtL-type proteins, genes orthologous to cruP also occur in plant genomes. The CruA- and CruP-type carotenoid cyclases are members of the FixC dehydrogenase superfamily and are distantly related to CrtL- and CrtY-type lycopene cyclases. Identification of these cyclases fills a major gap in the carotenoid biosynthetic pathways of green sulfur bacteria and cyanobacteria.

  15. The cyclase-associated protein FgCap1 has both protein kinase A-dependent and -independent functions during deoxynivalenol production and plant infection in Fusarium graminearum.

    Science.gov (United States)

    Yin, Tao; Zhang, Qiang; Wang, Jianhua; Liu, Huiquan; Wang, Chenfang; Xu, Jin-Rong; Jiang, Cong

    2017-01-31

    Fusarium graminearum is a causal agent of wheat scab and a producer of the trichothecene mycotoxin deoxynivalenol (DON). The expression of trichothecene biosynthesis (TRI) genes and DON production are mainly regulated by the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway and two pathway-specific transcription factors (TRI6 and TRI10). Interestingly, deletion mutants of TRI6 show reduced expression of several components of cAMP signalling, including the FgCAP1 adenylate-binding protein gene that has not been functionally characterized in F. graminearum. In this study, we show that FgCap1 interacts with Fac1 adenylate cyclase and that deletion of FgCAP1 reduces the intracellular cAMP level and PKA activity. The Fgcap1 deletion mutant is defective in vegetative growth, conidiogenesis and plant infection. It also shows significantly reduced DON production and TRI gene expression, which can be suppressed by exogenous cAMP, indicating a PKA-dependent regulation of DON biosynthesis by FgCap1. The wild-type, but not tri6 mutant, shows increased levels of intracellular cAMP and FgCAP1 expression under DON-producing conditions. Furthermore, the promoter of FgCAP1 contains one putative Tri6-binding site that is important for its function during DON biosynthesis, but is dispensable for hyphal growth, conidiogenesis and pathogenesis. In addition, FgCap1 shows an actin-like localization to the cortical patches at the apical region of hyphal tips. Phosphorylation of FgCap1 at S353 was identified by phosphoproteomics analysis. The S353A mutation in FgCAP1 has no effect on its functions during vegetative growth, conidiation and DON production. However, expression of the FgCAP1(S353A) allele fails to complement the defects of the Fgcap1 mutant in plant infection, indicating the importance of the phosphorylation of FgCap1 at S353 during pathogenesis. Taken together, our results suggest that FgCAP1 is involved in the regulation of DON production via cAMP signalling

  16. Inferring biological functions of guanylyl cyclases with computational methods

    KAUST Repository

    Alquraishi, May Majed

    2013-09-03

    A number of studies have shown that functionally related genes are often co-expressed and that computational based co-expression analysis can be used to accurately identify functional relationships between genes and by inference, their encoded proteins. Here we describe how a computational based co-expression analysis can be used to link the function of a specific gene of interest to a defined cellular response. Using a worked example we demonstrate how this methodology is used to link the function of the Arabidopsis Wall-Associated Kinase-Like 10 gene, which encodes a functional guanylyl cyclase, to host responses to pathogens. © Springer Science+Business Media New York 2013.

  17. Diadenosine Homodinucleotide Products of ADP-ribosyl Cyclases Behave as Modulators of the Purinergic Receptor P2X7*

    Science.gov (United States)

    Bruzzone, Santina; Basile, Giovanna; Chothi, Madhu Parakkottil; Nobbio, Lucilla; Usai, Cesare; Jacchetti, Emanuela; Schenone, Angelo; Guse, Andreas H.; Di Virgilio, Francesco; De Flora, Antonio; Zocchi, Elena

    2010-01-01

    ADP-ribosyl cyclases from both vertebrates and invertebrates were previously shown to produce two isomers of P1,P2 diadenosine 5′,5′"-P1, P2-diphosphate, P18 and P24, from cyclic ADP-ribose (cADPR) and adenine. P18 and P24 are characterized by an unusual N-glycosidic linkage in one of the adenylic mononucleotides (Basile, G., Taglialatela-Scafati, O., Damonte, G., Armirotti, A., Bruzzone, S., Guida, L., Franco, L., Usai, C., Fattorusso, E., De Flora, A., and Zocchi, E. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 14509–14514). P24, but not P18, proved to increase the intracellular Ca2+ concentration ([Ca2+]i) in HeLa cells and to negatively affect mitochondrial function. Here we show that micromolar P24, but not P18, triggers a slow and sustained influx of extracellular Ca2+ through the opening of the purinergic receptor/channel P2X7. On the other hand, P18 inhibits the Ca2+ influx induced by 0.6 mm ATP in HEK293 cells stably transfected with P2X7, with an IC50 of ∼1 μm. Thus, P18 is devoid of intrinsic P2X7 stimulatory activity and behaves as an ATP antagonist. A P2X7-mediated increase of the basal [Ca2+]i has been demonstrated to negatively affect Schwann cell (SC) function in rats with the inherited, peripheral neuropathy Charcot-Marie-Tooth 1A (CMT1A) (Nobbio, L., Sturla, L., Fiorese, F., Usai, C., Basile, G., Moreschi, I., Benvenuto, F., Zocchi, E., De Flora, A., Schenone, A., and Bruzzone S. (2009) J. Biol. Chem. 284, 23146–23158). Preincubation of CMT1A SC with 200 nm P18 restored the basal [Ca2+]i to values similar to those recorded in wild-type SC. These results identify P18 as a new P2X7 antagonist, potentially useful in the treatment of CMT1A. PMID:20439466

  18. Diadenosine homodinucleotide products of ADP-ribosyl cyclases behave as modulators of the purinergic receptor P2X7.

    Science.gov (United States)

    Bruzzone, Santina; Basile, Giovanna; Chothi, Madhu Parakkottil; Nobbio, Lucilla; Usai, Cesare; Jacchetti, Emanuela; Schenone, Angelo; Guse, Andreas H; Di Virgilio, Francesco; De Flora, Antonio; Zocchi, Elena

    2010-07-02

    ADP-ribosyl cyclases from both vertebrates and invertebrates were previously shown to produce two isomers of P1,P2 diadenosine 5',5'"-P1, P2-diphosphate, P18 and P24, from cyclic ADP-ribose (cADPR) and adenine. P18 and P24 are characterized by an unusual N-glycosidic linkage in one of the adenylic mononucleotides (Basile, G., Taglialatela-Scafati, O., Damonte, G., Armirotti, A., Bruzzone, S., Guida, L., Franco, L., Usai, C., Fattorusso, E., De Flora, A., and Zocchi, E. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 14509-14514). P24, but not P18, proved to increase the intracellular Ca(2+) concentration ([Ca(2+)](i)) in HeLa cells and to negatively affect mitochondrial function. Here we show that micromolar P24, but not P18, triggers a slow and sustained influx of extracellular Ca(2+) through the opening of the purinergic receptor/channel P2X7. On the other hand, P18 inhibits the Ca(2+) influx induced by 0.6 mm ATP in HEK293 cells stably transfected with P2X7, with an IC(50) of approximately 1 mum. Thus, P18 is devoid of intrinsic P2X7 stimulatory activity and behaves as an ATP antagonist. A P2X7-mediated increase of the basal [Ca(2+)](i) has been demonstrated to negatively affect Schwann cell (SC) function in rats with the inherited, peripheral neuropathy Charcot-Marie-Tooth 1A (CMT1A) (Nobbio, L., Sturla, L., Fiorese, F., Usai, C., Basile, G., Moreschi, I., Benvenuto, F., Zocchi, E., De Flora, A., Schenone, A., and Bruzzone S. (2009) J. Biol. Chem. 284, 23146-23158). Preincubation of CMT1A SC with 200 nm P18 restored the basal [Ca(2+)](i) to values similar to those recorded in wild-type SC. These results identify P18 as a new P2X7 antagonist, potentially useful in the treatment of CMT1A.

  19. Design, synthesis, and biological evaluation of α-hydroxyacyl-AMS inhibitors of amino acid adenylation enzymes.

    Science.gov (United States)

    Davis, Tony D; Mohandas, Poornima; Chiriac, Maria I; Bythrow, Glennon V; Quadri, Luis E N; Tan, Derek S

    2016-11-01

    Biosynthesis of bacterial natural-product virulence factors is emerging as a promising antibiotic target. Many such natural products are produced by nonribosomal peptide synthetases (NRPS) from amino acid precursors. To develop selective inhibitors of these pathways, we have previously described aminoacyl-AMS (sulfamoyladenosine) macrocycles that inhibit NRPS amino acid adenylation domains but not mechanistically-related aminoacyl-tRNA synthetases. To improve the cell permeability of these inhibitors, we explore herein replacement of the α-amino group with an α-hydroxy group. In both macrocycles and corresponding linear congeners, this leads to decreased biochemical inhibition of the cysteine adenylation domain of the Yersina pestis siderophore synthetase HMWP2, which we attribute to loss of an electrostatic interaction with a conserved active-site aspartate. However, inhibitory activity can be regained by installing a cognate β-thiol moiety in the linear series. This provides a path forward to develop selective, cell-penetrant inhibitors of the biosynthesis of virulence factors to probe their biological functions and potential as therapeutic targets.

  20. Structure of the adenylation domain of NAD(+)-dependent DNA ligase from Staphylococcus aureus.

    Science.gov (United States)

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

    2009-11-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-09-17

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

  2. High level expression of chicken muscle adenylate kinase in Escherichia coli.

    Science.gov (United States)

    Tanizawa, Y; Kishi, F; Kaneko, T; Nakazawa, A

    1987-05-01

    Chicken muscle adenylate kinase was produced in a large amount in Escherichia coli cells harboring an expression plasmid, pKK-cAKl-1. The plasmid was constructed by placing the cDNA sequence for chicken muscle adenylate kinase after the tac promoter. After induction by isopropyl-beta-D-thiogalactopyranoside, the enzyme protein amounted to about 10% of the bacterial proteins. The enzyme was readily purified in two steps by using phosphocellulose and Sephadex G-100 columns. The apparent molecular weight of the enzyme produced in E. coli was estimated to be 22,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in agreement with the value deduced from the cDNA sequence. Ten amino acids in the NH2-terminal region were determined, and were identical with the sequence deduced from the cDNA sequence except that the terminal methionine was absent. Michaelis constants for ATP, ADP, and AMP of the enzyme thus synthesized were essentially identical to those determined with the enzyme in crude extracts of chicken skeletal muscle.

  3. Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120.

    Science.gov (United States)

    Frébortová, Jitka; Greplová, Marta; Seidl, Michael F; Heyl, Alexander; Frébort, Ivo

    2015-01-01

    Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants.

  4. Purine and pyrimidine nucleosides preserve human astrocytoma cell adenylate energy charge under ischemic conditions.

    Science.gov (United States)

    Balestri, Francesco; Giannecchini, Michela; Sgarrella, Francesco; Carta, Maria Caterina; Tozzi, Maria Grazia; Camici, Marcella

    2007-02-01

    The brain depends on both glycolysis and mitochondrial oxidative phosphorylation for maintenance of ATP pools. Astrocytes play an integral role in brain functions providing trophic supports and energy substrates for neurons. In this paper, we report that human astrocytoma cells (ADF) undergoing ischemic conditions may use both purine and pyrimidine nucleosides as energy source to slow down cellular damage. The cells are subjected to metabolic stress conditions by exclusion of glucose and incubation with oligomycin (an inhibitor of oxidative phosphorylation). This treatment brings about a depletion of the ATP pool, with a concomitant increase in the AMP levels, which results in a significant decrease of the adenylate energy charge. The presence of purine nucleosides in the culture medium preserves the adenylate energy charge, and improves cell viability. Besides purine nucleosides, also pyrimidine nucleosides, such as uridine and, to a lesser extent, cytidine, are able to preserve the ATP pool. The determination of lactate in the incubation medium indicates that nucleosides can preserve the ATP pool through anaerobic glycolysis, thus pointing to a relevant role of the phosphorolytic cleavage of the N-glycosidic bond of nucleosides which generates, without energy expense, the phosphorylated pentose, which through the pentose phosphate pathway and glycolysis can be converted to energetic intermediates also in the absence of oxygen. In fact, ADF cells possess both purine nucleoside phosphorylase and uridine phosphorylase activities.

  5. Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains.

    Science.gov (United States)

    Ishikawa, Fumihiro; Miyamoto, Kengo; Konno, Sho; Kasai, Shota; Kakeya, Hideaki

    2015-12-18

    A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the Escherichia coli expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe-protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the kcat/Km value with the noncognate substrate Sal and a corresponding 48-fold decrease in the kcat/Km value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique

  6. 2'-Phosphate cyclase activity of RtcA: a potential rationale for the operon organization of RtcA with an RNA repair ligase RtcB in Escherichia coli and other bacterial taxa.

    Science.gov (United States)

    Das, Ushati; Shuman, Stewart

    2013-10-01

    RNA terminal phosphate cyclase catalyzes the ATP-dependent conversion of a 3'-phosphate RNA end to a 2',3'-cyclic phosphate via covalent enzyme-(histidinyl-Nε)-AMP and RNA(3')pp(5')A intermediates. Here, we report that Escherichia coli RtcA (and its human homolog Rtc1) are capable of cyclizing a 2'-phosphate RNA end in high yield. The rate of 2'-phosphate cyclization by RtcA is five orders of magnitude slower than 3'-phosphate cyclization, notwithstanding that RtcA binds with similar affinity to RNA3'p and RNA2'p substrates. These findings expand the functional repertoire of RNA cyclase and suggest that phosphate geometry during adenylate transfer to RNA is a major factor in the kinetics of cyclization. RtcA is coregulated in an operon with an RNA ligase, RtcB, that splices RNA 5'-OH ends to either 3'-phosphate or 2',3'-cyclic phosphate ends. Our results suggest that RtcA might serve an end healing function in an RNA repair pathway, by converting RNA 2'-phosphates, which cannot be spliced by RtcB, to 2',3'-cyclic phosphates that can be sealed. The rtcBA operon is controlled by the σ(54) coactivator RtcR encoded by an adjacent gene. This operon arrangement is conserved in diverse bacterial taxa, many of which have also incorporated the RNA-binding protein Ro (which is implicated in RNA quality control under stress conditions) as a coregulated component of the operon.

  7. [Biosynthesis of cyclic GMP in plant cells - new insight into guanylate cyclases].

    Science.gov (United States)

    Świeżawska, Brygida; Marciniak, Katarzyna; Szmidt-Jaworska, Adriana

    2015-01-01

    Cyclic 3',5'-guanosine monophosphate (cGMP) is involved in many physiological processes in plants. Concentration of this second messenger in plant cell is determined by guanylyl cyclases (GCs) responsible for cGMP synthesis and phosphodiesterases (PDEs) involved in cGMP inactivation. First discovered plant GCs were localized in cytosol, but few years ago a new family of plasma membrane proteins with guanylyl cyclase activity was identified in Arabidopsis thaliana. These proteins belong to the family of a leucine-rich repeat receptor-like kinases (LRR-RLK) with extracellular leucine-rich repeat domain, a transmembrane-spanning domain, and an intracellular kinase domain. A novel class of guanylyl cyclases contain the GC catalytic center encapsulated within the intracellular kinase domain. These molecules are different to animal GCs in that the GC catalytic center is nested within the kinase domain. In presented paper we summarized the most recent data concerning plant guanylyl cyclases.

  8. [Soluble guanylate cyclase in the molecular mechanism underlying the therapeutic action of drugs].

    Science.gov (United States)

    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.

  9. Genetic and biochemical analysis of the adenylyl cyclase-associated protein, cap, in Schizosaccharomyces pombe.

    OpenAIRE

    Kawamukai, M; Gerst, J; Field, J.; Riggs, M.; Rodgers, L; Wigler, M; Young, D

    1992-01-01

    We have identified, cloned, and studied a gene, cap, encoding a protein that is associated with adenylyl cyclase in the fission yeast Schizosaccharomyces pombe. This protein shares significant sequence homology with the adenylyl cyclase-associated CAP protein in the yeast Saccharomyces cerevisiae. CAP is a bifunctional protein; the N-terminal domain appears to be involved in cellular responsiveness to RAS, whereas loss of the C-terminal portion is associated with morphological and nutritional...

  10. Lycopene cyclase paralog CruP protects against reactive oxygen species in oxygenic photosynthetic organisms

    OpenAIRE

    Bradbury, Louis M. T.; Shumskaya, Maria; Tzfadia, Oren; Wu, Shi-Biao; Kennelly, Edward J.; Wurtzel, Eleanore T

    2012-01-01

    In photosynthetic organisms, carotenoids serve essential roles in photosynthesis and photoprotection. A previous report designated CruP as a secondary lycopene cyclase involved in carotenoid biosynthesis [Maresca J, et al. (2007) Proc Natl Acad Sci USA 104:11784–11789]. However, we found that cruP KO or cruP overexpression plants do not exhibit correspondingly reduced or increased production of cyclized carotenoids, which would be expected if CruP was a lycopene cyclase. Instead, we show that...

  11. Choreographing the adenylyl cyclase signalosome: sorting out the partners and the steps.

    Science.gov (United States)

    Ostrom, Rennolds S; Bogard, Amy S; Gros, Robert; Feldman, Ross D

    2012-01-01

    Adenylyl cyclases are a ubiquitous family of enzymes and are critical regulators of metabolic and cardiovascular function. Multiple isoforms of the enzyme are expressed in a range of tissues. However, for many processes, the adenylyl cyclase isoforms have been thought of as essentially interchangeable, with their impact more dependent on their common actions to increase intracellular cyclic adenosine monophosphate content regardless of the isoform involved. It has long been appreciated that each subfamily of isoforms demonstrate a specific pattern of "upstream" regulation, i.e., specific patterns of ion dependence (e.g., calcium-dependence) and specific patterns of regulation by kinases (protein kinase A (PKA), protein kinase C (PKC), raf). However, more recent studies have suggested that adenylyl cyclase isoform-selective patterns of signaling are a wide-spread phenomenon. The determinants of these selective signaling patterns relate to a number of factors, including: (1) selective coupling of specific adenylyl cyclase isoforms with specific G protein-coupled receptors, (2) localization of specific adenylyl cyclase isoforms in defined structural domains (AKAP complexes, caveolin/lipid rafts), and (3) selective coupling of adenylyl cyclase isoforms with specific downstream signaling cascades important in regulation of cell growth and contractility. The importance of isoform-specific regulation has now been demonstrated both in mouse models as well as in humans. Adenylyl cyclase has not been viewed as a useful target for therapeutic regulation, given the ubiquitous expression of the enzyme and the perceived high risk of off-target effects. Understanding which isoforms of adenylyl cyclase mediate distinct cellular effects would bring new significance to the development of isoform-specific ligands to regulate discrete cellular actions.

  12. Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W. (UIUC); (Iowa State); (Penn)

    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.

  13. Structure and Mechanism of the Diterpene Cyclase ent-Copalyl Diphosphate Synthase

    Science.gov (United States)

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W.

    2011-01-01

    The structure of ent-copalyl diphosphate synthase (CPS) reveals three α-helical domains (α, β, γ), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the βγ domains in CPS but exclusively in the α 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. PMID:21602811

  14. Dimerization Domain of Retinal Membrane Guanylyl Cyclase 1 (RetGC1) Is an Essential Part of Guanylyl Cyclase-activating Protein (GCAP) Binding Interface.

    Science.gov (United States)

    Peshenko, Igor V; Olshevskaya, Elena V; Dizhoor, Alexander M

    2015-08-01

    The photoreceptor-specific proteins guanylyl cyclase-activating proteins (GCAPs) bind and regulate retinal membrane guanylyl cyclase 1 (RetGC1) but not natriuretic peptide receptor A (NPRA). Study of RetGC1 regulation in vitro and its association with fluorescently tagged GCAP in transfected cells showed that R822P substitution in the cyclase dimerization domain causing congenital early onset blindness disrupted RetGC1 ability to bind GCAP but did not eliminate its affinity for another photoreceptor-specific protein, retinal degeneration 3 (RD3). Likewise, the presence of the NPRA dimerization domain in RetGC1/NPRA chimera specifically disabled binding of GCAPs but not of RD3. In subsequent mapping using hybrid dimerization domains in RetGC1/NPRA chimera, multiple RetGC1-specific residues contributed to GCAP binding by the cyclase, but the region around Met(823) was the most crucial. Either positively or negatively charged residues in that position completely blocked GCAP1 and GCAP2 but not RD3 binding similarly to the disease-causing mutation in the neighboring Arg(822). The specificity of GCAP binding imparted by RetGC1 dimerization domain was not directly related to promoting dimerization of the cyclase. The probability of coiled coil dimer formation computed for RetGC1/NPRA chimeras, even those incapable of binding GCAP, remained high, and functional complementation tests showed that the RetGC1 active site, which requires dimerization of the cyclase, was formed even when Met(823) or Arg(822) was mutated. These results directly demonstrate that the interface for GCAP binding on RetGC1 requires not only the kinase homology region but also directly involves the dimerization domain and especially its portion containing Arg(822) and Met(823).

  15. Use of adenylate kinase as a solubility tag for high level expression of T4 DNA ligase in Escherichia coli.

    Science.gov (United States)

    Liu, Xinxin; Huang, Anliang; Luo, Dan; Liu, Haipeng; Han, Huzi; Xu, Yang; Liang, Peng

    2015-05-01

    The discovery of T4 DNA ligase in 1960s was pivotal in the spread of molecular biotechnology. The enzyme has become ubiquitous for recombinant DNA routinely practiced in biomedical research around the globe. Great efforts have been made to express and purify T4 DNA ligase to meet the world demand, yet over-expression of soluble T4 DNA ligase in E. coli has been difficult. Here we explore the use of adenylate kinase to enhance T4 DNA ligase expression and its downstream purification. E.coli adenylate kinase, which can be expressed in active form at high level, was fused to the N-terminus of T4 DNA ligase. The resulting His-tagged AK-T4 DNA ligase fusion protein was greatly over-expressed in E. coli, and readily purified to near homogeneity via two purification steps consisting of Blue Sepharose and Ni-NTA chromatography. The purified AK-T4 DNA ligase not only is fully active for DNA ligation, but also can use ADP in addition to ATP as energy source since adenylate kinase converts ADP to ATP and AMP. Thus adenylate kinase may be used as a solubility tag to facilitate recombinant protein expression as well as their downstream purification.

  16. The pimeloyl-CoA synthetase BioW defines a new fold for adenylate-forming enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Estrada, Paola; Manandhar, Miglena; Dong, Shi-Hui; Deveryshetty, Jaigeeth; Agarwal, Vinayak; Cronan, John E.; Nair, Satish K.

    2017-04-17

    Reactions that activate carboxylates through acyl-adenylate intermediates are found throughout biology and include acyl- and aryl-CoA synthetases and tRNA synthetases. Here we describe the characterization of Aquifex aeolicus BioW, which represents a new protein fold within the superfamily of adenylating enzymes. Substrate-bound structures identified the enzyme active site and elucidated the mechanistic strategy for conjugating CoA to the seven-carbon α,ω-dicarboxylate pimelate, a biotin precursor. Proper position of reactive groups for the two half-reactions is achieved solely through movements of active site residues, as confirmed by site-directed mutational analysis. The ability of BioW to hydrolyze adenylates of noncognate substrates is reminiscent of pre-transfer proofreading observed in some tRNA synthetases, and we show that this activity can be abolished by mutation of a single residue. These studies illustrate how BioW can carry out three different biologically prevalent chemical reactions (adenylation, thioesterification, and proofreading) in the context of a new protein fold.

  17. Diazepam inhibits forskolin-stimulated adenylyl cyclase activity in human tumour cells.

    Science.gov (United States)

    Niles, L P; Wang, J

    1999-10-01

    Previous studies have shown that the benzodiazepine agonist, diazepam, suppresses adenylyl cyclase activity in rat brain, via a G protein-coupled benzodiazepine receptor. Since diazepam binding sites are also present in diverse non-neuronal tissues including tumour cells, its effects on adenylyl cyclase activity were examined in membranes from human MCF-7 (breast cancer) and M-6 (melanoma) cells. Diazepam caused a biphasic and concentration-dependent inhibition of forskolin-stimulated adenylyl cyclase activity in MCF-7 membranes. The first phase of inhibition, at picomolar to nanomolar drug concentrations (EC50=5.7 x 10(-12)M), is similar to the receptor mediated phase observed in the rat brain. At micromolar concentrations of diazepam (EC50= 1.8 x 10(-4)M), the steep decrease in adenylyl cyclase activity may involve a direct action on the enzyme itself, as detected previously in rat brain membranes. Diazepam-induced suppression of adenylyl cyclase activity was also detected in M-6 membranes. However, in contrast to MCF-7 findings, only micromolar concentrations of diazepam (EC50=5.2 x 10(-4)M) inhibited enzyme activity in M-6 membranes. These findings suggest that G protein-coupled benzodiazepine receptors, which mediate inhibition of the adenylyl cyclase-cAMP pathway in the brain, are also expressed in MCF-7 cells.

  18. The Cyclase-associated Protein CAP as Regulator of Cell Polarity and cAMP Signaling in Dictyostelium

    OpenAIRE

    Noegel, Angelika A; Blau-Wasser, Rosemarie; Sultana, Hameeda; Müller, Rolf; Israel, Lars; Schleicher, Michael; Patel, Hitesh; Weijer, Cornelis J

    2004-01-01

    Cyclase-associated protein (CAP) is an evolutionarily conserved regulator of the G-actin/F-actin ratio and, in yeast, is involved in regulating the adenylyl cyclase activity. We show that cell polarization, F-actin organization, and phototaxis are altered in a Dictyostelium CAP knockout mutant. Furthermore, in complementation assays we determined the roles of the individual domains in signaling and regulation of the actin cytoskeleton. We studied in detail the adenylyl cyclase activity and fo...

  19. Long-Term Exposure to High Corticosterone Levels Inducing a Decrease of Adenylate Kinase 1 Activity

    Institute of Scientific and Technical Information of China (English)

    ZHAO Yu'nan; SHEN Jia; SU Hui; HUANG Yufang; XING Dongming; DU Lijun

    2009-01-01

    Corticosterone, a principal glucocorticoid synthesized in the rodent adrenal cortex, can be cumula-tively toxic to hippocampal neurons, the cause of which is not known. The present study determined whether the cytosol adenylate kinase (AK) system was involved in the neuronal damage induced by long-term exposure to high corticosterone levels. We investigated the effects of long-term exposure to high corticosterone levels on AK1 activity, AK1 mRNA expression, and energy levels in cultured hippocampal neurons. The results show that long-term exposure to high corticosterone levels induces a reduction of the cultured hippocampal neuron viability, significantly reduces energy levels, and causes a time-dependant re-duction of the AK1 activity. These findings indicate that changes in the AK system might be the mechanism underlying neuronal damage induced by long-term exposure to high corticosterone levels.

  20. INVOLVEMENT OF THE Ca2+-PROTEIN KINASE C AND ADENYLATE CYCLACE SIGNAL PATHWAYS IN THE ACTIVATION OF THYMOCYTES IN RESPONSE TO WHOLE-BODY IRRADIATION WITH LOW DOSE X-RAYS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective. To study the molecular mechanism of the stimulatory effect of low dose radiation(LDR) on T cell activation.Methods. Thymocytes from Kunming mice exposed to whole-body irradiation(WBI) with different doses of X-rays were analyzed for the changes in signal molecules of the phospholipase C-phosphatidylinositol biphosphate(PLC-IP2) and G protein-adenylate cyclase(AC) pathways.Results.It was found that[Ca2+]i increased in response to doses within 0.2 Gy which was most marked after 0.075 Gy and the increase was accentuated in the presence of Con A. The changes in CD3 and calcineurin(CN) expression of the thymocytes followed the same pattern as the alterations in [Ca2+]i after LDR. The expression of α,β1 and β2 isoforms of protein kinase C(PKC) was all up-regulated after 0.075 Gy with the increase in PKC-β1 expression being most marked. The cAMP/cGMP ratio and PKA activity of the thymocytes was lowered after low dose radiation and increased after doses above 0.5 Gy in a dose-dependent manner, thus giving rise to J-shaped dose-response curves. The Ca antagonist TMB-8 and cAMP stimulant cholera toxin suppressed the augmented thymocyte proliferation induced by LDR.Conclusion.Data presented in the present paper suggest that activation of the PLC-PIP2 signal pathway and suppression of the AC-cAMP signal pathway are involved in the stimulation of the thymocytes following WBI with low dose X-rays.

  1. NMr studies of the AMP binding site and mechanism of adenylate kinase

    Energy Technology Data Exchange (ETDEWEB)

    Kuby, S.A.; Fry, D.C.; Mildvan, A.S.

    1986-05-01

    The authors recently located by NMR the MgATP binding site on adenylate kinase correcting the proposed location for this site based on X-ray studies of the binding of salicylate. To determine the conformation and location of the other substrate, they have determined distances from Cr/sup 3 +/ AMPPCP to 6 protons and to the phosphorus atom of AMP on adenylate kinase using the paramagnetic-probe-T/sub 1/ method. They have also used time-dependent NOEs to measure five interproton distances on AMP, permitting evaluation of the conformation of enzyme-bound AMP and its position with respect to metal-ATP. Enzyme-bound AMP exhibits a high-anti glycosyl torsional angle (X = 110/sup 0/), a 3'-endo sugar pucker (delta = 105/sup 0/), and a gauche-trans orientation about the C/sub 4/'-C/sub 5/' bond (..gamma.. = 180/sup 0/). The distance from Cr/sup 3 +/ to the phosphorus of AMP is 6.4 +/- 0.3 A, indicating a reaction coordinate distance of greater than or equal to A which is consistent with an associative SN2 mechanism for the phosphoryl transfer. Ten intermolecular NOEs, from protons of the enzyme to those of AMP were detected. These constraints, together with the conformation of AMP and the X-ray structure of the enzyme, suggest proximity (less than or equal to A) of AMP to leu 116, arg 171, val 173, gln 185, thr 188, and asp 191.

  2. Synthetic genes for human muscle-type adenylate kinase in Escherichia coli.

    Science.gov (United States)

    Kim, H J; Nishikawa, S; Tanaka, T; Uesugi, S; Takenaka, H; Hamada, M; Kuby, S A

    1989-01-01

    An artificial gene coding for the human muscle-type cytosolic adenylate kinase (hAK1) was chemically synthesized and directly expressed in Escherichia coli under the control of trp promoter. The DNA duplex of 596 bp was designed and constructed from 40 oligonucleotide fragments of typically 30 nucleotides in length. Twelve unique restriction sites were fairly evenly spaced in the synthetic gene to facilitate site-specific mutagenesis at any part of this recombinant protein. The genes for mutant hAK1 (Tyr 95----Phe 95, Y95F hAK1; Arg 97----Ala 97, R97A hAK1) were constructed by cassette mutagenesis and utilized restriction sites incorporated in the hAK1 gene. The recombinant hAK1 was purified to homogeneity by a two-step chromatographic procedure with a good yield, and showed the same adenylate kinase activity as that of authentic hAK1. Preliminary kinetic studies show that the enzymatic activity (Vmax app,cor/Et) of Y95F hAK1 was slightly greater than that of recombinant hAK1, whereas R97A hAK1 still possessed approximately 4% of recombinant hAK1 activity. These results suggest that the Arg-97 residue is important but not essential for catalytic activity, and that Tyr-95 can be replaced by phenylalanine without substantial effects on the enzymatic activity. Moreover, preliminary estimates of the apparent kinetic parameters suggest that these residues are not required for MgATP binding, and therefore they do not appear to be part of the MgATP binding site.

  3. New crystal structures of adenylate kinase from Streptococcus pneumoniae D39 in two conformations.

    Science.gov (United States)

    Thach, Trung Thanh; Lee, Sangho

    2014-11-01

    Adenylate kinases (AdKs; EC 2.7.3.4) play a critical role in intercellular homeostasis by the interconversion of ATP and AMP to two ADP molecules. Crystal structures of adenylate kinase from Streptococcus pneumoniae D39 (SpAdK) have recently been determined using ligand-free and inhibitor-bound crystals belonging to space groups P21 and P1, respectively. Here, new crystal structures of SpAdK in ligand-free and inhibitor-bound states determined at 1.96 and 1.65 Å resolution, respectively, are reported. The new ligand-free crystal belonged to space group C2, with unit-cell parameters a=73.5, b=54.3, c=62.7 Å, β=118.8°. The new ligand-free structure revealed an open conformation that differed from the previously determined conformation, with an r.m.s.d on Cα atoms of 1.4 Å. The new crystal of the complex with the two-substrate-mimicking inhibitor P1,P5-bis(adenosine-5'-)pentaphosphate (Ap5A) belonged to space group P1, with unit-cell parameters a=53.9, b=62.3, c=63.0 Å, α=101.9, β=112.6, γ=89.9°. Despite belonging to the same space group as the previously reported crystal, the new Ap5A-bound crystal contains four molecules in the asymmetric unit, compared with two in the previous crystal, and shows slightly different lattice contacts. These results demonstrate that SpAdK can crystallize promiscuously in different forms and that the open structure is flexible in conformation.

  4. Human recombinant soluble guanylyl cyclase: expression, purification, and regulation

    Science.gov (United States)

    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.

  5. Synthesis of arborane triterpenols by a bacterial oxidosqualene cyclase

    Science.gov (United States)

    Banta, Amy B.; Wei, Jeremy H.; Gill, Clare C. C.; Giner, José-Luis; Welander, Paula V.

    2017-01-01

    Cyclic triterpenoids are a broad class of polycyclic lipids produced by bacteria and eukaryotes. They are biologically relevant for their roles in cellular physiology, including membrane structure and function, and biochemically relevant for their exquisite enzymatic cyclization mechanism. Cyclic triterpenoids are also geobiologically significant as they are readily preserved in sediments and are used as biomarkers for ancient life throughout Earth's history. Isoarborinol is one such triterpenoid whose only known biological sources are certain angiosperms and whose diagenetic derivatives (arboranes) are often used as indicators of terrestrial input into aquatic environments. However, the occurrence of arborane biomarkers in Permian and Triassic sediments, which predates the accepted origin of angiosperms, suggests that microbial sources of these lipids may also exist. In this study, we identify two isoarborinol-like lipids, eudoraenol and adriaticol, produced by the aerobic marine heterotrophic bacterium Eudoraea adriatica. Phylogenetic analysis demonstrates that the E. adriatica eudoraenol synthase is an oxidosqualene cyclase homologous to bacterial lanosterol synthases and distinct from plant triterpenoid synthases. Using an Escherichia coli heterologous sterol expression system, we demonstrate that substitution of four amino acid residues in a bacterial lanosterol synthase enabled synthesis of pentacyclic arborinols in addition to tetracyclic sterols. This variant provides valuable mechanistic insight into triterpenoid synthesis and reveals diagnostic amino acid residues to differentiate between sterol and arborinol synthases in genomic and metagenomic datasets. Our data suggest that there may be additional bacterial arborinol producers in marine and freshwater environments that could expand our understanding of these geologically informative lipids.

  6. Structure of RNA 3'-phosphate cyclase bound to substrate RNA.

    Science.gov (United States)

    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.

  7. NCBI nr-aa BLAST: CBRC-GACU-11-0020 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-GACU-11-0020 ref|NP_001107.2| adenylate cyclase 9 [Homo sapiens] sp|O60503|ADCY9_HUMAN Adenylate cyclas...e type 9 (Adenylate cyclase type IX) (ATP pyrophosphate-lyase 9) (Adenylyl cyclase ...9) gb|AAK29464.1| adenylate cyclase type 9 [Homo sapiens] gb|AAY21237.1| adenylyl cyclase type 9 [Homo sapie...ns] gb|EAW85331.1| adenylate cyclase 9, isoform CRA_a [Homo sapiens] gb|EAW85332.1| adenylate cyclase... 9, isoform CRA_a [Homo sapiens] gb|AAI51208.1| Adenylate cyclase 9 [Homo sapiens] NP_001107.2 0.0 63% ...

  8. Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides.

    Science.gov (United States)

    Gagne, Steve J; Stout, Jake M; Liu, Enwu; Boubakir, Zakia; Clark, Shawn M; Page, Jonathan E

    2012-07-31

    Δ(9)-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.

  9. Cloning and Functional Characterization of a Lycopene β-Cyclase from Macrophytic Red Alga Bangia fuscopurpurea

    Directory of Open Access Journals (Sweden)

    Tian-Jun Cao

    2017-04-01

    Full Text Available Lycopene cyclases cyclize the open ends of acyclic lycopene (ψ,ψ-carotene into β- or ε-ionone rings in the crucial bifurcation step of carotenoid biosynthesis. Among all carotenoid constituents, β-carotene (β,β-carotene is found in all photosynthetic organisms, except for purple bacteria and heliobacteria, suggesting a ubiquitous distribution of lycopene β-cyclase activity in these organisms. In this work, we isolated a gene (BfLCYB encoding a lycopene β-cyclase from Bangia fuscopurpurea, a red alga that is considered to be one of the primitive multicellular eukaryotic photosynthetic organisms and accumulates carotenoid constituents with both β- and ε-rings, including β-carotene, zeaxanthin, α-carotene (β,ε-carotene and lutein. Functional complementation in Escherichia coli demonstrated that BfLCYB is able to catalyze cyclization of lycopene into monocyclic γ-carotene (β,ψ-carotene and bicyclic β-carotene, and cyclization of the open end of monocyclic δ-carotene (ε,ψ-carotene to produce α-carotene. No ε-cyclization activity was identified for BfLCYB. Sequence comparison showed that BfLCYB shares conserved domains with other functionally characterized lycopene cyclases from different organisms and belongs to a group of ancient lycopene cyclases. Although B. fuscopurpurea also synthesizes α-carotene and lutein, its enzyme-catalyzing ε-cyclization is still unknown.

  10. Cloning and Functional Characterization of a Lycopene β-Cyclase from Macrophytic Red Alga Bangia fuscopurpurea.

    Science.gov (United States)

    Cao, Tian-Jun; Huang, Xing-Qi; Qu, Yuan-Yuan; Zhuang, Zhong; Deng, Yin-Yin; Lu, Shan

    2017-04-11

    Lycopene cyclases cyclize the open ends of acyclic lycopene (ψ,ψ-carotene) into β- or ε-ionone rings in the crucial bifurcation step of carotenoid biosynthesis. Among all carotenoid constituents, β-carotene (β,β-carotene) is found in all photosynthetic organisms, except for purple bacteria and heliobacteria, suggesting a ubiquitous distribution of lycopene β-cyclase activity in these organisms. In this work, we isolated a gene (BfLCYB) encoding a lycopene β-cyclase from Bangia fuscopurpurea, a red alga that is considered to be one of the primitive multicellular eukaryotic photosynthetic organisms and accumulates carotenoid constituents with both β- and ε-rings, including β-carotene, zeaxanthin, α-carotene (β,ε-carotene) and lutein. Functional complementation in Escherichia coli demonstrated that BfLCYB is able to catalyze cyclization of lycopene into monocyclic γ-carotene (β,ψ-carotene) and bicyclic β-carotene, and cyclization of the open end of monocyclic δ-carotene (ε,ψ-carotene) to produce α-carotene. No ε-cyclization activity was identified for BfLCYB. Sequence comparison showed that BfLCYB shares conserved domains with other functionally characterized lycopene cyclases from different organisms and belongs to a group of ancient lycopene cyclases. Although B. fuscopurpurea also synthesizes α-carotene and lutein, its enzyme-catalyzing ε-cyclization is still unknown.

  11. Estradiol rapidly inhibits soluble guanylyl cyclase expression in rat uterus

    Science.gov (United States)

    Krumenacker, J. S.; Hyder, S. M.; Murad, F.

    2001-01-01

    Previous reports that investigated the regulation of the NO/soluble guanylyl cyclase (sGC)/cGMP pathway by estrogenic compounds have focused primarily on the levels of NO, NO-producing enzymes, and cGMP in various tissues. In this study, we demonstrate that 17beta-estradiol (E2) regulates the alpha(1) and beta(1) subunits of the NO receptor, sGC, at the mRNA and protein levels in rat uterus. Using real-time quantitative PCR, we found that within 1 h of in vivo E2 administration to rats, sGC mRNA levels begin to diminish. After 3 h, there is a maximal diminution of sGC mRNA expression (sGC alpha(1) 10% and sGC beta(1) 33% of untreated). This effect was blocked by the estrogen receptor antagonist, ICI 182,780, indicating that estrogen receptor is required. The effect of E2 also was observed in vitro with incubations of uterine tissue, indicating that the response does not depend on the secondary release of other hormones or factors from other tissues. Puromycin did not block the effect, suggesting the effects occur because of preexisting factors in uterine tissues and do not require new protein synthesis. Using immunoblot analysis, we found that sGC protein levels also were reduced by E2 over a similar time course as the sGC mRNA. We conclude that sGC plays a vital role in the NO/sGC/cGMP regulatory pathway during conditions of elevated estrogen levels in the rat uterus as a result of the reduction of sGC expression.

  12. A cost-effective method for Illumina small RNA-Seq library preparation using T4 RNA ligase 1 adenylated adapters

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    Chen Yun-Ru

    2012-09-01

    Full Text Available Abstract Background Deep sequencing is a powerful tool for novel small RNA discovery. Illumina small RNA sequencing library preparation requires a pre-adenylated 3’ end adapter containing a 5’,5’-adenyl pyrophosphoryl moiety. In the absence of ATP, this adapter can be ligated to the 3’ hydroxyl group of small RNA, while RNA self-ligation and concatenation are repressed. Pre-adenylated adapters are one of the most essential and costly components required for library preparation, and few are commercially available. Results We demonstrate that DNA oligo with 5’ phosphate and 3’ amine groups can be enzymatically adenylated by T4 RNA ligase 1 to generate customized pre-adenylated adapters. We have constructed and sequenced a small RNA library for tomato (Solanum lycopersicum using the T4 RNA ligase 1 adenylated adapter. Conclusion We provide an efficient and low-cost method for small RNA sequencing library preparation, which takes two days to complete and costs around $20 per library. This protocol has been tested in several plant species for small RNA sequencing including sweet potato, pepper, watermelon, and cowpea, and could be readily applied to any RNA samples.

  13. Structure of the adenylation-peptidyl carrier protein didomain of the Microcystis aeruginosa microcystin synthetase McyG.

    Science.gov (United States)

    Tan, Xiao-Feng; Dai, Ya-Nan; Zhou, Kang; Jiang, Yong-Liang; Ren, Yan-Min; Chen, Yuxing; Zhou, Cong-Zhao

    2015-04-01

    Microcystins, which are the most common cause of hepatotoxicity associated with cyanobacterial water blooms, are assembled in vivo on a large multienzyme complex via a mixed nonribosomal peptide synthetase/polyketide synthetase (NRPS/PKS). The biosynthesis of microcystin in Microcystis aeruginosa PCC 7806 starts with the enzyme McyG, which contains an adenylation-peptidyl carrier protein (A-PCP) didomain for loading the starter unit to assemble the side chain of an Adda residue. However, the catalytic mechanism remains unclear. Here, the 2.45 Å resolution crystal structure of the McyG A-PCP didomain complexed with the catalytic intermediate L-phenylalanyl-adenylate (L-Phe-AMP) is reported. Each asymmetric unit contains two protein molecules, one of which consists of the A-PCP didomain and the other of which comprises only the A domain. Structural analyses suggest that Val227 is likely to be critical for the selection of hydrophobic substrates. Moreover, two distinct interfaces demonstrating variable crosstalk between the PCP domain and the A domain were observed. A catalytic cycle for the adenylation and peptide transfer of the A-PCP didomain is proposed.

  14. Simultaneous stimulation of GABA and beta adrenergic receptors stabilizes isotypes of activated adenylyl cyclase heterocomplex

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    Robichon Alain

    2004-06-01

    Full Text Available Abstract Background We investigated how the synthesis of cAMP, stimulated by isoproterenol acting through β-adrenoreceptors and Gs, is strongly amplified by simultaneous incubation with baclofen. Baclofen is an agonist of δ-aminobutyric acid type B receptors [GABAB], known to inhibit adenylyl cyclase via Gi. Because these agents have opposite effects on cAMP levels, the unexpected increase in cAMP synthesis when they are applied simultaneously has been intensively investigated. From previous reports, it appears that cyclase type II contributes most significantly to this phenomenon. Results We found that simultaneous application of isoproterenol and baclofen specifically influences the association/dissociation of molecules involved in the induction and termination of cyclase activity. Beta/gamma from [GABA]B receptor-coupled Gi has a higher affinity for adenylyl cyclase isoform(s when these isoforms are co-associated with Gs. Our data also suggest that, when beta/gamma and Gαs are associated with adenylyl cyclase isoform(s, beta/gamma from [GABA]B receptor-coupled Gi retards the GTPase activity of Gαs from adrenergic receptor. These reciprocal regulations of subunits of the adenylyl cyclase complex might be responsible for the drastic increase of cAMP synthesis in response to the simultaneous signals. Conclusions Simultaneous signals arriving at a particular synapse converge on molecular detectors of coincidence and trigger specific biochemical events. We hypothesize that this phenomenon comes from the complex molecular architectures involved, including scaffolding proteins that make reciprocal interactions between associated molecules possible. The biochemistry of simultaneous signaling is addressed as a key to synaptic function.

  15. Highly cooperative feedback control of retinal rod guanylate cyclase by calcium ions.

    Science.gov (United States)

    Koch, K W; Stryer, L

    1988-07-07

    Visual excitation in retinal rod cells is mediated by a cascade that leads to the amplified hydrolysis of cyclic GMP (cGMP) and the consequent closure of cGMP-activated cation-specific channels in the plasma membrane. Recovery of the dark state requires the resynthesis of cGMP, which is catalysed by guanylate cyclase, an axoneme-associated enzyme. The lowering of the cytosolic calcium concentration (Cai) following illumination is thought to be important in stimulating cyclase activity. This hypothesis is supported by the finding that the cGMP content of rod outer segments increases several-fold when Cai is lowered to less than 10 nM. It is evident that cGMP and Cai levels are reciprocally controlled by negative feedback. Guanylate cyclase from toad ROS is strongly stimulated when the calcium level is lowered from 10 microM to 10 nM, but only if they are excited by light. We show here that the guanylate cyclase activity of unilluminated bovine rod outer segments increases markedly (5 to 20-fold) when the calcium level is lowered from 200 nM to 50 nM. This steep dependence of guanylate cyclase activity on the calcium level in the physiological range has a Hill coefficient of 3.9. Stimulation at low calcium levels is mediated by a protein that can be released from the outer segment membranes by washing with a low salt buffer. Calcium sensitivity is partially restored by adding the soluble extract back to the washed membranes. The highly cooperative activation of guanylate cyclase by the light-induced lowering of Cai is likely to be a key event in restoring the dark current after excitation.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  17. Multifunctional oxidosqualene cyclases and cytochrome P450involved in the biosynthesis of apple fruit triterpenic acids

    OpenAIRE

    Andre, Christelle; Legay, Sylvain; Deleruelle, Amélie; Nieuwenhuizen, Niels; Punter, Matthew; Brendolise, Cyril; M.Cooney, Janine; Lateur, Marc; Hausman, Jean-François; Larondelle, Yvan; A.Laing, William

    2016-01-01

    Summary 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 co...

  18. Identification of a fourth family of lycopene cyclases in photosynthetic bacteria

    OpenAIRE

    Maresca, Julia A.; Graham, Joel E.; Wu, Martin; Eisen, Jonathan A; Bryant, Donald A.

    2007-01-01

    A fourth and large family of lycopene cyclases was identified in photosynthetic prokaryotes. The first member of this family, encoded by the cruA gene of the green sulfur bacterium Chlorobium tepidum, was identified in a complementation assay with a lycopene-producing strain of Escherichia coli. Orthologs of cruA are found in all available green sulfur bacterial genomes and in all cyanobacterial genomes that lack genes encoding CrtL- or CrtY-type lycopene cyclases. The cyanobacterium Synechoc...

  19. Purification and assay of cell-invasive form of calmodulin-sensitive adenylyl cyclase from Bordetella pertussis

    Energy Technology Data Exchange (ETDEWEB)

    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.

  20. Multiple lineage specific expansions within the guanylyl cyclase gene family

    Directory of Open Access Journals (Sweden)

    O'Halloran Damien M

    2006-03-01

    Full Text Available Abstract Background Guanylyl cyclases (GCs are responsible for the production of the secondary messenger cyclic guanosine monophosphate, which plays important roles in a variety of physiological responses such as vision, olfaction, muscle contraction, homeostatic regulation, cardiovascular and nervous function. There are two types of GCs in animals, soluble (sGCs which are found ubiquitously in cell cytoplasm, and receptor (rGC forms which span cell membranes. The complete genomes of several vertebrate and invertebrate species are now available. These data provide a platform to investigate the evolution of GCs across a diverse range of animal phyla. Results In this analysis we located GC genes from a broad spectrum of vertebrate and invertebrate animals and reconstructed molecular phylogenies for both sGC and rGC proteins. The most notable features of the resulting phylogenies are the number of lineage specific rGC and sGC expansions that have occurred during metazoan evolution. Among these expansions is a large nematode specific rGC clade comprising 21 genes in C. elegans alone; a vertebrate specific expansion in the natriuretic receptors GC-A and GC-B; a vertebrate specific expansion in the guanylyl GC-C receptors, an echinoderm specific expansion in the sperm rGC genes and a nematode specific sGC clade. Our phylogenetic reconstruction also shows the existence of a basal group of nitric oxide (NO insensitive insect and nematode sGCs which are regulated by O2. This suggests that the primordial eukaryotes probably utilized sGC as an O2 sensor, with the ligand specificity of sGC later switching to NO which provides a very effective local cell-to-cell signalling system. Phylogenetic analysis of the sGC and bacterial heme nitric oxide/oxygen binding protein domain supports the hypothesis that this domain originated from a cyanobacterial source. Conclusion The most salient feature of our phylogenies is the number of lineage specific expansions

  1. Gi proteins regulate adenylyl cyclase activity independent of receptor activation.

    Science.gov (United States)

    Melsom, Caroline Bull; Ørstavik, Øivind; Osnes, Jan-Bjørn; Skomedal, Tor; Levy, Finn Olav; Krobert, Kurt Allen

    2014-01-01

    Despite the view that only β2- as opposed to β1-adrenoceptors (βARs) couple to G(i), some data indicate that the β1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes. We used the Gs-selective (R,R)- and the Gs- and G(i)-activating (R,S)-fenoterol to selectively activate β2ARs (β1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats. PTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response. Together, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic G(i) and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents.

  2. Adenylate nucleotide levels and energy charge in Arthrobacter crystallopoietes during growth and starvation.

    Science.gov (United States)

    Leps, W T; Ensign, J C

    1979-07-01

    The adenylate nucleotide concentrations, based on internal water space, were determined in cells of Arthrobacter crystallopoietes during growth and starvation and the energy charge of the cells was calculated. The energy charge of spherical cells rose during the first 10 h of growth, then remained nearly constant for as long as 20 h into the stationary phase. The energy charge of rod-shaped cells rose during the first 4 h of growth, then remained constant during subsequent growth and decreased in the stationary growth phase. Both spherical and rod-shaped cells excreted adenosine monophosphate but not adenosine triphosphate or adenosine diphosphate during starvation. The intracellular energy charge of spherical cells declined during the initial 10 h and then remained constant for 1 week of starvation at a value of 0.78. The intracellular energy charge of rod-shaped cells declined during the first 24 h of starvation, remained constant for the next 80 h, then decreased to a value of 0.73 after a total of 168 h starvation. Both cell forms remained more than 90% viable during this time. Addition of a carbon and energy source to starving cells resulted in an increase in the ATP concentration and as a result the energy charge increased to the smae levels as found during growth.

  3. Structure and function of adenylate kinase isozymes in normal humans and muscular dystrophy patients.

    Science.gov (United States)

    Hamada, M; Takenaka, H; Fukumoto, K; Fukamachi, S; Yamaguchi, T; Sumida, M; Shiosaka, T; Kurokawa, Y; Okuda, H; Kuby, S A

    1987-01-01

    Two isozymes of adenylate kinase from human Duchenne muscular dystrophy serum, one of which was an aberrant form specific to DMD patients, were separated by Blue Sepharose CL-6B affinity chromatography. The separated aberrant form possessed a molecular weight of 98,000 +/- 1,500, whereas the normal serum isozyme had a weight of 87,000 +/- 1,600, as determined by SDS-polyacrylamide gel electrophoresis, gel filtration, and sedimentation equilibrium. The sedimentation coefficients were 5.8 S and 5.6 S for the aberrant form and the normal form, respectively. Both serum isozymes are tetramers. The subunit size of the aberrant isozyme (Mr = 24,700) was very similar to that of the normal human liver isozyme, and the subunit size of the normal isozyme (Mr = 21,700) was very similar to that of the normal human muscle enzyme. The amino acid composition of the normal serum isozyme was similar to that of the muscle-type enzyme, and that of the aberrant isozyme was similar to that of the liver enzyme, with some exceptions in both cases.

  4. Influence of sampling, storage, processing and optimal experimental conditions on adenylate energy charge in penaeid shrimp

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    Robles-Romo Arlett

    2014-01-01

    Full Text Available Adenylate energy charge (AEC has been used as a practical index of the physiological status and health in several disciplines, such as ecotoxicology and aquaculture. This study standardizes several procedures for AEC determination in penaeid shrimp that are very sensitive to sampling. We concluded that shrimp can be frozen in liquid nitrogen and then stored at -76°C for up to two years for further analysis, or freshly dissected and immediately homogenized in acid. Other cooling procedures, such as immersion in cold water or placing shrimp on ice for 15 min resulted in 50% and 73% decreases in ATP levels, and 9-fold and 10-fold increases in IMP levels, respectively. Optimal values of AEC (0.9 were obtained in shrimp recently transferred from ponds to indoor conditions, but decreased to 0.77 after one month in indoor tanks when stocked at high densities; the AEC re-established to 0.85 when the shrimps were transferred to optimal conditions (lower density and dark tanks. While the levels of arginine phosphate followed the same pattern, its levels did not fully re-establish. Comparison of different devices for sample homogenization indicated that a cryogenic ball mill mixer is the more suitable procedure.

  5. Adenylate kinase 2 (AK2 promotes cell proliferation in insect development

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    Chen Ru-Ping

    2012-09-01

    Full Text Available Abstract Background Adenylate kinase 2 (AK2 is a phosphotransferase that catalyzes the reversible reaction 2ADP(GDP ↔ ATP(GTP + AMP and influences cellular energy homeostasis. However, the role of AK2 in regulating cell proliferation remains unclear because AK2 has been reported to be involved in either cell proliferation or cell apoptosis in different cell types of various organisms. Results This study reports AK2 promotion of cell proliferation using the lepidopteran insect Helicoverpa armigera and its epidermal cell line HaEpi as models. Western blot analysis indicates that AK2 constitutively expresses in various tissues during larval development. Immunocytochemistry analysis indicates that AK2 localizes in the mitochondria. The recombinant expressed AK2 in E. coli promotes cell growth and viability of HaEpi cell line by 3-(4, 5-Dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide (MTT assay. AK2 knockdown in larvae by RNA interference causes larval growth defects, including body weight decrease and development delay. AK2 knockdown in larvae also decreases the number of circulating haemocytes. The mechanism for such effects might be the suppression of gene transcription involved in insect development caused by AK2 knockdown. Conclusion These results show that AK2 regulates cell growth, viability, and proliferation in insect growth and development.

  6. The energy profiles of atomic conformational transition intermediates of adenylate kinase.

    Science.gov (United States)

    Feng, Yaping; Yang, Lei; Kloczkowski, Andrzej; Jernigan, Robert L

    2009-11-15

    The elastic network interpolation (ENI) (Kim et al., Biophys J 2002;83:1620-1630) is a computationally efficient and physically realistic method to generate conformational transition intermediates between two forms of a given protein. However it can be asked whether these calculated conformations provide good representatives for these intermediates. In this study, we use ENI to generate conformational transition intermediates between the open form and the closed form of adenylate kinase (AK). Based on C(alpha)-only intermediates, we construct atomic intermediates by grafting all the atoms of known AK structures onto the C(alpha) atoms and then perform CHARMM energy minimization to remove steric conflicts and optimize these intermediate structures. We compare the energy profiles for all intermediates from both the CHARMM force-field and from knowledge-based energy functions. We find that the CHARMM energies can successfully capture the two energy minima representing the open AK and closed AK forms, while the energies computed from the knowledge-based energy functions can detect the local energy minimum representing the closed AK form and show some general features of the transition pathway with a somewhat similar energy profile as the CHARMM energies. The combinatorial extension structural alignment (Shindyalov et al., 1998;11:739-747) and the k-means clustering algorithm are then used to show that known PDB structures closely resemble computed intermediates along the transition pathway.

  7. On the Roles of Substrate Binding and Hinge Unfolding in Conformational Changes of Adenylate Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Brokaw, Jason B.; Chu, Jhih-wei

    2010-11-17

    We characterized the conformational change of adenylate kinase (AK) between open and closed forms by conducting five all-atom molecular-dynamics simulations, each of 100 ns duration. Different initial structures and substrate binding configurations were used to probe the pathways of AK conformational change in explicit solvent, and no bias potential was applied. A complete closed-to-open and a partial open-to-closed transition were observed, demonstrating the direct impact of substrate-mediated interactions on shifting protein conformation. The sampled configurations suggest two possible pathways for connecting the open and closed structures of AK, affirming the prediction made based on available x-ray structures and earlier works of coarse-grained modeling. The trajectories of the all-atom molecular-dynamics simulations revealed the complexity of protein dynamics and the coupling between different domains during conformational change. Calculations of solvent density and density fluctuations surrounding AK did not show prominent variation during the transition between closed and open forms. Finally, we characterized the effects of local unfolding of an important hinge near Pro177 on the closed-to-open transition of AK and identified a novel mechanism by which hinge unfolding modulates protein conformational change. The local unfolding of Pro177 hinge induces alternative tertiary contacts that stabilize the closed structure and prevent the opening transition.

  8. Phylogenetic relationships of 18 passerines based on Adenylate Kinase Intron 5 sequences

    Institute of Scientific and Technical Information of China (English)

    GUO Hui-yan; YU Hui-xin; BAI Su-ying; MA Yu-kun

    2008-01-01

    The 18 species of bird studied originally are known to belong to muscicapids, robins and sylviids of passerines, but some disputations are always present in their classification systems. In this experiment, phylogenetic relationships of 18 species of passerines were studied using Adenylate Kinase Intron 5 (AK5) sequences and DNA techniques. Through sequences analysis in comparison with each other, phylogenetic tree figures of 18 species of passerines were constructed using Neighbor-Joining (NJ) and Maximum-Parsimony (MP) methods . The results showed that sylviids should be listed as an independent family, while robins and flycatchers should be listed into Muscicapidae. Since the phylogenetic relationships between long-tailed tits and old world warblers are closer than that between long-tailed tits and parids, the long-tailed tits should be independent of paridae and be categorized into aegithalidae. Muscicapidae and Paridae are known to be two monophylitic families, but Sylviidae is not a monophyletic group. AK5 sequences had better efficacy in resolving close relationships of interspecies among intrageneric groups.

  9. Riboswitch control of induction of aminoglycoside resistance acetyl and adenyl-transferases.

    Science.gov (United States)

    He, Weizhi; Zhang, Xuhui; Zhang, Jun; Jia, Xu; Zhang, Jing; Sun, Wenxia; Jiang, Hengyi; Chen, Dongrong; Murchie, Alastair I H

    2013-08-01

    The acquisition of antibiotic resistance by human pathogens poses a significant threat to public health. The mechanisms that control the proliferation and expression of antibiotic resistance genes are not yet completely understood. The aminoglycosides are a historically important class of antibiotics that were introduced in the 1940s. Aminoglycoside resistance is conferred most commonly through enzymatic modification of the drug or enzymatic modification of the target rRNA through methylation or through the overexpression of efflux pumps. In our recent paper, we reported that expression of the aminoglycoside resistance genes encoding the aminoglycoside acetyl transferase (AAC) and aminoglycoside adenyl transferase (AAD) enzymes was controlled by an aminoglycoside-sensing riboswitch RNA. This riboswitch is embedded in the leader RNA of the aac/aad genes and is associated with the integron cassette system. The leader RNA can sense and bind specific aminoglycosides such that the binding causes a structural transition in the leader RNA, which leads to the induction of aminoglycoside antibiotic resistance. Specific aminoglycosides induce reporter gene expression mediated by the leader RNA. Aminoglycoside RNA binding was measured directly and, aminoglycoside-induced changes in RNA structure monitored by chemical probing. UV cross-linking and mutational analysis identified potential aminoglycoside binding sites on the RNA.

  10. Ectopic expression of cyclase associated protein CAP restores the streaming and aggregation defects of adenylyl cyclase a deficient Dictyostelium discoideum cells

    Directory of Open Access Journals (Sweden)

    Sultana Hameeda

    2012-01-01

    Full Text Available Abstract Background Cell adhesion, an integral part of D. discoideum development, is important for morphogenesis and regulated gene expression in the multicellular context and is required to trigger cell-differentiation. G-protein linked adenylyl cyclase pathways are crucially involved and a mutant lacking the aggregation specific adenylyl cyclase ACA does not undergo multicellular development. Results Here, we have investigated the role of cyclase-associated protein (CAP, an important regulator of cell polarity and F-actin/G-actin ratio in the aca- mutant. We show that ectopic expression of GFP-CAP improves cell polarization, streaming and aggregation in aca- cells, but it fails to completely restore development. Our studies indicate a requirement of CAP in the ACA dependent signal transduction for progression of the development of unicellular amoebae into multicellular structures. The reduced expression of the cell adhesion molecule DdCAD1 together with csA is responsible for the defects in aca- cells to initiate multicellular development. Early development was restored by the expression of GFP-CAP that enhanced the DdCAD1 transcript levels and to a lesser extent the csA mRNA levels. Conclusions Collectively, our data shows a novel role of CAP in regulating cell adhesion mechanisms during development that might be envisioned to unravel the functions of mammalian CAP during animal embryogenesis.

  11. Soluble guanylyl cyclase is involved in PDT-induced injury of crayfish glial cells

    Science.gov (United States)

    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.

  12. Impairment of adenylyl cyclase signal transduction in mecobalamin-deficient rats.

    Science.gov (United States)

    Hatta, S; Watanabe, M; Ikeda, H; Kamada, H; Saito, T; Ohshika, H

    1995-11-30

    This study examined alterations in the beta-adrenoceptor-G5-adenylyl cyclase system in cerebral cortex membranes from vitamin B12-deficient rats fed a diet lacking vitamin B12 (mecobalamin) for 15 weeks. Basal, 5(7)-guanylylimidodiphosphate (GppNHp)-, isoproterenol-, and forskolin-stimulated adenylyl cyclase activities were significantly reduced in mecobalamin-deficient rats compared with those in control rats. However, no significant differences were observed in the amount and function of G5- estimated by immunoblotting and guanine nucleotide photoaffinity labeling, respectively, or in the densities and the dissociation constants of beta-adrenoceptors, estimated by [125I] pindolol binding, between control and the deficient rats. These results indicate that vitamin B12 deficiency results in the impairment of the coupling among the beta-adrenoceptor, G5- and the catalytic subunit of adenylyl cyclase, and in dysfunction of the catalytic subunit of the enzyme, suggesting that vitamin B12 participates in the regulation of neuronal adenylyl cyclase signal transduction.

  13. A novel Myb homolog initiates Dictyostelium development by induction of adenylyl cyclase expression

    NARCIS (Netherlands)

    Otsuka, Hideshi; Haastert, Peter J.M. van

    1998-01-01

    Dictyostelium development is induced by starvation. The adenylyl cyclase gene ACA is one of the first genes expressed upon starvation. ACA produces extracellular cAMP that induces chemotaxis, aggregation, and differentiation in neighboring cells. Using insertional mutagenesis we have isolated a muta

  14. A short history of cGMP, guanylyl cyclases, and cGMP-dependent protein kinases.

    Science.gov (United States)

    Kots, Alexander Y; Martin, Emil; Sharina, Iraida G; Murad, Ferid

    2009-01-01

    Here, we review the early studies on cGMP, guanylyl cyclases, and cGMP-dependent protein kinases to facilitate understanding of development of this exciting but complex field of research encompassing pharmacology, biochemistry, physiology, and molecular biology of these important regulatory molecules.

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

    DEFF Research Database (Denmark)

    Chen, Yicai; Yuan, Mingjun; Mohanty, Anee

    2015-01-01

    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 intri...

  16. Comparison of human CAP and CAP2, homologs of the yeast adenylyl cyclase-associated proteins.

    Science.gov (United States)

    Yu, G; Swiston, J; Young, D

    1994-06-01

    We previously reported the identification of human CAP, a protein that is related to the Saccharomyces cerevisiae and Schizosaccharomyces pombe adenylyl cyclase-associated CAP proteins. The two yeast CAP proteins have similar functions: the N-terminal domains are required for the normal function of adenylyl cyclase, while loss of the C-terminal domains result in morphological and nutritional defects that are unrelated to the cAMP pathways. We have amplified and cloned cDNAs from a human glioblastoma library that encode a second CAP-related protein, CAP2. The human CAP and CAP2 proteins are 64% identical. Expression of either human CAP or CAP2 in S. cerevisiae cap- strains suppresses phenotypes associated with deletion of the C-terminal domain of CAP, but does not restore hyper-activation of adenylyl cyclase by RAS2val19. Similarly, expression of either human CAP or CAP2 in S. pombe cap- strains suppresses the morphological and temperature-sensitive phenotypes associated with deletion of the C-terminal domain of CAP in this yeast. In addition, expression of human CAP, but not CAP2, suppresses the propensity to sporulate due to deletion of the N-terminal domain of CAP in S. pombe. This latter observation suggests that human CAP restores normal adenylyl cyclase activity in S. pombe cap- cells. Thus, functional properties of both N-terminal and C-terminal domains are conserved between the human and S. pombe CAP proteins.

  17. Cryptic indole hydroxylation by a non-canonical terpenoid cyclase parallels bacterial xenobiotic detoxification

    Science.gov (United States)

    Kugel, Susann; Baunach, Martin; Baer, Philipp; Ishida-Ito, Mie; Sundaram, Srividhya; Xu, Zhongli; Groll, Michael; Hertweck, Christian

    2017-06-01

    Terpenoid natural products comprise a wide range of molecular architectures that typically result from C-C bond formations catalysed by classical type I/II terpene cyclases. However, the molecular diversity of biologically active terpenoids is substantially increased by fully unrelated, non-canonical terpenoid cyclases. Their evolutionary origin has remained enigmatic. Here we report the in vitro reconstitution of an unusual flavin-dependent bacterial indoloterpenoid cyclase, XiaF, together with a designated flavoenzyme-reductase (XiaP) that mediates a key step in xiamycin biosynthesis. The crystal structure of XiaF with bound FADH2 (at 2.4 Å resolution) and phylogenetic analyses reveal that XiaF is, surprisingly, most closely related to xenobiotic-degrading enzymes. Biotransformation assays show that XiaF is a designated indole hydroxylase that can be used for the production of indigo and indirubin. We unveil a cryptic hydroxylation step that sets the basis for terpenoid cyclization and suggest that the cyclase has evolved from xenobiotics detoxification enzymes.

  18. Differential calcium signaling by cone specific guanylate cyclase-activating proteins from the zebrafish retina.

    Directory of Open Access Journals (Sweden)

    Alexander Scholten

    Full Text Available Zebrafish express in their retina a higher number of guanylate cyclase-activating proteins (zGCAPs than mammalians pointing to more complex guanylate cyclase signaling systems. All six zGCAP isoforms show distinct and partial overlapping expression profiles in rods and cones. We determined critical Ca(2+-dependent parameters of their functional properties using purified zGCAPs after heterologous expression in E.coli. Isoforms 1-4 were strong, 5 and 7 were weak activators of membrane bound guanylate cyclase. They further displayed different Ca(2+-sensitivities of guanylate cyclase activation, which is half maximal either at a free Ca(2+ around 30 nM (zGCAP1, 2 and 3 or around 400 nM (zGCAP4, 5 and 7. Zebrafish GCAP isoforms showed also differences in their Ca(2+/Mg(2+-dependent conformational changes and in the Ca(2+-dependent monomer-dimer equilibrium. Direct Ca(2+-binding revealed that all zGCAPs bound at least three Ca(2+. The corresponding apparent affinity constants reflect binding of Ca(2+ with high (≤ 100 nM, medium (0.1-5 µM and/or low (≥ 5 µM affinity, but were unique for each zGCAP isoform. Our data indicate a Ca(2+-sensor system in zebrafish rod and cone cells supporting a Ca(2+-relay model of differential zGCAP operation in these cells.

  19. A novel Myb homolog initiates Dictyostelium development by induction of adenylyl cyclase expression

    NARCIS (Netherlands)

    Otsuka, Hideshi; Haastert, Peter J.M. van

    1998-01-01

    Dictyostelium development is induced by starvation. The adenylyl cyclase gene ACA is one of the first genes expressed upon starvation. ACA produces extracellular cAMP that induces chemotaxis, aggregation, and differentiation in neighboring cells. Using insertional mutagenesis we have isolated a muta

  20. Studies on adenosine triphosphate transphosphorylases. Human isoenzymes of adenylate kinase: isolation and physicochemical comparison of the crystalline human ATP-AMP transphosphorylases from muscle and liver.

    Science.gov (United States)

    Kuby, S A; Fleming, G; Frischat, A; Cress, M C; Hamada, M

    1983-02-10

    Procedures are described for the isolation, in crystalline form, of the adenylate kinases from autopsy samples of human muscle and from human liver. Weight average molecular weights were determined by sedimentation equilibrium to be 22,000 (+/- 700) and 25,450 (+/- 160) for the human muscle and liver isoenzymes, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their molecular weights were estimated to be 21,700 and 26,500 for the muscle and liver enzymes, respectively. Both isoenzymes are accordingly monomeric proteins in their native state. Amino acid analyses are reported here for the normal human liver, calf liver, and rabbit liver adenylate kinases and compared with the normal human muscle, calf muscle, and rabbit muscle myokinases. The liver types as a group and the muscle types as a group show a great deal of homology, but some distinct differences are evident between the liver and muscle enzyme groups, especially in the number of residues of His, Pro, half-cystine, and the presence of tryptophan in the liver enzymes. The normal human liver adenylate kinase, as isolated in this report, has proved to be similar in its properties, if not identical, to the adenylate kinase isolated directly from human liver mitochondria (Hamada, M., Sumida, M., Okuda, H., Watanabe, T., Nojima, M., and Kuby, S. A. (1982) J. Biol. Chem. 257, 13120-13128). Therefore, the liver-type adenylate kinase may be considered a mitochondrial type.

  1. Bioinformatics Analysis of Adenylate Cyclase (AC)in Col letotrichum graminicola%禾谷炭疽菌腺苷酸环化酶的生物信息学分析

    Institute of Scientific and Technical Information of China (English)

    韩长志

    2014-01-01

    The typical AC sequence was compared with on-line C.protein databank by Blastp comparison,then the bioinformatics of conserved domain,physicochemical property,cell signal peptide, structure of transmembrane domain and secondary structure was analyzed by SMART and the genetic relationships between typical AC sequence in C.graminicola and the homologous sequence of other species were compared to provide a reference for development and utilization of new medicines with drug target of C.graminicola.The results showed that CgCap1in C.graminicola is a homologous protein with Srv2 in S. cerevisiae.CgCap 1 located in the plasma membrane with conserved CARP domain and isoeletric point with 5~6 range belongs to the sequence of hydrophily,instable protein and without signal peptide.The proportion of βfold is low in the secondary structure.The genetic relationship between CgCap 1 and Colletoreichum is close.%为了给禾谷炭疽菌新靶标药物的研究开发提供参考,以酿酒酵母的典型腺苷酸环化酶(AC)序列为基础,利用炭疽菌属蛋白数据库在线对其进行 Blastp 比对及关键词搜索,通过 SMART 对该菌典型AC 蛋白的保守结构域、理化性质、细胞信号肽、跨膜区结构以及二级结构等生物信息学进行分析,同时比较禾谷炭疽菌中典型 AC 氨基酸序列与其他物种同源序列的遗传关系。结果表明:禾谷炭疽菌中存在一个与酿酒酵母 Srv2同源的序列,命名为 CgCap1腺苷酸环化酶蛋白,该酶蛋白包含有530个氨基酸,基因序列长1906 bp 具有保守 CARP 结构域,等电点蛋白在5~6范围,属亲水性、不稳定蛋白,不含信号肽序列,定位于质膜上,二级结构中的β折叠所占比例较低,与同属于炭疽菌属真菌胶孢炭疽菌、西瓜炭疽菌的亲缘关系较近。

  2. Combinatorial effects of genistein and sex-steroids on the level of cystic fibrosis transmembrane regulator (CFTR), adenylate cyclase (AC) and cAMP in the cervix of ovariectomised rats.

    Science.gov (United States)

    Salleh, Naguib; Ismail, Nurain; Muniandy, Sekaran; Korla, Praveen Kumar; Giribabu, Nelli

    2015-12-01

    The combinatorial effects of genistein and estrogen (E) or estrogen plus progesterone (E+P) on CFTR, AC and cAMP levels in cervix were investigated. Ovariectomised adult female rats received 50 or 100mg/kg/day genistein with E or E followed by E+P [E+(E+P)] for seven consecutive days. Cervixes were harvested and analyzed for CFTR mRNA levels by Real-time PCR. Distribution of AC and CFTR proteins in endocervix were observed by immunohistochemistry. Levels of cAMP were measured by enzyme-immunoassay. Molecular docking predicted interaction between genistein and AC. Our results indicate that levels of CFTR, AC and cAMP in cervix of rats receiving genistein plus E were higher than E-only treatment (pCFTR, AC and cAMP in cervix of E and E+(E+P)-treated rats by genistein could affect the cervical secretory function which could influence the female reproductive processes.

  3. 希金斯炭疽菌腺苷酸环化酶相关蛋白生物信息学分析%Bioinformatics analysis of adenylate cyclase associated protein in Colletotrichum higginsianum

    Institute of Scientific and Technical Information of China (English)

    韩长志

    2014-01-01

    [目的]希金斯炭疽菌可引起菜心、萝卜等十字花科植物的炭疽病,给经济产生巨大损失.目前,炭疽病菌对苯并咪唑类杀菌剂所产生的抗性问题引起了学者广泛关注,有待于开发新的作用靶标的化学药剂.腺苷酸环化酶(AC)在G蛋白信号传导途径上发挥着重要作用.[方法]基于酿酒酵母中Srv2序列,通过Blastp比对及关键词搜索,并利用SMART进行保守结构域分析,同时,通过对该菌中AC相关蛋白序列进行理化性质、信号肽、跨膜结构域等分析.[结果]明确该菌含有2个与Srv2同源的AC相关蛋白CgCap1、CgCap2,就理化性质、二级结构等特征方面而言,CgCap1、CgCap2与Srv2有着较大的差异.此外,通过对Srv2与CgCap1、CgCap2及其同源序列进行比对分析,发现CgCap1、CgCap2序列分别与其同源序列及Srv2的C端、N端具有较大的相似性,推测上述序列分别为希金斯炭疽菌AC的C端和N端.[结论]该研究为深入开展该菌AC的研究打下坚实的理论基础.

  4. 百日咳杆菌腺苷酸环化酶毒素基因的克隆及原核表达%Cloning and Prokaryotic Expression of Bordetella pertussis Adenylate Cyclase Toxin Gene

    Institute of Scientific and Technical Information of China (English)

    石碧珠; 张华捷; 孟民杰; 张庶民

    2010-01-01

    目的 克隆百日咳杆菌腺苷酸环化酶毒素(CyaA,ACT)基因,表达并纯化重组CyaA蛋白.方法 从百日咳杆菌CS株的基因组DNA中PCR扩增CyaA编码基因,克隆人载体pET30a,构建重组原核表达质粒pET30a/cyaA,转化感受态大肠杆菌BL21(DE3),IPTG诱导表达.表达的重组蛋白经8 mol/L尿素变性、透析复性、DEAE阴离子交换柱纯化后,采用Western blot法鉴定其反应原性.结果 重组原核表达质粒pET30a/cyaA经PCR、双酶切及测序证明构建正确;表达的重组蛋白主要以包涵体形式存在,表达量约占菌体总蛋白的20%;纯化的重组蛋白纯度达90%左右,可与全细胞百日咳疫苗和无细胞百日咳疫苗免疫血清结合.结论 已成功克隆了百日咳杆菌cyaA基因,并在大肠杆菌中表达了重组CyaA蛋白,为进一步开展CyaA的应用研究奠定了基础.

  5. Identification of the chlE gene encoding oxygen-independent Mg-protoporphyrin IX monomethyl ester cyclase in cyanobacteria.

    Science.gov (United States)

    Yamanashi, Kaori; Minamizaki, Kei; Fujita, Yuichi

    2015-08-07

    The fifth ring (E-ring) of chlorophyll (Chl) a is produced by Mg-protoporphyrin IX monomethyl ester (MPE) cyclase. There are two evolutionarily unrelated MPE cyclases: oxygen-independent (BchE) and oxygen-dependent (ChlA/AcsF) MPE cyclases. Although ChlA is the sole MPE cyclase in Synechocystis PCC 6803, it is yet unclear whether BchE exists in cyanobacteria. A BLAST search suggests that only few cyanobacteria possess bchE. Here, we report that two bchE candidate genes from Cyanothece strains PCC 7425 and PCC 7822 restore the photosynthetic growth and bacteriochlorophyll production in a bchE-lacking mutant of Rhodobacter capsulatus. We termed these cyanobacterial bchE orthologs "chlE."

  6. Overexpression of guanylate cyclase activating protein 2 in rod photoreceptors in vivo leads to morphological changes at the synaptic ribbon

    OpenAIRE

    Natalia López-del Hoyo; Lucrezia Fazioli; Santiago López-Begines; Laura Fernández-Sánchez; Nicolás Cuenca; Jordi Llorens; Pedro de la Villa; Ana Méndez

    2012-01-01

    Guanylate cyclase activating proteins are EF-hand containing proteins that confer calcium sensitivity to retinal guanylate cyclase at the outer segment discs of photoreceptor cells. By making the rate of cGMP synthesis dependent on the free intracellular calcium levels set by illumination, GCAPs play a fundamental role in the recovery of the light response and light adaptation. The main isoforms GCAP1 and GCAP2 also localize to the synaptic terminal, where their function is not known. Based o...

  7. Comparative effect of methioninyl adenylate on the growth of Salmonella typhimurium and Pseudomonas aeruginosa.

    Science.gov (United States)

    Enouf, J; Laurence, F; Farrugia, G; Blanchard, P; Robert-Gero, M

    1976-10-11

    The bacteriostatic effect of methioninyl adenylate(MAMP)--a specific inhibitor of the enzyme methionyl-tRNA synthetase--was investigated on Salmonella typhimurium and Pseudomonas aeruginosa. 0.1 mM of this molecule added to the culture, inhibits the growth of S. typhimurium. The inhibition is specifically reversible by 0.1 mM L-methionine. In the same conditions even 1-2 mM MAMP has a very slight effect on the growth rate of P. aeruginosa and only during the first two generations. The same observation was made with the two other members of the fluorescens group P.fluorescens and P.putida. The growth rate of P. testosteroni with 1 mM MAMP in the medium is similar to the growth rate of P. aeruginosa but the other member of the acidovorans group P. acidovorans is much more affected by the smae concentration of the inhibitor. --P. multivorans is inhibited by MAMP like P. acidovorans but with a somewhat higher yield at the end of the culture. --MAMP has no effect on P. alcaligenes. The possible reasons for the weak bacteriostatic effect of MAMP on P. aeruginosa were investigated. It was established that the inhibitor enters the cells and is not used as a carbon and energy source. The intracellular methionine concentration in S. typhimurium and in P. aeruginosa is about the same and does not increase when bacteria are cultivated with MAMP. The MTS of the two microorganisms is inhibited by MAMP in vitro to about the same extent. Furthermore the tRNAmet from P. aeruginosa are fully acylated after 3 to 4 generations with this compound. Nevertheless MAMP elicits higher MTS activity in P. aeruginosa and in P. acidovorans after 1 h of incubation. The most striking difference between S. typhimurium and P. aeruginosa is that the intra and extracellular level of 5'phosphodiesterase which degrades MAMP is 10-20 fold higher in the second than in the first species.

  8. Characterization of the acyl-adenylate linked metabolite of mefenamic Acid.

    Science.gov (United States)

    Horng, Howard; Benet, Leslie Z

    2013-03-18

    Mefenamic acid, (MFA), a carboxylic acid-containing nonsteroidal anti-inflammatory drug (NSAID), is metabolized into the chemically reactive conjugates MFA-1-O-acyl-glucuronide (MFA-1-O-G) and MFA-S-acyl-CoA (MFA-CoA), which are both implicated in the formation of MFA-S-acyl-glutathione (MFA-GSH) conjugates, protein-adduct formation, and thus the potential toxicity of the drug. However, current studies suggest that an additional acyl-linked metabolite may be implicated in the formation of MFA-GSH. In the present study, we investigated the ability of MFA to become bioactivated into the acyl-linked metabolite, mefenamyl-adenylate (MFA-AMP). In vitro incubations in rat hepatocytes with MFA (100 μM), followed by LC-MS/MS analyses of extracts, led to the detection of MFA-AMP. In these incubations, the initial rate of MFA-AMP formation was rapid, leveling off at a maximum concentration of 90.1 nM (20 s), while MFA-GSH formation increased linearly, reaching a concentration of 1.7 μM after 60 min of incubation. In comparison, MFA-CoA was undetectable in incubation extracts until the 4 min time point, achieving a concentration of 45.6 nM at the 60 min time point, and MFA-1-O-G formation was linear, attaining a concentration of 42.2 μM after 60 min of incubation. In vitro incubation in buffer with the model nucleophile glutathione (GSH) under physiological conditions showed MFA-AMP to be reactive toward GSH, but 11-fold less reactive than MFA-CoA, while MFA-1-O-G exhibited little reactivity. However, in the presence of glutathione-S-transferase (GST), MFA-AMP mediated formation of MFA-GSH increased 6-fold, while MFA-CoA mediated formation of MFA-GSH only increased 1.4-fold. Collectively, in addition to the MFA-1-O-G, these results demonstrate that mefenamic acid does become bioactivated by acyl-CoA synthetase enzyme(s) in vitro in rat hepatocytes into the reactive transacylating derivatives MFA-AMP and MFA-CoA, both of which contribute to the transacylation of GSH and may

  9. Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding surface.

    Science.gov (United States)

    Williamson, Adele; Rothweiler, Ulli; Leiros, Hanna Kirsti Schrøder

    2014-11-01

    DNA ligases are a structurally diverse class of enzymes which share a common catalytic core and seal breaks in the phosphodiester backbone of double-stranded DNA via an adenylated intermediate. Here, the structure and activity of a recombinantly produced ATP-dependent DNA ligase from the bacterium Psychromonas sp. strain SP041 is described. This minimal-type ligase, like its close homologues, is able to ligate singly nicked double-stranded DNA with high efficiency and to join cohesive-ended and blunt-ended substrates to a more limited extent. The 1.65 Å resolution crystal structure of the enzyme-adenylate complex reveals no unstructured loops or segments, and suggests that this enzyme binds the DNA without requiring full encirclement of the DNA duplex. This is in contrast to previously characterized minimal DNA ligases from viruses, which use flexible loop regions for DNA interaction. The Psychromonas sp. enzyme is the first structure available for the minimal type of bacterial DNA ligases and is the smallest DNA ligase to be crystallized to date.

  10. Receptor-type guanylate cyclase is required for carbon dioxide sensation by Caenorhabditis elegans.

    Science.gov (United States)

    Hallem, Elissa A; Spencer, W Clay; McWhirter, Rebecca D; Zeller, Georg; Henz, Stefan R; Rätsch, Gunnar; Miller, David M; Horvitz, H Robert; Sternberg, Paul W; Ringstad, Niels

    2011-01-04

    CO(2) is both a critical regulator of animal physiology and an important sensory cue for many animals for host detection, food location, and mate finding. The free-living soil nematode Caenorhabditis elegans shows CO(2) avoidance behavior, which requires a pair of ciliated sensory neurons, the BAG neurons. Using in vivo calcium imaging, we show that CO(2) specifically activates the BAG neurons and that the CO(2)-sensing function of BAG neurons requires TAX-2/TAX-4 cyclic nucleotide-gated ion channels and the receptor-type guanylate cyclase GCY-9. Our results delineate a molecular pathway for CO(2) sensing and suggest that activation of a receptor-type guanylate cyclase is an evolutionarily conserved mechanism by which animals detect environmental CO(2).

  11. The metabolic/pH sensor soluble adenylyl cyclase is a tumor suppressor protein

    Science.gov (United States)

    Ramos-Espiritu, Lavoisier; Diaz, Ana; Nardin, Charlee; Saviola, Anthony J.; Shaw, Fiona; Plitt, Tamar; Yang, Xia; Wolchok, Jedd; Pirog, Edyta C.; Desman, Garrett; Sboner, Andrea; Zhang, Tuo; Xiang, Jenny; Merghoub, Taha; Levin, Lonny R.; Buck, Jochen; Zippin, Jonathan H.

    2016-01-01

    cAMP signaling pathways can both stimulate and inhibit the development of cancer; however, the sources of cAMP important for tumorigenesis remain poorly understood. Soluble adenylyl cyclase (sAC) is a non-canonical, evolutionarily conserved, nutrient- and pH-sensing source of cAMP. sAC has been implicated in the metastatic potential of certain cancers, and it is differentially localized in human cancers as compared to benign tissues. We now show that sAC expression is reduced in many human cancers. Loss of sAC increases cellular transformation in vitro and malignant progression in vivo. These data identify the metabolic/pH sensor soluble adenylyl cyclase as a previously unappreciated tumor suppressor protein. PMID:27323809

  12. Elevation of lutein content in tomato: a biochemical tug-of-war between lycopene cyclases.

    Science.gov (United States)

    Giorio, Giovanni; Yildirim, Arzu; Stigliani, Adriana Lucia; D'Ambrosio, Caterina

    2013-11-01

    Lutein is becoming increasingly important in preventive medicine due to its possible role in maintaining good vision and in preventing age-related maculopathy. Average daily lutein intake in developed countries is often below suggested daily consumption levels, and lutein supplementation could be beneficial. Lutein is also valuable in the food and feed industries and is emerging in nutraceutical and pharmaceutical markets. Currently, lutein is obtained at high cost from marigold petals, and synthesis alternatives are thus desirable. Tomato constitutes a promising starting system for production as it naturally accumulates high levels of lycopene. To develop tomato for lutein synthesis, the tomato Red Setter cultivar was transformed with the tomato lycopene ε-cyclase-encoding gene under the control of a constitutive promoter, and the HighDelta (HD) line, characterised by elevated lutein and δ-carotene content in ripe fruits, was selected. HD was crossed to the transgenic HC line and to RS(B) with the aim of converting all residual fruit δ-carotene to lutein. Fruits of both crosses were enriched in lutein and presented unusual carotenoid profiles. The unique genetic background of the crosses used in this study permitted an unprecedented analysis of the role and regulation of the lycopene cyclase enzymes in tomato. A new defined biochemical index, the relative cyclase activity ratio, was used to discern post-transcriptional regulation of cyclases, and will help in the study of carotenoid biosynthesis in photosynthetic plant species and particularly in those, like tomato, that have been domesticated for the production of food, feed or useful by-products.

  13. Bicarbonate-responsive “soluble” adenylyl cyclase defines a nuclear cAMP microdomain

    Science.gov (United States)

    Zippin, Jonathan H.; Farrell, Jeanne; Huron, David; Kamenetsky, Margarita; Hess, Kenneth C.; Fischman, Donald A.; Levin, Lonny R.; Buck, Jochen

    2004-01-01

    Bicarbonate-responsive “soluble” adenylyl cyclase resides, in part, inside the mammalian cell nucleus where it stimulates the activity of nuclear protein kinase A to phosphorylate the cAMP response element binding protein (CREB). The existence of this complete and functional, nuclear-localized cAMP pathway establishes that cAMP signals in intracellular microdomains and identifies an alternate pathway leading to CREB activation. PMID:14769862

  14. Bicarbonate-responsive “soluble” adenylyl cyclase defines a nuclear cAMP microdomain

    OpenAIRE

    2004-01-01

    Bicarbonate-responsive “soluble” adenylyl cyclase resides, in part, inside the mammalian cell nucleus where it stimulates the activity of nuclear protein kinase A to phosphorylate the cAMP response element binding protein (CREB). The existence of this complete and functional, nuclear-localized cAMP pathway establishes that cAMP signals in intracellular microdomains and identifies an alternate pathway leading to CREB activation.

  15. Interaction Of GCAP1 With Retinal Guanylyl Cyclase And Calcium: Sensitivity to Fatty Acylation

    Directory of Open Access Journals (Sweden)

    Igor V. Peshenko

    2012-02-01

    Full Text Available Guanylyl cyclase activating proteins (GCAP1 are calcium/magnesium binding proteins within neuronal calcium sensor proteins group (NCS of the EF-hand proteins superfamily. GCAPs activate retinal guanylyl cyclase (RetGC in vertebrate photoreceptors in response to light-dependent fall of the intracellular free Ca2+ concentrations. GCAPs consist of four EF-hand domains and contain N-terminal fatty acylated glycine, which in GCAP1 is required for the normal activation of RetGC. We analyzed the effects of a substitution prohibiting N-myristoylation (Gly2 → Ala on the ability of the recombinant GCAP1 to co-localize with its target enzyme when heterologously expressed in HEK293 cells. We also compared Ca2+ binding and RetGC-activating properties of the purified non-acylated G2A mutant and C14:0 acylated GCAP1 in vitro. The G2A GCAP1 expressed with a C-terminal GFP tag was able to co-localize with the cyclase, albeit less efficiently than the wild type, but much less effectively stimulated cyclase activity in vitro. Ca2+ binding isotherm of the G2A GCAP1 was slightly shifted toward higher free Ca2+ concentrations and so was Ca2+ sensitivity of RetGC reconstituted with the non-acylated mutant. At the same time, myristoylation had little effect on the high-affinity Ca2+-binding in the EF-hand that is proximal to the myristoyl residue in the three-dimensional GCAP1 structure. These data indicate that the N-terminal fatty acyl group may alter the activity of EF-hands in the distal portion of the GCAP1 molecule via presently unknown intramolecular mechanism.

  16. Cyclase-associated proteins: CAPacity for linking signal transduction and actin polymerization.

    Science.gov (United States)

    Hubberstey, Andrew V; Mottillo, Emilio P

    2002-04-01

    Many extracellular signals elicit changes in the actin cytoskeleton, which are mediated through an array of signaling proteins and pathways. One family of proteins that plays a role in regulating actin remodeling in response to cellular signals are the cyclase-associated proteins (CAPs). CAPs are highly conserved monomeric actin binding proteins present in a wide range of organisms including yeast, fly, plants, and mammals. The original CAP was isolated as a component of the Saccharomyces cerevisiae adenylyl cyclase complex that serves as an effector of Ras during nutritional signaling. CAPs are multifunctional molecules that contain domains involved in actin binding, adenylyl cyclase association in yeast, SH3 binding, and oligomerization. Genetic studies in yeast have implicated CAPs in vesicle trafficking and endocytosis. CAPs play a developmental role in multicellular organisms, and studies of Drosophila have illuminated the importance of the actin cytoskeleton during eye development and in establishing oocyte polarity. This review will highlight the critical structural and functional domains of CAPs, describe recent studies that have implied important roles for these proteins in linking cell signaling with actin polymerization, and highlight their roles in vesicle trafficking and development.

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

    Science.gov (United States)

    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.

  18. Identification of photoactivated adenylyl cyclases in Naegleria australiensis and BLUF-containing protein in Naegleria fowleri.

    Science.gov (United States)

    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.

  19. Genetic reduction of chronic muscle pain in mice lacking calcium/calmodulin-stimulated adenylyl cyclases

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    Petrovic Michele J

    2006-02-01

    Full Text Available Abstract Background The Ca2+/calmodulin-stimulated adenylyl cyclase (AC isoforms AC1 and AC8, couple NMDA receptor activation to cAMP signaling pathways in neurons and are important for development, learning and memory, drug addiction and persistent pain. AC1 and AC8 in the anterior cingulate cortex (ACC and the spinal cord were previously shown to be important in subcutaneous inflammatory pain. Muscle pain is different from cutaneous pain in its characteristics as well as conducting fibers. Therefore, we conducted the present work to test the role of AC1 and AC8 in both acute persistent and chronic muscle pain. Results Using an acute persistent inflammatory muscle pain model, we found that the behavioral nociceptive responses of both the late phase of acute muscle pain and the chronic muscle inflammatory pain were significantly reduced in AC1 knockout (KO and AC1&8 double knockout (DKO mice. Activation of other adenylyl cyclases in these KO mice by microinjection of forskolin into the ACC or spinal cord, but not into the peripheral tissue, rescued the behavioral nociceptive responses. Additionally, intra-peritoneal injection of an AC1 inhibitor significantly reduced behavioral responses in both acute persistent and chronic muscle pain. Conclusion The results of the present study demonstrate that neuronal Ca2+/calmodulin-stimulated adenylyl cyclases in the ACC and spinal cord are important for both late acute persistent and chronic inflammatory muscle pain.

  20. Soluble guanylate cyclase α1-deficient mice: a novel murine model for primary open angle glaucoma.

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    Emmanuel S Buys

    Full Text Available Primary open angle glaucoma (POAG is a leading cause of blindness worldwide. The molecular signaling involved in the pathogenesis of POAG remains unknown. Here, we report that mice lacking the α1 subunit of the nitric oxide receptor soluble guanylate cyclase represent a novel and translatable animal model of POAG, characterized by thinning of the retinal nerve fiber layer and loss of optic nerve axons in the context of an open iridocorneal angle. The optic neuropathy associated with soluble guanylate cyclase α1-deficiency was accompanied by modestly increased intraocular pressure and retinal vascular dysfunction. Moreover, data from a candidate gene association study suggests that a variant in the locus containing the genes encoding for the α1 and β1 subunits of soluble guanylate cyclase is associated with POAG in patients presenting with initial paracentral vision loss, a disease subtype thought to be associated with vascular dysregulation. These findings provide new insights into the pathogenesis and genetics of POAG and suggest new therapeutic strategies for POAG.

  1. Microarray evidence of glutaminyl cyclase gene expression in melanoma: implications for tumor antigen specific immunotherapy

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    Gillis John

    2006-07-01

    Full Text Available Abstract Background In recent years encouraging progress has been made in developing vaccine treatments for cancer, particularly with melanoma. However, the overall rate of clinically significant results has remained low. The present research used microarray datasets from previous investigations to examine gene expression patterns in cancer cell lines with the goal of better understanding the tumor microenvironment. Methods Principal Components Analyses with Promax rotational transformations were carried out with 90 cancer cell lines from 3 microarray datasets, which had been made available on the internet as supplementary information from prior publications. Results In each of the analyses a well defined melanoma component was identified that contained a gene coding for the enzyme, glutaminyl cyclase, which was as highly expressed as genes from a variety of well established biomarkers for melanoma, such as MAGE-3 and MART-1, which have frequently been used in clinical trials of melanoma vaccines. Conclusion Since glutaminyl cyclase converts glutamine and glutamic acid into a pyroglutamic form, it may interfere with the tumor destructive process of vaccines using peptides having glutamine or glutamic acid at their N-terminals. Finding ways of inhibiting the activity of glutaminyl cyclase in the tumor microenvironment may help to increase the effectiveness of some melanoma vaccines.

  2. Crystallization and preliminary X-ray diffraction studies of the glutaminyl cyclase from Carica papaya latex

    Energy Technology Data Exchange (ETDEWEB)

    Azarkan, Mohamed [Laboratoire de Chimie Générale I, Faculté de Médecine-ULB CP609, 808 Route de Lennik, B-1070 Brussels (Belgium); Clantin, Bernard; Bompard, Coralie [CNRS-UMR 8525, Institut de Biologie de Lille, BP 477, 1 Rue du Professeur Calmette, F-59021 Lille (France); Belrhali, Hassan [EMBL Grenoble Outstation, 6 Rue Jules Horowitz, BP 181, F-38042 Grenoble CEDEX 9 (France); Baeyens-Volant, Danielle [Laboratoire de Chimie Générale I, Faculté de Médecine-ULB CP609, 808 Route de Lennik, B-1070 Brussels (Belgium); Looze, Yvan [Laboratoire de Chimie Générale, Institut de Pharmacie-ULB CP206/04, Boulevard du Triomphe, B-1050 Brussels (Belgium); Villeret, Vincent, E-mail: vincent.villeret@ibl.fr [CNRS-UMR 8525, Institut de Biologie de Lille, BP 477, 1 Rue du Professeur Calmette, F-59021 Lille (France); Wintjens, René, E-mail: vincent.villeret@ibl.fr [Laboratoire de Chimie Générale, Institut de Pharmacie-ULB CP206/04, Boulevard du Triomphe, B-1050 Brussels (Belgium); Laboratoire de Chimie Générale I, Faculté de Médecine-ULB CP609, 808 Route de Lennik, B-1070 Brussels (Belgium)

    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.

  3. The first structure of a bacterial diterpene cyclase: CotB2.

    Science.gov (United States)

    Janke, Ronja; Görner, Christian; Hirte, Max; Brück, Thomas; Loll, Bernhard

    2014-06-01

    Sesquiterpenes and diterpenes are a diverse class of secondary metabolites that are predominantly derived from plants and some prokaryotes. The properties of these natural products encompass antitumor, antibiotic and even insecticidal activities. Therefore, they are interesting commercial targets for the chemical and pharmaceutical industries. Owing to their structural complexity, these compounds are more efficiently accessed by metabolic engineering of microbial systems than by chemical synthesis. This work presents the first crystal structure of a bacterial diterpene cyclase, CotB2 from the soil bacterium Streptomyces melanosporofaciens, at 1.64 Å resolution. CotB2 is a diterpene cyclase that catalyzes the cyclization of the linear geranylgeranyl diphosphate to the tricyclic cyclooctat-9-en-7-ol. The subsequent oxidation of cyclooctat-9-en-7-ol by two cytochrome P450 monooxygenases leads to bioactive cyclooctatin. Plasticity residues that decorate the active site of CotB2 have been mutated, resulting in alternative monocyclic, dicyclic and tricyclic compounds that show bioactivity. These new compounds shed new light on diterpene cyclase reaction mechanisms. Furthermore, the product of mutant CotB2(W288G) produced the new antibiotic compound (1R,3E,7E,11S,12S)-3,7,18-dolabellatriene, which acts specifically against multidrug-resistant Staphylococcus aureus. This opens a sustainable route for the industrial-scale production of this bioactive compound.

  4. Effects of dopamine on adenylyl cyclase activity and amylase secretion in rat parotid tissue.

    Science.gov (United States)

    Hatta, S; Amemiya, N; Takemura, H; Ohshika, H

    1995-06-01

    Several previous studies have shown that dopamine causes amylase secretion from rat parotid tissue. However, the mechanism of this dopamine action is still unclear. The present study was designed to characterize dopamine action in rat parotid gland tissue by examining the effects of dopamine on cyclic AMP accumulation, adenylyl cyclase activity, and amylase release. Dopamine significantly enhanced accumulation of cyclic AMP in parotid slices and stimulated adenylyl cyclase activity in parotid membrane preparations. It also significantly stimulated amylase release from parotid slices. The stimulatory effects of dopamine on cyclic AMP accumulation, adenylyl cyclase activity, and amylase release were effectively blocked with propranolol, a beta-adrenergic antagonist, but not by either SCH 23390, a preferential D1 antagonist, or butaclamol, a preferential D2 antagonist. No substantial specific binding sites for D1 receptors were detectable by [3H]SCH 23390 binding in parotid membranes. These results suggest that the stimulatory effect of dopamine on amylase secretion in rat parotid tissue is not mediated through specific D1 dopamine receptors but rather through beta-adrenergic receptors.

  5. Trichomonas transmembrane cyclases result from massive gene duplication and concomitant development of pseudogenes.

    Directory of Open Access Journals (Sweden)

    Jike Cui

    2010-08-01

    Full Text Available Trichomonas vaginalis has an unusually large genome (approximately 160 Mb encoding approximately 60,000 proteins. With the goal of beginning to understand why some Trichomonas genes are present in so many copies, we characterized here a family of approximately 123 Trichomonas genes that encode transmembrane adenylyl cyclases (TMACs.The large family of TMACs genes is the result of recent duplications of a small set of ancestral genes that appear to be unique to trichomonads. Duplicated TMAC genes are not closely associated with repetitive elements, and duplications of flanking sequences are rare. However, there is evidence for TMAC gene replacements by homologous recombination. A high percentage of TMAC genes (approximately 46% are pseudogenes, as they contain stop codons and/or frame shifts, or the genes are truncated. Numerous stop codons present in the genome project G3 strain are not present in orthologous genes of two other Trichomonas strains (S1 and B7RC2. Each TMAC is composed of a series of N-terminal transmembrane helices and a single C-terminal cyclase domain that has adenylyl cyclase activity. Multiple TMAC genes are transcribed by Trichomonas cloned by limiting dilution.We conclude that one reason for the unusually large genome of Trichomonas is the presence of unstable families of genes such as those encoding TMACs that are undergoing massive gene duplication and concomitant development of pseudogenes.

  6. Laboratory Evaluation of Adenylate Energy Charge as a Test for Stress in Mytilus edulis and Nephtys incisa Treated with Dredged Material.

    Science.gov (United States)

    1985-02-01

    concentrations of three adenine nucleotides, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP), which are...that all trace metals but iron were eliminated and the concentration of the vitamins thiamin and B12 were doubled. Adenylate Extraction 13. The adductor

  7. Detection of somatic coliphages through a bioluminescence assay measuring phage mediated release of adenylate kinase and adenosine 5'-triphosphate.

    Science.gov (United States)

    Guzmán Luna, Carolina; Costán-Longares, Ana; Lucena, Francisco; Jofre, Joan

    2009-10-01

    The feasibility of detecting somatic coliphages by phage infection of Escherichia coli WG5 and measurement of phage propagation by the lysis mediated release of the bacterial host adenylate kinase (AK) and adenosine 5'-triphosphate (ATP) detected by a bioluminescent signal was evaluated. After 2h of incubation, all cultures infected with reference bacteriophage phiX174 showed a significant increase in the bioluminescent signal, even with number of phages as low as less of 10 plaque forming units (PFU). Naturally occurring somatic coliphages ensured a significant bioluminescent signal after 3h of infection when >10 PFU were inoculated. These results indicate that an easy and reliable method to detect low numbers of coliphages in less than 3h is feasible.

  8. The cyclase-associated protein CAP as regulator of cell polarity and cAMP signaling in Dictyostelium.

    Science.gov (United States)

    Noegel, Angelika A; Blau-Wasser, Rosemarie; Sultana, Hameeda; Müller, Rolf; Israel, Lars; Schleicher, Michael; Patel, Hitesh; Weijer, Cornelis J

    2004-02-01

    Cyclase-associated protein (CAP) is an evolutionarily conserved regulator of the G-actin/F-actin ratio and, in yeast, is involved in regulating the adenylyl cyclase activity. We show that cell polarization, F-actin organization, and phototaxis are altered in a Dictyostelium CAP knockout mutant. Furthermore, in complementation assays we determined the roles of the individual domains in signaling and regulation of the actin cytoskeleton. We studied in detail the adenylyl cyclase activity and found that the mutant cells have normal levels of the aggregation phase-specific adenylyl cyclase and that receptor-mediated activation is intact. However, cAMP relay that is responsible for the generation of propagating cAMP waves that control the chemotactic aggregation of starving Dictyostelium cells was altered, and the cAMP-induced cGMP production was significantly reduced. The data suggest an interaction of CAP with adenylyl cyclase in Dictyostelium and an influence on signaling pathways directly as well as through its function as a regulatory component of the cytoskeleton.

  9. Adenylyl cyclase 3/adenylyl cyclase-associated protein 1 (CAP1) complex mediates the anti-migratory effect of forskolin in pancreatic cancer cells.

    Science.gov (United States)

    Quinn, Sierra N; Graves, Sarai H; Dains-McGahee, Clayton; Friedman, Emilee M; Hassan, Humma; Witkowski, Piotr; Sabbatini, Maria E

    2017-04-01

    Pancreatic cancer is one of the most lethal human malignancies. A better understanding of the intracellular mechanism of migration and invasion is urgently needed to develop treatment that will suppress metastases and improve overall survival. Cyclic adenosine monophosphate (cyclic AMP) is a second messenger that has shown to regulate migration and invasion of pancreatic cancer cells. The rise of cyclic AMP suppressed migration and invasion of pancreatic ductal adenocarcinoma cells. Cyclic AMP is formed from cytosolic ATP by the enzyme adenylyl cyclase (AC). There are ten isoforms of ACs; nine are anchored in the plasma membrane and one is soluble. What remains unknown is the extent to which the expression of transmembrane AC isoforms is both modified in pancreatic cancer and mediates the inhibitory effect of forskolin on cell motility. Using real-time PCR analysis, ADCY3 was found to be highly expressed in pancreatic tumor tissues, resulting in a constitutive increase in cyclic AMP levels. On the other hand, ADCY2 was down-regulated. Migration, invasion, and filopodia formation in two different pancreatic adenocarcinoma cell lines, HPAC and PANC-1 deficient in AC1 or AC3, were studied. We found that AC3, upon stimulation with forskolin, enhanced cyclic AMP levels and inhibited cell migration and invasion. Unlikely to be due to a cytotoxic effect, the inhibitory effects of forskolin involved the quick formation of AC3/adenylyl cyclase-associated protein 1 (CAP1)/G-actin complex, which inhibited filopodia formation and cell motility. Using Western blotting analysis, forskolin, through AC3 activation, caused phosphorylation of CREB, but not ERK. The effect of CREB phosphorylation is likely to be associated with long-term signaling changes. © 2016 Wiley Periodicals, Inc.

  10. Association of yeast adenylyl cyclase with cyclase-associated protein CAP forms a second Ras-binding site which mediates its Ras-dependent activation.

    Science.gov (United States)

    Shima, F; Okada, T; Kido, M; Sen, H; Tanaka, Y; Tamada, M; Hu, C D; Yamawaki-Kataoka, Y; Kariya, K; Kataoka, T

    2000-01-01

    Posttranslational modification, in particular farnesylation, of Ras is crucial for activation of Saccharomyces cerevisiae adenylyl cyclase (CYR1). Based on the previous observation that association of CYR1 with cyclase-associated protein (CAP) is essential for its activation by posttranslationally modified Ras, we postulated that the associated CAP might contribute to the formation of a Ras-binding site of CYR1, which mediates CYR1 activation, other than the primary Ras-binding site, the leucine-rich repeat domain. Here, we observed a posttranslational modification-dependent association of Ras with a complex between CAP and CYR1 C-terminal region. When CAP mutants defective in Ras signaling but retaining the CYR1-binding activity were isolated by screening of a pool of randomly mutagenized CAP, CYR1 complexed with two of the obtained three mutants failed to be activated efficiently by modified Ras and exhibited a severely impaired ability to bind Ras, providing a genetic evidence for the importance of the physical association with Ras at the second Ras-binding site. On the other hand, CYR1, complexed with the other CAP mutant, failed to be activated by Ras but exhibited a greatly enhanced binding to Ras. Conversely, a Ras mutant E31K, which exhibits a greatly enhanced binding to the CYR1-CAP complex, failed to activate CYR1 efficiently. Thus, the strength of interaction at the second Ras-binding site appears to be a critical determinant of CYR1 regulation by Ras: too-weak and too-strong interactions are both detrimental to CYR1 activation. These results, taken together with those obtained with mammalian Raf, suggest the importance of the second Ras-binding site in effector regulation.

  11. Multifunctional oxidosqualene cyclases and cytochrome P450 involved in the biosynthesis of apple fruit triterpenic acids.

    Science.gov (United States)

    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.

  12. Structure, signaling mechanism and regulation of the natriuretic peptide receptor guanylate cyclase.

    Energy Technology Data Exchange (ETDEWEB)

    Misono, K. S.; Philo, J. S.; Arakawa, T.; Ogata, C. M.; Qiu, Y.; Ogawa, H.; Young, H. S. (Biosciences Division); (Univ. of Nevada); (Alliance Protein Labs.)

    2011-06-01

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

  13. Identification of Glutaminyl Cyclase Genes Involved in Pyroglutamate Modification of Fungal Lignocellulolytic Enzymes

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    Vincent W. Wu

    2017-01-01

    Full Text Available The breakdown of plant biomass to simple sugars is essential for the production of second-generation biofuels and high-value bioproducts. Currently, enzymes produced from filamentous fungi are used for deconstructing plant cell wall polysaccharides into fermentable sugars for biorefinery applications. A post-translational N-terminal pyroglutamate modification observed in some of these enzymes occurs when N-terminal glutamine or glutamate is cyclized to form a five-membered ring. This modification has been shown to confer resistance to thermal denaturation for CBH-1 and EG-1 cellulases. In mammalian cells, the formation of pyroglutamate is catalyzed by glutaminyl cyclases. Using the model filamentous fungus Neurospora crassa, we identified two genes (qc-1 and qc-2 that encode proteins homologous to mammalian glutaminyl cyclases. We show that qc-1 and qc-2 are essential for catalyzing the formation of an N-terminal pyroglutamate on CBH-1 and GH5-1. CBH-1 and GH5-1 produced in a Δqc-1 Δqc-2 mutant, and thus lacking the N-terminal pyroglutamate modification, showed greater sensitivity to thermal denaturation, and for GH5-1, susceptibility to proteolytic cleavage. QC-1 and QC-2 are endoplasmic reticulum (ER-localized proteins. The pyroglutamate modification is predicted to occur in a number of additional fungal proteins that have diverse functions. The identification of glutaminyl cyclases in fungi may have implications for production of lignocellulolytic enzymes, heterologous expression, and biotechnological applications revolving around protein stability.

  14. Identification of Glutaminyl Cyclase Genes Involved in Pyroglutamate Modification of Fungal Lignocellulolytic Enzymes

    Science.gov (United States)

    Wu, Vincent W.; Dana, Craig M.; Iavarone, Anthony T.; Clark, Douglas S.

    2017-01-01

    ABSTRACT The breakdown of plant biomass to simple sugars is essential for the production of second-generation biofuels and high-value bioproducts. Currently, enzymes produced from filamentous fungi are used for deconstructing plant cell wall polysaccharides into fermentable sugars for biorefinery applications. A post-translational N-terminal pyroglutamate modification observed in some of these enzymes occurs when N-terminal glutamine or glutamate is cyclized to form a five-membered ring. This modification has been shown to confer resistance to thermal denaturation for CBH-1 and EG-1 cellulases. In mammalian cells, the formation of pyroglutamate is catalyzed by glutaminyl cyclases. Using the model filamentous fungus Neurospora crassa, we identified two genes (qc-1 and qc-2) that encode proteins homologous to mammalian glutaminyl cyclases. We show that qc-1 and qc-2 are essential for catalyzing the formation of an N-terminal pyroglutamate on CBH-1 and GH5-1. CBH-1 and GH5-1 produced in a Δqc-1 Δqc-2 mutant, and thus lacking the N-terminal pyroglutamate modification, showed greater sensitivity to thermal denaturation, and for GH5-1, susceptibility to proteolytic cleavage. QC-1 and QC-2 are endoplasmic reticulum (ER)-localized proteins. The pyroglutamate modification is predicted to occur in a number of additional fungal proteins that have diverse functions. The identification of glutaminyl cyclases in fungi may have implications for production of lignocellulolytic enzymes, heterologous expression, and biotechnological applications revolving around protein stability. PMID:28096492

  15. Inhibition of adenylyl and guanylyl cyclase isoforms by the antiviral drug foscarnet.

    Science.gov (United States)

    Kudlacek, O; Mitterauer, T; Nanoff, C; Hohenegger, M; Tang, W J; Freissmuth, M; Kleuss, C

    2001-02-02

    The pyrophosphate (PP(i)) analog foscarnet inhibits viral DNA-polymerases and is used to treat cytomegalovirus and human immunodeficiency vius infections. Nucleotide cyclases and DNA-polymerases catalyze analogous reactions, i.e. a phosphodiester bond formation, and have similar topologies in their active sites. Inhibition by foscarnet of adenylyl cyclase isoforms was therefore tested with (i) purified catalytic domains C1 and C2 of types I and VII (IC1 and VIIC1) and of type II (IIC2) and (ii) membrane-bound holoenzymes (from mammalian tissues and types I, II, and V heterologously expressed in Sf9 cell membranes). Foscarnet was more potent than PP(i) in suppressing forskolin-stimulated catalysis by both, IC1/IIC2 and VIIC1/IIC2. Stimulation of VIIC1/IIC2 by Galpha(s) relieved the inhibition by foscarnet but not that by PP(i). The IC(50) of foscarnet on membrane-bound adenylyl cyclases also depended on their mode of regulation. These findings predict that receptor-dependent cAMP formation is sensitive to inhibition by foscarnet in some, but not all, cells. This was verified with two cell lines; foscarnet blocked cAMP accumulation after A(2A)-adenosine receptor stimulation in PC12 but not in HEK-A(2A) cells. Foscarnet also inhibited soluble and, to a lesser extent, particulate guanylyl cylase. Thus, foscarnet interferes with the generation of cyclic nucleotides, an effect which may give rise to clinical side effects. The extent of inhibition varies with the enzyme isoform and with the regulatory input.

  16. From Kinase to Cyclase: An Unusual Example of Catalytic Promiscuity Modulated by Metal Switching

    OpenAIRE

    Sánchez-Moreno, Israel; Iturrate Montoya, Laura; Martín-Hoyos, Rocio; Jimeno, M. Luisa; Mena, Montaña; Bastida, Ágatha; García-Junceda, Eduardo

    2009-01-01

    “This is the pre-peer reviewed version of the following article: Sánchez-Moreno, I., Iturrate, L., Martín-Hoyos, R., Jimeno, M. L., Mena, M., Bastida, A. and García-Junceda, E. (2009) From Kinase to Cyclase: An Unusual Example of Catalytic Promiscuity Modulated by Metal Switching. ChemBioChem. 10, 225-229, which has been published in final form at http://www3.interscience.wiley.com/journal/121544668/abstract?CRETRY=1&SRETRY=0.”

  17. A kinase-anchoring proteins and adenylyl cyclase in cardiovascular physiology and pathology.

    Science.gov (United States)

    Efendiev, Riad; Dessauer, Carmen W

    2011-10-01

    3'-5'-Cyclic adenosine monophosphate (cAMP), generated by adenylyl cyclase (AC), serves as a second messenger in signaling pathways regulating many aspects of cardiac physiology, including contraction rate and action potential duration, and in the pathophysiology of hypertrophy and heart failure. A kinase-anchoring proteins localize the effect of cAMP in space and time by organizing receptors, AC, protein kinase A, and other components of the cAMP cascade into multiprotein complexes. In this review, we discuss how the interaction of A kinase-anchoring proteins with distinct AC isoforms affects cardiovascular physiology.

  18. Adenylyl Cyclase-Associated Protein 1 in the Development of Head and Neck Squamous Cell Carcinomas.

    Science.gov (United States)

    Kakurina, G V; Kondakova, I V; Cheremisina, O V; Shishkin, D A; Choinzonov, E L

    2016-03-01

    We compared the content of adenylyl cyclase-associated protein 1 (CAP1) in the blood and tissues of patients with head and neck squamous cell carcinomas (with and without regional metastases), patients with chronic inflammatory diseases aggravated by laryngeal and laryngopharyngeal dysplasia, and healthy individuals. The data suggest that serum CAP1 concentration correlated with the depth of primary tumor invasion and the presence of regional metastases. In cancer patients, the serum level of CAP1 was lower than in patients with laryngeal and laryngopharyngeal dysplasia, which can be of importance for differential and timely diagnostics of malignant tumors.

  19. Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter

    Energy Technology Data Exchange (ETDEWEB)

    Morris, D.I.; Robbins, J.D.; Ruoho, A.E.; Sutkowski, E.M.; Seamon, K.B. (Division of Biochemistry and Biophysics, Food and Drug Administration, Bethesda, MD (USA))

    1991-07-15

    Two photolabels, N-(3-(4-azido-3-125I-phenyl)-propionamide)-6- aminoethylcarbamylforskolin(125I-6-AIPP-Fsk) and N-(3-(4-azido-3-125I-phenyl)propionamide)-7-aminoethylcarbamyl-7- desacetylforskolin (125I-7-AIPP-Fsk) were synthesized with specific activities of 2200 Ci/mmol and used to label adenylyl cyclase and the glucose transporter. The affinities of the photolabels for adenylyl cyclase were determined by their inhibition of (3H)forskolin binding to bovine brain membranes. 6-AIPP-Fsk and 7-AIPP-Fsk inhibited (3H)forskolin binding with IC50 values of 15 nM and 200 nM, respectively. 125I-6-AIPP-Fsk labeled a 115-kDa protein in control and GTP {gamma} S-preactivated bovine brain membranes. This labeling was inhibited by forskolin but not by 1,9-dideoxyforskolin or cytochalasin B. 125I-6-AIPP-Fsk labeling of partially purified adenylyl cyclase was inhibited by forskolin but not by 1,9-dideoxyforskolin. 125I-7-AIPP-Fsk specifically labeled a 45-kDa protein and not a 115-kDa protein in control and GTP {gamma} S-preactivated brain membranes. This labeling was inhibited by forskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose but not cytochalasin E or L-glucose. Human erythrocyte membranes were photolyzed with 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk. 125I-7-AIPP-Fsk, but not 125I-6-AIPP-Fsk, strongly labeled a broad 45-70-kDa band. Forskolin, 7-bromoacetyl-7-desacetylforskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose, but not cytochalasin E or L-glucose, inhibited 125I-7-AIPP-Fsk labeling of the 45-70-kDa band. 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk are high affinity photolabels with specificity for adenylyl cyclase and the glucose transporter, respectively.

  20. Structural basis for olivetolic acid formation by a polyketide cyclase from Cannabis sativa.

    Science.gov (United States)

    Yang, Xinmei; Matsui, Takashi; Kodama, Takeshi; Mori, Takahiro; Zhou, Xiaoxi; Taura, Futoshi; Noguchi, Hiroshi; Abe, Ikuro; Morita, Hiroyuki

    2016-03-01

    In polyketide biosynthesis, ring formation is one of the key diversification steps. Olivetolic acid cyclase (OAC) from Cannabis sativa, involved in cannabinoid biosynthesis, is the only known plant polyketide cyclase. In addition, it is the only functionally characterized plant α+β barrel (DABB) protein that catalyzes the C2-C7 aldol cyclization of the linear pentyl tetra-β-ketide CoA as the substrate, to generate olivetolic acid (OA). Herein, we solved the OAC apo and OAC-OA complex binary crystal structures at 1.32 and 1.70 Å resolutions, respectively. The crystal structures revealed that the enzyme indeed belongs to the DABB superfamily, as previously proposed, and possesses a unique active-site cavity containing the pentyl-binding hydrophobic pocket and the polyketide binding site, which have never been observed among the functionally and structurally characterized bacterial polyketide cyclases. Furthermore, site-directed mutagenesis studies indicated that Tyr72 and His78 function as acid/base catalysts at the catalytic center. Structural and/or functional studies of OAC suggested that the enzyme lacks thioesterase and aromatase activities. These observations demonstrated that OAC employs unique catalytic machinery utilizing acid/base catalytic chemistry for the formation of the precursor of OA. The structural and functional insights obtained in this work thus provide the foundation for analyses of the plant polyketide cyclases that will be discovered in the future. Structural data reported in this paper are available in the Protein Data Bank under the accession numbers 5B08 for the OAC apo, 5B09 for the OAC-OA binary complex and 5B0A, 5B0B, 5B0C, 5B0D, 5B0E, 5B0F and 5B0G for the OAC His5Q, Ile7F, Tyr27F, Tyr27W, Val59M, Tyr72F and His78S mutant enzymes, respectively. © 2016 Federation of European Biochemical Societies.

  1. Enzymatic 13C Labeling and Multidimensional NMR Analysis of Miltiradiene Synthesized by Bifunctional Diterpene Cyclase in Selaginella moellendorffii*

    Science.gov (United States)

    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

  2. Human glutaminyl cyclase and bacterial zinc aminopeptidase share a common fold and active site

    Directory of Open Access Journals (Sweden)

    Misquitta Stephanie A

    2004-02-01

    Full Text Available Abstract Background Glutaminyl cyclase (QC forms the pyroglutamyl residue at the amino terminus of numerous secretory peptides and proteins. We previously proposed the mammalian QC has some features in common with zinc aminopeptidases. We now have generated a structural model for human QC based on the aminopeptidase fold (pdb code 1AMP and mutated the apparent active site residues to assess their role in QC catalysis. Results The structural model proposed here for human QC, deposited in the protein databank as 1MOI, is supported by a variety of fold prediction programs, by the circular dichroism spectrum, and by the presence of the disulfide. Mutagenesis of the six active site residues present in both 1AMP and QC reveal essential roles for the two histidines (140 and 330, QC numbering and the two glutamates (201 and 202, while the two aspartates (159 and 248 appear to play no catalytic role. ICP-MS analysis shows less than stoichiometric zinc (0.3:1 in the purified enzyme. Conclusions We conclude that human pituitary glutaminyl cyclase and bacterial zinc aminopeptidase share a common fold and active site residues. In contrast to the aminopeptidase, however, QC does not appear to require zinc for enzymatic activity.

  3. Expression, purification and crystallization of a plant polyketide cyclase from Cannabis sativa.

    Science.gov (United States)

    Yang, Xinmei; Matsui, Takashi; Mori, Takahiro; Taura, Futoshi; Noguchi, Hiroshi; Abe, Ikuro; Morita, Hiroyuki

    2015-12-01

    Plant polyketides are a structurally diverse family of natural products. In the biosynthesis of plant polyketides, the construction of the carbocyclic scaffold is a key step in diversifying the polyketide structure. Olivetolic acid cyclase (OAC) from Cannabis sativa L. is the only known plant polyketide cyclase that catalyzes the C2-C7 intramolecular aldol cyclization of linear pentyl tetra-β-ketide-CoA to generate olivetolic acid in the biosynthesis of cannabinoids. The enzyme is also thought to belong to the dimeric α+β barrel (DABB) protein family. However, because of a lack of functional analysis of other plant DABB proteins and low sequence identity with the functionally distinct bacterial DABB proteins, the catalytic mechanism of OAC has remained unclear. To clarify the intimate catalytic mechanism of OAC, the enzyme was overexpressed in Escherichia coli and crystallized using the vapour-diffusion method. The crystals diffracted X-rays to 1.40 Å resolution and belonged to space group P3121 or P3221, with unit-cell parameters a = b = 47.3, c = 176.0 Å. Further crystallographic analysis will provide valuable insights into the structure-function relationship and catalytic mechanism of OAC.

  4. Phosphorylation-independent regulation of the diguanylate cyclase WspR.

    Directory of Open Access Journals (Sweden)

    Nabanita De

    2008-03-01

    Full Text Available 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.

  5. G protein β1γ2 subunits purification and their interaction with adenylyl cyclase

    Institute of Scientific and Technical Information of China (English)

    CHEN; Julian; (陈巨莲); NI; Hanxiang; (倪汉祥); SUN; Jingrui; (孙京瑞); WENG; Gezhi

    2003-01-01

    A preliminary study on the interaction of G protein (guanine triphosphate binding pro- tein) β1γ2 subunits and their coupled components in cell signal transduction was conducted in vitro. The insect cell lines, Sf9 (Spodoptera frugiperda) and H5 (Trichoplusia ni) were used to express the recombinant protein Gβ1γ2. The cell membrane containing Gβ1γ2 was isolated through affinity chromatography column with Ni-NTA agarose by FPLC method, and the highly purified protein was obtained. The adenylyl cyclase 2 (AC2) activity assay showed that the purified Gβ1γ2 could significantly stimulate AC2 activity. The interaction of β1γ2 subunits of G protein with the cytoplasmic tail of various mammalian adenylyl cyclases was monitored by BIAcore technology using NTA sensor chip, which relies on the phenomenon of surface plasmon resonance (SPR). The experiments showed the direct binding of Gβ1γ2 to the cytoplasmic tail C2 domain of AC2. The specific binding domain of AC2 with Gβ1γ2 was the same as AC2 activity domain which was stimulated by β1γ2.

  6. Adenylyl cyclase 3 haploinsufficiency confers susceptibility to diet-induced obesity and insulin resistance in mice

    Science.gov (United States)

    Tong, Tao; Shen, Ying; Lee, Han-Woong; Yu, Rina; Park, Taesun

    2016-01-01

    Adenylyl cyclase 3 (Adcy3), a member of the mammalian adenylyl cyclase family responsible for generating the second messenger cAMP, has long been known to play an essential role in olfactory signal transduction. Here, we demonstrated that Adcy3 heterozygous null mice displayed increased visceral adiposity in the absence of hyperphagia and developed abnormal metabolic features characterized by impaired insulin sensitivity, dyslipidemia, and increased plasma levels of proinflammatory cytokines on both chow and high-fat diet (HFD). Of note, HFD decreased the Adcy3 expression in white adipose tissue, liver, and muscle. We also report for the first time that Adcy3 haploinsufficiency resulted in reduced expression of genes involved in thermogenesis, fatty acid oxidation, and insulin signaling, with enhanced expression of genes related to adipogenesis in peripheral tissues of mice. In conclusion, these findings suggest that cAMP signals generated by Adcy3 in peripheral tissues may play a pivotal role in modulating obesity and insulin sensitivity. PMID:27678003

  7. Mechanistic studies of sesquiterpene cyclases based on their carbon isotope ratios at natural abundance.

    Science.gov (United States)

    Tan, Wenhua; Bartram, Stefan; Boland, Wilhelm

    2017-01-03

    During the process of terpene biosynthesis, C-C bond breaking and forming steps are subjected to kinetic carbon isotope effects, leading to distinct carbon isotopic signatures of the products. Accordingly, carbon isotopic signatures could be used to reveal the 'biosynthetic history' of the produced terpenoids. Five known sesquiterpene cyclases, regulating three different pathways, representing simple to complex biosynthetic sequences, were heterologously expressed and used for in vitro assays with farnesyl diphosphate as substrate. Compound specific isotope ratio mass spectrometry measurements of the enzyme substrate farnesyl diphosphate (FDP) and the products of all the five cyclases were performed. The calculated δ(13) C value for FDP, based on δ(13) C values and relative amounts of the products, was identical with its measured δ(13) C value, confirming the reliability of the approach and the precision of measurements. The different carbon isotope ratios of the products reflect the complexity of their structure and are correlated with the frequency of carbon-carbon bond forming and breaking steps on their individual biosynthetic pathways. Thus, the analysis of carbon isotopic signatures of terpenes at natural abundance can be used as a powerful tool in elucidation of associated biosynthetic mechanisms of terpene synthases and in future in vivo studies even without 'touching' the plant. © 2017 John Wiley & Sons Ltd.

  8. Characterization of Plasmodium falciparum adenylyl cyclase-β and its role in erythrocytic stage parasites.

    Directory of Open Access Journals (Sweden)

    Eric Salazar

    Full Text Available The most severe form of human malaria is caused by the parasite Plasmodium falciparum. The second messenger cAMP has been shown to be important for the parasite's ability to infect the host's liver, but its role during parasite growth inside erythrocytes, the stage responsible for symptomatic malaria, is less clear. The P. falciparum genome encodes two adenylyl cyclases, the enzymes that synthesize cAMP, PfACα and PfACβ. We now show that one of these, PfACβ, plays an important role during the erythrocytic stage of the P. falciparum life cycle. Biochemical characterization of PfACβ revealed a marked pH dependence, and sensitivity to a number of small molecule inhibitors. These inhibitors kill parasites growing inside red blood cells. One particular inhibitor is selective for PfACβ relative to its human ortholog, soluble adenylyl cyclase (sAC; thus, PfACβ represents a potential target for development of safe and effective antimalarial therapeutics.

  9. Modification of a bi-functional diguanylate cyclase-phosphodiesterase to efficiently produce cyclic diguanylate monophosphate

    Directory of Open Access Journals (Sweden)

    Natasha M. Nesbitt

    2015-09-01

    Full Text Available Cyclic-diGMP is a bacterial messenger that regulates many physiological processes, including many attributed to pathogenicity. Bacteria synthesize cyclic-diGMP from GTP using diguanylate cyclases; its hydrolysis is catalyzed by phosphodiesterases. Here we report the over-expression and purification of a bi-functional diguanylate cyclase-phosphodiesterase from Agrobacterium vitis S4. Using homology modeling and primary structure alignment, we identify several amino acids predicted to participate in the phosphodiesterase reaction. Upon altering selected residues, we obtain variants of the enzyme that efficiently and quantitatively catalyze the synthesis of cyclic-diGMP from GTP without hydrolysis to pGpG. Additionally, we identify a variant that produces cyclic-diGMP while immobilized to NiNTA beads and can catalyze the conversion of [α-32P]-GTP to [32P]-cyclic-diGMP. In short, we characterize a novel cyclic-diGMP processing enzyme and demonstrate its utility for efficient and cost-effective production of cyclic-diGMP, as well as modified cyclic-diGMP molecules, for use as probes in studying the many important biological processes mediated by cyclic-diGMP.

  10. Bicarbonate-sensing soluble adenylyl cyclase is an essential sensor for acid/base homeostasis.

    Science.gov (United States)

    Tresguerres, Martin; Parks, Scott K; Salazar, Eric; Levin, Lonny R; Goss, Greg G; Buck, Jochen

    2010-01-05

    pH homeostasis is essential for life, yet it remains unclear how animals sense their systemic acid/base (A/B) status. Soluble adenylyl cyclase (sAC) is an evolutionary conserved signaling enzyme that produces the second messenger cAMP in response to bicarbonate ions (HCO(3)(-)). We cloned the sAC ortholog from the dogfish, a shark that regulates blood A/B by absorbing and secreting protons (H(+)) and HCO(3)(-) at its gills. Similar to mammalian sAC, dogfish soluble adenylyl cyclase (dfsAC) is activated by HCO(3)(-) and can be inhibited by two structurally and mechanistically distinct small molecule inhibitors. dfsAC is expressed in the gill epithelium, where the subset of base-secreting cells resides. Injection of inhibitors into animals under alkaline stress confirmed that dfsAC is essential for maintaining systemic pH and HCO(3)(-) levels in the whole organism. One of the downstream effects of dfsAC is to promote the insertion of vacuolar proton pumps into the basolateral membrane to absorb H(+) into the blood. sAC orthologs are present throughout metazoans, and mammalian sAC is expressed in A/B regulatory organs, suggesting that systemic A/B sensing via sAC is widespread in the animal kingdom.

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

    Science.gov (United States)

    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. Copyright © 2016 the American Physiological Society.

  12. Structural evidence for variable oligomerization of the N-terminal domain of cyclase-associated protein (CAP).

    Science.gov (United States)

    Yusof, Adlina Mohd; Hu, Nien-Jen; Wlodawer, Alexander; Hofmann, Andreas

    2005-02-01

    Cyclase-associated protein (CAP) is a highly conserved and widely distributed protein that links the nutritional response signaling to cytoskeleton remodeling. In yeast, CAP is a component of the adenylyl cyclase complex and helps to activate the Ras-mediated catalytic cycle of the cyclase. While the N-terminal domain of CAP (N-CAP) provides a binding site for adenylyl cyclase, the C-terminal domain (C-CAP) possesses actin binding activity. Our attempts to crystallize full-length recombinant CAP from Dictyostelium discoideum resulted in growth of orthorhombic crystals containing only the N-terminal domain (residues 42-227) due to auto-proteolytic cleavage. The structure was solved by molecular replacement with data at 2.2 A resolution. The present crystal structure allows the characterization of a head-to-tail N-CAP dimer in the asymmetric unit and a crystallographic side-to-side dimer. Comparison with previously published structures of N-CAP reveals variable modes of dimerization of this domain, but the presence of a common interface for the side-to-side dimer.

  13. The magnesium-protoporphyrin IX (oxidative) cyclase system. Studies on the mechanism and specificity of the reaction sequence.

    Science.gov (United States)

    Walker, C J; Mansfield, K E; Rezzano, I N; Hanamoto, C M; Smith, K M; Castelfranco, P A

    1988-10-15

    Mg-protoporphyrin IX monomethyl ester cyclase activity was assayed in isolated developing cucumber (Cucumis sativus L. var. Beit Alpha) chloroplasts [Chereskin, Wong & Castelfranco (1982) Plant Physiol. 70, 987-993]. The presence of both 6- and 7-methyl esterase activities was detected, which permitted the use of diester porphyrins in a substrate-specificity study. It was found that: (1) the 6-methyl acrylate derivative of Mg-protoporphyrin monomethyl ester was inactive as a substrate for cyclization; (2) only one of the two enantiomers of 6-beta-hydroxy-Mg-protoporphyrin dimethyl ester had detectable activity as a substrate for the cyclase; (3) the 2-vinyl-4-ethyl-6-beta-oxopropionate derivatives of Mg-protoporphyrin mono- or di-methyl ester were approx. 4 times more active as substrates for cyclization than the corresponding divinyl forms; (4) at the level of Mg-protoporphyrin there was no difference in cyclase activity between the 4-vinyl and 4-ethyl substrates; (5) reduction of the side chain of Mg-protoporphyrin in the 2-position from a vinyl to an ethyl resulted in a partial loss of cyclase activity. This work suggests that the original scheme for cyclization proposed by Granick [(1950) Harvey Lect. 44, 220-245] should now be modified by the omission of the 6-methyl acrylate derivative of Mg-protoporphyrin monomethyl ester and the introduction of stereo-specificity at the level of the hydroxylated intermediate.

  14. Molecular Cloning,Expression,and Characterization of an Adenylyl Cyclase-associated Protein from Gossypium arboreum Fuzzless Mutant

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    CAP,an adenylyl cyclase-associated protein,is predicted to be involved in cytoskeletal organization and signal transduction.Recently,we found that CAP may play an important role in fuzz-like fiber cell initiation in cotton.For the further research,we isolated two CAP homologues from wild type

  15. Isolation of a cotton CAP gene: a homologue of adenylyl cyclase-associated protein highly expressed during fiber elongation.

    Science.gov (United States)

    Kawai, M; Aotsuka, S; Uchimiya, H

    1998-12-01

    The cDNA encoding CAP (adenylyl cyclase-associated protein) was isolated from a cotton (Gossypium hirsutum) fiber cDNA library. The cDNA (GhCAP) contained an open reading frame that encoded 471 amino acid residues. RNA blot analysis showed that the cotton CAP gene was expressed mainly in young fibers.

  16. Membrane Guanylate Cyclase catalytic Subdomain: Structure and Linkage with Calcium Sensors and Bicarbonate.

    Science.gov (United States)

    Ravichandran, Sarangan; Duda, Teresa; Pertzev, Alexandre; Sharma, Rameshwar K

    2017-01-01

    Membrane guanylate cyclase (MGC) is a ubiquitous multi-switching cyclic GMP generating signaling machine linked with countless physiological processes. In mammals it is encoded by seven distinct homologous genes. It is a single transmembrane spanning multi-modular protein; composed of integrated blocks and existing in homo-dimeric form. Its core catalytic domain (CCD) module is a common transduction center where all incoming signals are translated into the production of cyclic GMP, a cellular signal second messenger. Crystal structure of the MGC's CCD does not exist and its precise identity is ill-defined. Here, we define it at a sub-molecular level for the phototransduction-linked MGC, the rod outer segment guanylate cyclase type 1, ROS-GC1. (1) The CCD is a conserved 145-residue structural unit, represented by the segment V(820)-P(964). (2) It exists as a homo-dimer and contains seven conserved catalytic elements (CEs) wedged into seven conserved motifs. (3) It also contains a conserved 21-residue neurocalcin δ-modulated structural domain, V(836)-L(857). (4) Site-directed mutagenesis documents that each of the seven CEs governs the cyclase's catalytic activity. (5) In contrast to the soluble and the bacterium MGC which use Mn(2+)-GTP substrate for catalysis, MGC CCD uses the natural Mg(2+)-GTP substrate. (6) Strikingly, the MGC CCD requires anchoring by the Transmembrane Domain (TMD) to exhibit its major (∼92%) catalytic activity; in isolated form the activity is only marginal. This feature is not linked with any unique sequence of the TMD; there is minimal conservation in TMD. Finally, (7) the seven CEs control each of four phototransduction pathways- -two Ca(2+)-sensor GCAPs-, one Ca(2+)-sensor, S100B-, and one bicarbonate-modulated. The findings disclose that the CCD of ROS-GC1 has built-in regulatory elements that control its signal translational activity. Due to conservation of these regulatory elements, it is proposed that these elements also control the

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

    Directory of Open Access Journals (Sweden)

    Jürgen Ulrich Linder

    Full Text Available 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.

  18. Molecular cloning, subcellular localization and characterization of two adenylate kinases from cassava, Manihot esculenta Crantz cv. KU50.

    Science.gov (United States)

    Boonrueng, Channarong; Tangpranomkorn, Surachat; Yazhisai, Uthaman; Sirikantaramas, Supaart

    2016-10-01

    Adenylate kinase (ADK) is a phosphotransferase that plays an important role in cellular energy homeostasis. Many isozymes located in different subcellular compartments have been reported. In this study, we focus on the characterization of cassava (Manihot esculenta) ADKs. We found 15 ADKs that are publicly available in the African cassava genome database. We cloned two ADKs, namely MeADK1 and MeADK2, which are phylogenetically grouped together with the plastidial ADK in potato. Both MeADK1 and MeADK2 showed 66% identity in the amino acid sequences with plastidial ADK in potato. However, we demonstrated that they are localized to mitochondria using GFP fusions of MeADK1 and MeADK2. The Escherichia coli-produced recombinant MeADK1 and MeADK2 preferred forward reactions that produce ATP. They exhibited similar specific activities. The semi-quantitative RT-PCR analysis showed that MeADK1 and MeADK2 in 2-month-old leaves have similar expression patterns under a diurnal light-dark cycle. However, MeADK2 transcripts were expressed at much higher levels than MeADK1 in 5-month-old leaves and roots. Thus, we conclude that MeADK2 might play a vital role in energy homeostasis in cassava mitochondria.

  19. From endoplasmic reticulum to mitochondria: absence of the Arabidopsis ATP antiporter endoplasmic Reticulum Adenylate Transporter1 perturbs photorespiration.

    Science.gov (United States)

    Hoffmann, Christiane; Plocharski, Bartolome; Haferkamp, Ilka; Leroch, Michaela; Ewald, Ralph; Bauwe, Hermann; Riemer, Jan; Herrmann, Johannes M; Neuhaus, H Ekkehard

    2013-07-01

    The carrier Endoplasmic Reticulum Adenylate Transporter1 (ER-ANT1) resides in the endoplasmic reticulum (ER) membrane and acts as an ATP/ADP antiporter. Mutant plants lacking ER-ANT1 exhibit a dwarf phenotype and their seeds contain reduced protein and lipid contents. In this study, we describe a further surprising metabolic peculiarity of the er-ant1 mutants. Interestingly, Gly levels in leaves are immensely enhanced (26×) when compared with that of wild-type plants. Gly accumulation is caused by significantly decreased mitochondrial glycine decarboxylase (GDC) activity. Reduced GDC activity in mutant plants was attributed to oxidative posttranslational protein modification induced by elevated levels of reactive oxygen species (ROS). GDC activity is crucial for photorespiration; accordingly, morphological and physiological defects in er-ant1 plants were nearly completely abolished by application of high environmental CO(2) concentrations. The latter observation demonstrates that the absence of ER-ANT1 activity mainly affects photorespiration (maybe solely GDC), whereas basic cellular metabolism remains largely unchanged. Since ER-ANT1 homologs are restricted to higher plants, it is tempting to speculate that this carrier fulfils a plant-specific function directly or indirectly controlling cellular ROS production. The observation that ER-ANT1 activity is associated with cellular ROS levels reveals an unexpected and critical physiological connection between the ER and other organelles in plants.

  20. Moonlighting kinases with guanylate cyclase activity can tune regulatory signal networks

    KAUST Repository

    Irving, Helen R.

    2012-02-01

    Guanylate cyclase (GC) catalyzes the formation of cGMP and it is only recently that such enzymes have been characterized in plants. One family of plant GCs contains the GC catalytic center encapsulated within the intracellular kinase domain of leucine rich repeat receptor like kinases such as the phytosulfokine and brassinosteroid receptors. In vitro studies show that both the kinase and GC domain have catalytic activity indicating that these kinase-GCs are examples of moonlighting proteins with dual catalytic function. The natural ligands for both receptors increase intracellular cGMP levels in isolated mesophyll protoplast assays suggesting that the GC activity is functionally relevant. cGMP production may have an autoregulatory role on receptor kinase activity and/or contribute to downstream cell expansion responses. We postulate that the receptors are members of a novel class of receptor kinases that contain functional moonlighting GC domains essential for complex signaling roles.

  1. Oxygen promotes biofilm formation of Shewanella putrefaciens CN32 through a diguanylate cyclase and an adhesin.

    Science.gov (United States)

    Wu, Chao; Cheng, Yuan-Yuan; Yin, Hao; Song, Xiang-Ning; Li, Wen-Wei; Zhou, Xian-Xuan; Zhao, Li-Ping; Tian, Li-Jiao; Han, Jun-Cheng; Yu, Han-Qing

    2013-01-01

    Although oxygen has been reported to regulate biofilm formation by several Shewanella species, the exact regulatory mechanism mostly remains unclear. Here, we identify a direct oxygen-sensing diguanylate cyclase (DosD) and reveal its regulatory role in biofilm formation by Shewanella putrefaciens CN32 under aerobic conditions. In vitro and in vivo analyses revealed that the activity of DosD culminates to synthesis of cyclic diguanylate (c-di-GMP) in the presence of oxygen. DosD regulates the transcription of bpfA operon which encodes seven proteins including a large repetitive adhesin BpfA and its cognate type I secretion system (TISS). Regulation of DosD in aerobic biofilms is heavily dependent on an adhesin BpfA and the TISS. This study offers an insight into the molecular mechanism of oxygen-stimulated biofilm formation by S. putrefaciens CN32.

  2. The roles of cysteines in the heme domain of human soluble guanylate cyclase

    Institute of Scientific and Technical Information of China (English)

    Fang Fang Zhong; Xiao Xiao Liu; Jie Pan; Zhong Xian Huang; Xiang Shi Tan

    2012-01-01

    Soluble guanylate cyclase (sGC) is a critical heme-containing enzyme involved in NO signaling.The dimerization of sGC subunits is necessary for its bioactivity and its mechanism is a striiking and an indistinct issue.The roles of heme domain cysteines of the sGC on the dimerization and heme binding were investigated herein.The site-directed mutations of three conserved cysteines (C78A,C 122A and C 174S) were studied systematically and the three mutants were characterized by gel filtration analysis,UV-vis spectroscopy and heime transfer examination.Cys78 was involved in heme binding but not referred to the dimerization,while Cys174 was demonstrated to be involved in the homodimerization.These results provide new insights into the cysteine-related dimerization regulation of sGC.

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

    Science.gov (United States)

    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.

  4. Adenylyl Cyclase Signaling in the Developing Chick Heart: The Deranging Effect of Antiarrhythmic Drugs

    Directory of Open Access Journals (Sweden)

    Lucie Hejnova

    2014-01-01

    Full Text Available The adenylyl cyclase (AC signaling system plays a crucial role in the regulation of cardiac contractility. Here we analyzed the key components of myocardial AC signaling in the developing chick embryo and assessed the impact of selected β-blocking agents on this system. Application of metoprolol and carvedilol, two commonly used β-blockers, at embryonic day (ED 8 significantly downregulated (by about 40% expression levels of AC5, the dominant cardiac AC isoform, and the amount of Gsα protein at ED9. Activity of AC stimulated by forskolin was also significantly reduced under these conditions. Interestingly, when administered at ED4, these drugs did not produce such profound changes in the myocardial AC signaling system, except for markedly increased expression of Giα protein. These data indicate that β-blocking agents can strongly derange AC signaling during the first half of embryonic heart development.

  5. Isoform-specific regulation of adenylyl cyclase: a potential target in future pharmacotherapy.

    Science.gov (United States)

    Iwatsubo, Kousaku; Tsunematsu, Takashi; Ishikawa, Yoshihiro

    2003-06-01

    Adenylyl cyclase (AC) is a target enzyme of multiple G-protein-coupled receptors (GPCRs). In the past decade, the cloning, structure and biochemical properties of nine AC isoforms were reported, and each isoform of AC shows distinct patterns of tissue distribution and biochemical/pharmacological properties. In addition to the conventional regulators of this enzyme, such as calmodulin (CaM) or PKC, novel regulators, for example, caveolin, have been identified. Most importantly, these regulators work on AC in an isoform dependent manner. Recent studies have demonstrated that certain classic AC inhibitors, i.e., P-site inhibitors, show an isoform-dependent inhibition of AC. The side chain modifications of forskolin, a diterpene extract from Coleus forskolii, markedly enhance its isoform selectivity. When taken together, these findings suggest that it is feasible to develop new pharmacotherapeutic agents that target AC isoforms to regulate various neurohormonal signals in a highly tissue-/organ-specific manner.

  6. New structural forms of a mycobacterial adenylyl cyclase Rv1625c

    Directory of Open Access Journals (Sweden)

    Deivanayaga Barathy

    2014-09-01

    Full Text Available Rv1625c is one of 16 adenylyl cyclases encoded in the genome of Mycobacterium tuberculosis. In solution Rv1625c exists predominantly as a monomer, with a small amount of dimer. It has been shown previously that the monomer is active and the dimeric fraction is inactive. Both fractions of wild-type Rv1625c crystallized as head-to-head inactive domain-swapped dimers as opposed to the head-to-tail dimer seen in other functional adenylyl cyclases. About half of the molecule is involved in extensive domain swapping. The strain created by a serine residue located on a hinge loop and the crystallization condition might have led to this unusual domain swapping. The inactivity of the dimeric form of Rv1625c could be explained by the absence of the required catalytic site in the swapped dimer. A single mutant of the enzyme was also generated by changing a phenylalanine predicted to occur at the functional dimer interface to an arginine. This single mutant exists as a dimer in solution but crystallized as a monomer. Analysis of the structure showed that a salt bridge formed between a glutamate residue in the N-terminal segment and the mutated arginine residue hinders dimer formation by pulling the N-terminal region towards the dimer interface. Both structures reported here show a change in the dimerization-arm region which is involved in formation of the functional dimer. It is concluded that the dimerization arm along with other structural elements such as the N-terminal region and certain loops are vital for determining the oligomeric nature of the enzyme, which in turn dictates its activity.

  7. Guanylyl Cyclase C Hormone Axis at the Intersection of Obesity and Colorectal Cancer.

    Science.gov (United States)

    Blomain, Erik S; Merlino, Dante J; Pattison, Amanda M; Snook, Adam E; Waldman, Scott A

    2016-09-01

    Obesity has emerged as a principal cause of mortality worldwide, reflecting comorbidities including cancer risk, particularly in colorectum. Although this relationship is established epidemiologically, molecular mechanisms linking colorectal cancer and obesity continue to be refined. Guanylyl cyclase C (GUCY2C), a membrane-bound guanylyl cyclase expressed in intestinal epithelial cells, binds the paracrine hormones guanylin and uroguanylin, inducing cGMP signaling in colorectum and small intestine, respectively. Guanylin is the most commonly lost gene product in sporadic colorectal cancer, and its universal loss early in transformation silences GUCY2C, a tumor suppressor, disrupting epithelial homeostasis underlying tumorigenesis. In small intestine, eating induces endocrine secretion of uroguanylin, the afferent limb of a novel gut-brain axis that activates hypothalamic GUCY2C-cGMP signaling mediating satiety opposing obesity. Recent studies revealed that diet-induced obesity suppressed guanylin and uroguanylin expression in mice and humans. Hormone loss reflects reversible calorie-induced endoplasmic reticulum stress and the associated unfolded protein response, rather than the endocrine, adipokine, or inflammatory milieu of obesity. Loss of intestinal uroguanylin secretion silences the hypothalamic GUCY2C endocrine axis, creating a feed-forward loop contributing to hyperphagia in obesity. Importantly, calorie-induced guanylin loss silences the GUCY2C-cGMP paracrine axis underlying obesity-induced epithelial dysfunction and colorectal tumorigenesis. Indeed, genetically enforced guanylin replacement eliminated diet-induced intestinal tumorigenesis in mice. Taken together, these observations suggest that GUCY2C hormone axes are at the intersection of obesity and colorectal cancer. Moreover, they suggest that hormone replacement that restores GUCY2C signaling may be a novel therapeutic paradigm to prevent both hyperphagia and intestinal tumorigenesis in obesity.

  8. Lycopene cyclase paralog CruP protects against reactive oxygen species in oxygenic photosynthetic organisms.

    Science.gov (United States)

    Bradbury, Louis M T; Shumskaya, Maria; Tzfadia, Oren; Wu, Shi-Biao; Kennelly, Edward J; Wurtzel, Eleanore T

    2012-07-03

    In photosynthetic organisms, carotenoids serve essential roles in photosynthesis and photoprotection. A previous report designated CruP as a secondary lycopene cyclase involved in carotenoid biosynthesis [Maresca J, et al. (2007) Proc Natl Acad Sci USA 104:11784-11789]. However, we found that cruP KO or cruP overexpression plants do not exhibit correspondingly reduced or increased production of cyclized carotenoids, which would be expected if CruP was a lycopene cyclase. Instead, we show that CruP aids in preventing accumulation of reactive oxygen species (ROS), thereby reducing accumulation of β-carotene-5,6-epoxide, a ROS-catalyzed autoxidation product, and inhibiting accumulation of anthocyanins, which are known chemical indicators of ROS. Plants with a nonfunctional cruP accumulate substantially higher levels of ROS and β-carotene-5,6-epoxide in green tissues. Plants overexpressing cruP show reduced levels of ROS, β-carotene-5,6-epoxide, and anthocyanins. The observed up-regulation of cruP transcripts under photoinhibitory and lipid peroxidation-inducing conditions, such as high light stress, cold stress, anoxia, and low levels of CO(2), fits with a role for CruP in mitigating the effects of ROS. Phylogenetic distribution of CruP in prokaryotes showed that the gene is only present in cyanobacteria that live in habitats characterized by large variation in temperature and inorganic carbon availability. Therefore, CruP represents a unique target for developing resilient plants and algae needed to supply food and biofuels in the face of global climate change.

  9. A reduced susceptibility to chemoconvulsant stimulation in adenylyl cyclase 8 knockout mice

    Science.gov (United States)

    Chen, Xia; Dong, Guoying; Zheng, Changhong; Wang, Hongbing; Yun, Wenwei; Zhou, Xianju

    2015-01-01

    Objective Adenylyl cyclases (ACs) catalyze the synthesis of cAMP from ATP, and cAMP signaling affects a large number of neuronal processes. Ca2+-stimualted adenylyl cyclase 8 (AC8) expressed in the CNS plays a role in synaptic plasticity, drug addiction and ethanol sensitivity, and chronic pain. This study was to aim at examining the contributions of AC8 to epileptogenesis. Methods In this study, we observed the seizure behavior induced by kainic acid (20mg/kg or 30mg/kg) or pilocarpine (350mg/kg) in AC8 KO and wild-type mice. Next we injected kainic acid or pilocarpine to induce status epilepticus (SE), and examined neuronal degeneration (by Fluoro-Jade B staining) and mossy fiber sprouting (by Timm staining) 24 hr and 2 weeks after SE termination in the hippocampus, respectively. Finally, 15min after intraperitoneal injection of kainic acid (30mg/kg), we examined phosphor-ERK1/2 in the hippocampus by western blot and immunochemistry staining. Results We first observed that AC8 KO mutants display reduced susceptibility (including seizure latency and episodes) to two chemoconvulsants, kainic acid and pilocarpine. Moreover, we found that degenerative neurons and mossy fiber sprouting induced by chemoconvulsants were significant decreased in the hippocampus. Further, western blot and immunochemistry analysis revealed that the MAPK signaling in the hippocampus was attenuated in kainic acid-injected AC8 KO mice. Conclusion AC8 is involved in epileptogenesis, and may serve as a potential target for the treatment of epilepsy. PMID:26656781

  10. Interaction of retinal guanylate cyclase with the alpha subunit of transducin: potential role in transducin localization.

    Science.gov (United States)

    Rosenzweig, Derek H; Nair, K Saidas; Levay, Konstantin; Peshenko, Igor V; Crabb, John W; Dizhoor, Alexander M; Slepak, Vladlen Z

    2009-02-01

    Vertebrate phototransduction is mediated by cGMP, which is generated by retGC (retinal guanylate cyclase) and degraded by cGMP phosphodiesterase. Light stimulates cGMP hydrolysis via the G-protein transducin, which directly binds to and activates phosphodiesterase. Bright light also causes relocalization of transducin from the OS (outer segments) of the rod cells to the inner compartments. In the present study, we show experimental evidence for a previously unknown interaction between G(alphat) (the transducin alpha subunit) and retGC. G(alphat) co-immunoprecipitates with retGC from the retina or from co-transfected COS-7 cells. The retGC-G(alphat) complex is also present in cones. The interaction also occurs in mice lacking RGS9 (regulator of G-protein signalling 9), a protein previously shown to associate with both G(alphat) and retGC. The G(alphat)-retGC interaction is mediated primarily by the kinase homology domain of retGC, which binds GDP-bound G(alphat) stronger than the GTP[S] (GTPgammaS; guanosine 5'-[gamma-thio]triphosphate) form. Neither G(alphat) nor G(betagamma) affect retGC-mediated cGMP synthesis, regardless of the presence of GCAP (guanylate cyclase activating protein) and Ca2+. The rate of light-dependent transducin redistribution from the OS to the inner segments is markedly accelerated in the retGC-1-knockout mice, while the migration of transducin to the OS after the onset of darkness is delayed. Supplementation of permeabilized photoreceptors with cGMP does not affect transducin translocation. Taken together, these results suggest that the protein-protein interaction between G(alphat) and retGC represents a novel mechanism regulating light-dependent translocation of transducin in rod photoreceptors.

  11. Stimulation of renin secretion by catecholamines is dependent on adenylyl cyclases 5 and 6.

    Science.gov (United States)

    Aldehni, Fadi; Tang, Tong; Madsen, Kirsten; Plattner, Michael; Schreiber, Andrea; Friis, Ulla G; Hammond, H Kirk; Han, Pyung Lim; Schweda, Frank

    2011-03-01

    The sympathetic nervous system stimulates renin release from juxtaglomerular cells via the β-adrenoreceptor-cAMP pathway. Recent in vitro studies have suggested that the calcium-inhibited adenylyl cyclases (ACs) 5 and 6 possess key roles in the control of renin exocytosis. To investigate the relative contribution of AC5 and AC6 to the regulation of renin release in vivo we performed experiments using AC5 and AC6 knockout mice. Male AC5(-/-) mice exhibited normal plasma renin concentrations, renal renin synthesis (mRNA and renin content), urinary volume, and systolic blood pressure. In male AC6(-/-) mice, plasma renin concentration (AC6(-/-): 732 ± 119; AC6 (+/+): 436 ± 78 ng of angiotensin I per hour*mL(-1); Prenin synthesis were stimulated associated with an increased excretion of dilute urine (1.55-fold; Pplasma renin concentration by a single injection of the β-adrenoreceptor agonist isoproterenol (10 mg/kg IP) was significantly attenuated in AC5(-/-) (male: -20%; female: -33%) compared with wild-type mice in vivo. The mitigation of the plasma renin concentration response to isoproterenol was even more pronounced in AC6(-/-) (male: -63%; female: -50% versus AC6(+/+)). Similarly, the effects of isoproterenol, prostaglandin E2, and pituitary adenylyl cyclase-activating polypeptide on renin release from isolated perfused kidneys were attenuated to a higher extent in AC6(-/-) (-51% to -98% versus AC6(+/+)) than in AC5(-/-) (-31% to 46% versus AC5(+/+)). In conclusion, both AC5 and AC6 are involved in the stimulation of renin secretion in vivo, and AC6 is the dominant isoforms in this process.

  12. Membrane Guanylyl Cyclase Complexes Shape the Photoresponses of Retinal Rods and Cones

    Directory of Open Access Journals (Sweden)

    Xiao-Hong eWen

    2014-06-01

    Full Text Available In vertebrate rods and cones, photon capture by rhodopsin leads to the destruction of cyclic GMP (cGMP and the subsequent closure of cyclic nucleotide gated (CNG ion channels in the outer segment plasma membrane. Replenishment of cGMP and reopening of the channels limit the growth of the photon response and are requisite for its recovery. In different vertebrate retinas, there may be as many as four types of membrane guanylyl cyclases (GCs for cGMP synthesis. Ten neuronal Ca2+ sensor proteins could potentially modulate their activities. The mouse is proving to be an effective model for characterizing the roles of individual components because its relative simplicity can be reduced further by genetic engineering. There are two types of guanylyl cyclase activating proteins (GCAPs and two types of GCs in mouse rods, whereas cones express one type of GCAP and one type of GC. Mutant mouse rods and cones bereft of both GCAPs have large, long lasting photon responses. Thus, GCAPs normally mediate negative feedback tied to the light-induced decline in intracellular Ca2+ that accelerates GC activity to curtail the growth and duration of the photon response. Rods from other mutant mice that express a single GCAP type reveal how the two GCAPs normally work together as a team. Because of its lower Ca2+ affinity, GCAP1 is the first responder that senses the initial decrease in Ca2+ following photon absorption and acts to limit response amplitude. GCAP2, with a higher Ca2+ affinity, is recruited later during the course of the photon response as Ca2+ levels continue to decline further. The main role of GCAP2 is to provide for a timely response recovery and it is particularly important after exposure to very bright light. The multiplicity of GC isozymes and GCAP homologs in the retinas of other vertebrates confers greater flexibility in shaping the photon responses in order to tune visual sensitivity, dynamic range and frequency response.

  13. In vitro metacyclogenesis of Trypanosoma cruzi induced by starvation correlates with a transient adenylyl cyclase stimulation as well as with a constitutive upregulation of adenylyl cyclase expression.

    Science.gov (United States)

    Hamedi, Afsaneh; Botelho, Larisse; Britto, Constança; Fragoso, Stenio Perdigão; Umaki, Adriana Castilhos Souza; Goldenberg, Samuel; Bottu, Guy; Salmon, Didier

    2015-01-01

    The Trypanosoma cruzi adenylyl cyclase (AC) multigene family encodes different isoforms (around 15) sharing a variable large N-terminal domain, which is extracellular and receptor-like, followed by a transmembrane helix and a conserved C-terminal catalytic domain. It was proposed that these key enzymes in the cAMP signalling pathway allow the parasite to sense its changing extracellular milieu in order to rapidly adapt to its new environment, which is generally achieved through a differentiation process. One of the critical differentiation events the parasitic protozoan T. cruzi undergoes during its life cycle, known as metacyclogenesis, occurs in the digestive tract of the insect and corresponds to the differentiation from noninfective epimastigotes to infective metacyclic trypomastigote forms. By in vitro monitoring the activity of AC during metacyclogenesis, we showed that both the activity of AC and the intracellular cAMP content follow a similar pattern of transient stimulation in a two-step process, with a first activation peak occurring during the first hours of nutritional stress and a second peak between 6 and 48 h, corresponding to the cellular adhesion. During this differentiation process, a general mechanism of upregulation of AC expression of both mRNA and protein is triggered and in particular for a major subclass of these enzymes that are present in various gene copies commonly associated to the THT gene clusters. Although the scattered genome distribution of these gene copies is rather unusual in trypanosomatids and seems to be a recent acquisition in the evolution of the T. cruzi clade, their encoded product redistributed on the flagellum of the parasite upon differentiation could be important to sense the extracellular milieu.

  14. Adenylyl cyclase 2 selectively couples to E prostanoid type 2 receptors, whereas adenylyl cyclase 3 is not receptor-regulated in airway smooth muscle.

    Science.gov (United States)

    Bogard, Amy S; Adris, Piyatilake; Ostrom, Rennolds S

    2012-08-01

    Adenylyl cyclases (ACs) are important regulators of airway smooth muscle function, because β-adrenergic receptor (βAR) agonists stimulate AC activity and cAMP production. We have previously shown in a number of cell types that AC6 selectively couples to βAR and these proteins are coexpressed in lipid rafts. We overexpressed AC2, AC3, and AC6 in mouse bronchial smooth muscle cells (mBSMCs) and human embryonic kidney (HEK)-293 cells by using recombinant adenoviruses and assessed their localization and regulation by various G protein-coupled receptors (GPCRs). AC3 and AC6 were expressed primarily in caveolin-rich fractions, whereas AC2 expression was excluded from these domains. AC6 expression enhanced cAMP production in response to isoproterenol but did not increase responses to butaprost, reflecting the colocalization of AC6 with β(2)AR but not E prostanoid type 2 receptor (EP(2)R) in lipid raft fractions. AC2 expression enhanced butaprost-stimulated cAMP production but had no effect on the β(2)AR-mediated response. AC3 did not couple to any GPCR tested. Forskolin-induced arborization of mBSMCs was assessed as a functional readout of cAMP signaling. Arborization was enhanced by overexpression of AC6 and AC3, but AC2 had no effect. GPCR-stimulated arborization mirrored the selective coupling observed for cAMP production. With the addition of the phosphodiesterase 4 (PDE4) inhibitor rolipram AC2 accelerated forskolin-stimulated arborization. Thus, AC2 selectively couples to EP(2)R, but signals from this complex are limited by PDE4 activity. AC3 does not seem to couple to GPCR in either mBSMCs or HEK-293 cells, so it probably exists in a distinct signaling domain in these cells.

  15. ADPase activity of recombinantly expressed thermotolerant ATPases may be caused by copurification of adenylate kinase of Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Baoyu; Sysoeva, Tatyana A.; Chowdhury, Saikat; Guo, Liang; Nixon, B.Tracy; (IIT); (Penn)

    2009-10-06

    Except for apyrases, ATPases generally target only the {gamma}-phosphate of a nucleotide. Some non-apyrase ATPases from thermophilic microorganisms are reported to hydrolyze ADP as well as ATP, which has been described as a novel property of the ATPases from extreme thermophiles. Here, we describe an apparent ADP hydrolysis by highly purified preparations of the AAA+ ATPase NtrC1 from an extremely thermophilic bacterium, Aquifex aeolicus. This activity is actually a combination of the activities of the ATPase and contaminating adenylate kinase (AK) from Escherichia coli, which is present at 1/10 000 of the level of the ATPase. AK catalyzes conversion of two molecules of ADP into AMP and ATP, the latter being a substrate for the ATPase. We raise concern that the observed thermotolerance of E. coli AK and its copurification with thermostable proteins by commonly used methods may confound studies of enzymes that specifically catalyze hydrolysis of nucleoside diphosphates or triphosphates. For example, contamination with E. coli AK may be responsible for reported ADPase activities of the ATPase chaperonins from Pyrococcus furiosus, Pyrococcus horikoshii, Methanococcus jannaschii and Thermoplasma acidophilum; the ATP/ADP-dependent DNA ligases from Aeropyrum pernix K1 and Staphylothermus marinus; or the reported ATP-dependent activities of ADP-dependent phosphofructokinase of P. furiosus. Purification methods developed to separate NtrC1 ATPase from AK also revealed two distinct forms of the ATPase. One is tightly bound to ADP or GDP and able to bind to Q but not S ion exchange matrixes. The other is nucleotide-free and binds to both Q and S ion exchange matrixes.

  16. A HCO(3)(-)-dependent mechanism involving soluble adenylyl cyclase for the activation of Ca²⁺ currents in locus coeruleus neurons.

    Science.gov (United States)

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

    2014-12-01

    Hypercapnic acidosis activates Ca²⁺ channels and increases intracellular Ca²⁺ levels in neurons of the locus coeruleus, a known chemosensitive region involved in respiratory control. We have also shown that large conductance Ca²⁺-activated K⁺ channels, in conjunction with this pathway, limits the hypercapnic-induced increase in firing rate in locus coeruleus neurons. Here, we present evidence that the Ca²⁺ current is activated by a HCO(3)(-)-sensitive pathway. The increase in HCO(3)(-) associated with hypercapnia activates HCO(3)(-)-sensitive adenylyl cyclase (soluble adenylyl cyclase). This results in an increase in cyclic adenosine monophosphate levels and activation of Ca²⁺ channels via cyclic adenosine monophosphate-activated protein kinase A. We also show the presence of soluble adenylyl cyclase in the cytoplasm of locus coeruleus neurons, and that the cyclic adenosine monophosphate analogue db-cyclic adenosine monophosphate increases Ca²⁺i. Disrupting this pathway by decreasing HCO(3)(-) levels during acidification or inhibiting either soluble adenylyl cyclase or protein kinase A, but not transmembrane adenylyl cyclase, can increase the magnitude of the firing rate response to hypercapnia in locus coeruleus neurons from older neonates to the same extent as inhibition of K⁺ channels. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. NMR structural characterization of the N-terminal domain of the adenylyl cyclase-associated protein (CAP) from Dictyostelium discoideum

    Energy Technology Data Exchange (ETDEWEB)

    Mavoungou, Chrystelle [Max Planck Institute for Biochemistry (Germany); Israel, Lars [Ludwig Maximilians-University, Adolf Butenandt Institute, Cell Biology (Germany); Rehm, Till; Ksiazek, Dorota; Krajewski, Marcin; Popowicz, Grzegorz [Max Planck Institute for Biochemistry (Germany); Noegel, Angelika A. [University of Cologne, Institute for Biochemistry (Germany); Schleicher, Michael [Ludwig Maximilians-University, Adolf Butenandt Institute, Cell Biology (Germany); Holak, Tad A. [Max Planck Institute for Biochemistry (Germany)

    2004-05-15

    Cyclase-associated proteins (CAPs) are highly conserved, ubiquitous actin binding proteins that are involved in microfilament reorganization. The N-termini of CAPs play a role in Ras signaling and bind adenylyl cyclase; the C-termini bind to G-actin. We report here the NMR characterization of the amino-terminal domain of CAP from Dictyostelium discoideum (CAP(1-226)). NMR data, including the steady state {sup 1}H-{sup 15}N heteronuclear NOE experiments, indicate that the first 50 N-terminal residues are unstructured and that this highly flexible serine-rich fragment is followed by a stable, folded core starting at Ser 51. The NMR structure of the folded core is an {alpha}-helix bundle composed of six antiparallel helices, in a stark contrast to the recently determined CAP C-terminal domain structure, which is solely built by {beta}-strands.

  18. Structure of the N-terminal domain of the adenylyl cyclase-associated protein (CAP) from Dictyostelium discoideum.

    Science.gov (United States)

    Ksiazek, Dorota; Brandstetter, Hans; Israel, Lars; Bourenkov, Gleb P; Katchalova, Galina; Janssen, Klaus-Peter; Bartunik, Hans D; Noegel, Angelika A; Schleicher, Michael; Holak, Tad A

    2003-09-01

    Cyclase-associated proteins (CAPs) are widely distributed and highly conserved proteins that regulate actin remodeling in response to cellular signals. The N termini of CAPs play a role in Ras signaling and bind adenylyl cyclase; the C termini bind to G-actin and thereby alter the dynamic rearrangements of the microfilament system. We report here the X-ray structure of the core of the N-terminal domain of the CAP from Dictyostelium discoideum, which comprises residues 51-226, determined by a combination of single isomorphous replacement with anomalous scattering (SIRAS). The overall structure of this fragment is an alpha helix bundle composed of six antiparallel helices. Results from gel filtration and crosslinking experiments for CAP(1-226), CAP(255-464), and the full-length protein, together with the CAP N-terminal domain structure and the recently determined CAP C-terminal domain structure, provide evidence that the functional structure of CAP is multimeric.

  19. NMR structural characterization of the N-terminal domain of the adenylyl cyclase-associated protein (CAP) from Dictyostelium discoideum.

    Science.gov (United States)

    Mavoungou, Chrystelle; Israel, Lars; Rehm, Till; Ksiazek, Dorota; Krajewski, Marcin; Popowicz, Grzegorz; Noegel, Angelika A; Schleicher, Michael; Holak, Tad A

    2004-05-01

    Cyclase-associated proteins (CAPs) are highly conserved, ubiquitous actin binding proteins that are involved in microfilament reorganization. The N-termini of CAPs play a role in Ras signaling and bind adenylyl cyclase; the C-termini bind to G-actin. We report here the NMR characterization of the amino-terminal domain of CAP from Dictyostelium discoideum (CAP(1-226)). NMR data, including the steady state (1)H-(15)N heteronuclear NOE experiments, indicate that the first 50 N-terminal residues are unstructured and that this highly flexible serine-rich fragment is followed by a stable, folded core starting at Ser 51. The NMR structure of the folded core is an alpha-helix bundle composed of six antiparallel helices, in a stark contrast to the recently determined CAP C-terminal domain structure, which is solely built by beta-strands.

  20. Somatic 'soluble' adenylyl cyclase isoforms are unaffected in Sacy tm1Lex/Sacy tm1Lex 'knockout' mice.

    Science.gov (United States)

    Farrell, Jeanne; Ramos, Lavoisier; Tresguerres, Martin; Kamenetsky, Margarita; Levin, Lonny R; Buck, Jochen

    2008-09-22

    Mammalian Soluble adenylyl cyclase (sAC, Adcy10, or Sacy) represents a source of the second messenger cAMP distinct from the widely studied, G protein-regulated transmembrane adenylyl cyclases. Genetic deletion of the second through fourth coding exons in Sacy(tm1Lex)/Sacy(tm1Lex) knockout mice results in a male sterile phenotype. The absence of any major somatic phenotype is inconsistent with the variety of somatic functions identified for sAC using pharmacological inhibitors and RNA interference. We now use immunological and molecular biological methods to demonstrate that somatic tissues express a previously unknown isoform of sAC, which utilizes a unique start site, and which 'escapes' the design of the Sacy(tm1Lex) knockout allele. These studies reveal increased complexity at the sAC locus, and they suggest that the known isoforms of sAC play a unique function in male germ cells.

  1. Covalent aspartylation of aspartyl-tRNA synthetase from Bakers' yeast by its cognat aspartyl adenylate: identification of the labeled residues

    Energy Technology Data Exchange (ETDEWEB)

    Mejdoub, H.; Kern, D.; Giege, R.; Ebel, J.P.; Boulanger, Y.; Reinbolt, J.

    1987-04-07

    Aspartyl-tRNA synthetase from bakers' yeast gives an unstable complex with the cognate adenylate, which reacts after dissociation with amino acid side chains of the protein. This leads to a covalent incorporation of (/sup 14/C)-aspartic acid into aspartyl-tRNA synthetase via amide or ester bonds formed between the ..cap alpha..-carboxyl group of activated aspartic acid and accessible lysines, serines, and threonines. This property is used to label the peptides at the surface of the enzyme. The main labeled residues have been identified, and their location in the primary structure is discussed in relation to structural properties of aspartyl-tRNA synthetase.

  2. Crystal structures at 2.5 Angstrom resolution of seryl-tRNA synthetase complexed with two analogs of seryl adenylate

    DEFF Research Database (Denmark)

    Belrhali, H.; Yaremchuk, A.; Tukalo, M.;

    1994-01-01

    Crystal structures of seryl-tRNA synthetase from Thermus thermophilus complexed with two different analogs of seryl adenylate have been determined at 2.5 Angstrom resolution. The first complex is between the enzyme and seryl-hydroxamate-AMP (adenosine monophosphate), produced enzymatically...... in a deep hydrophilic cleft formed by the antiparallel beta sheet and surrounding loops of the synthetase catalytic domain. Four regions in the primary sequence are involved in the interactions, including the motif 2 and 3 regions of class 2 synthetases. Apart from the specific recognition of the serine...

  3. The role of cyclase-associated protein in regulating actin filament dynamics – more than a monomer-sequestration factor

    OpenAIRE

    Ono, Shoichiro

    2013-01-01

    Dynamic reorganization of the actin cytoskeleton is fundamental to a number of cell biological events. A variety of actin-regulatory proteins modulate polymerization and depolymerization of actin and contribute to actin cytoskeletal reorganization. Cyclase-associated protein (CAP) is a conserved actin-monomer-binding protein that has been studied for over 20 years. Early studies have shown that CAP sequesters actin monomers; recent studies, however, have revealed more active roles of CAP in a...

  4. Cyclase-associated Protein 1 (CAP1) Promotes Cofilin-induced Actin Dynamics in Mammalian Nonmuscle CellsV⃞

    OpenAIRE

    Bertling, Enni; Hotulainen, Pirta; Mattila, Pieta K.; Matilainen, Tanja; Salminen, Marjo; Lappalainen, Pekka

    2004-01-01

    Cyclase-associated proteins (CAPs) are highly conserved actin monomer binding proteins present in all eukaryotes. However, the mechanism by which CAPs contribute to actin dynamics has been elusive. In mammals, the situation is further complicated by the presence of two CAP isoforms whose differences have not been characterized. Here, we show that CAP1 is widely expressed in mouse nonmuscle cells, whereas CAP2 is the predominant isoform in developing striated muscles. In cultured NIH3T3 and B1...

  5. Temporal and Regional Regulation of Gene Expression by Calcium-Stimulated Adenylyl Cyclase Activity during Fear Memory

    OpenAIRE

    Lindsay Wieczorek; James W Maas; Muglia, Lisa M.; Vogt, Sherri K.; Muglia, Louis J.

    2010-01-01

    BACKGROUND: The Ca2+-stimulated adenylyl cyclases (ACs), AC1 and AC8, are key components of long-term memory processing. AC1 and AC8 double knockout mice (Adcy1(-/-)Adcy8(-/-); DKO) display impaired fear memory processing; the mechanism of this impairment is largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We hypothesize that the Ca2+-stimulated ACs modulate long-lasting transcriptional changes essential for fear memory consolidation and maintenance. Here, we report a genome-wide study of gen...

  6. Atrial natriuretic factor receptor guanylate cyclase, ANF-RGC, transduces two independent signals, ANF and Ca2+

    Directory of Open Access Journals (Sweden)

    Teresa eDuda

    2014-03-01

    Full Text Available Atrial natriuretic factor receptor guanylate cyclase, ANF-RGC, was the first discovered member of the mammalian membrane guanylate cyclase family. The hallmark feature of the family is that a single protein contains both the site for recognition of the regulatory signal and the ability to transduce it into the production of the second messenger, cyclic GMP. For over two decades, the family has been classified into two subfamilies, the hormone receptor subfamily with ANF-RGC being its paramount member, and the Ca2+ modulated subfamily, which includes the rod outer segment guanylate cyclases, ROS-GC1 and 2, and the olfactory neuroepithelial guanylate cyclase, ONE-GC. ANF-RGC is the receptor and the signal transducer of the most hypotensive hormones, atrial natriuretic factor (ANF and B-type natriuretic peptide (BNP. After binding these hormones at the extracellular domain it, at its intracellular domain, signals activation of the C-terminal catalytic module and accelerates the production of cyclic GMP. Cyclic GMP then serves the second messenger role in biological responses of ANF and BNP such as natriuresis, diuresis, vasorelaxation and anti-proliferation. Very recently another modus operandi for ANF-RGC was revealed. Its crux is that ANF-RGC activity is also regulated by Ca2+. The Ca2+ sensor neurocalcin  mediates this signaling mechanism. Strikingly, the Ca2+ and ANF signaling mechanisms employ separate structural motifs of ANF-RGC in modulating its core catalytic domain in accelerating the production of cyclic GMP. In this review the biochemistry and physiology of these mechanisms with emphasis on cardiovascular regulation will be discussed.

  7. The CD38-independent ADP-ribosyl cyclase from mouse brain synaptosomes: a comparative study of neonate and adult brain.

    Science.gov (United States)

    Ceni, Claire; Pochon, Nathalie; Villaz, Michel; Muller-Steffner, Hélène; Schuber, Francis; Baratier, Julie; De Waard, Michel; Ronjat, Michel; Moutin, Marie-Jo

    2006-04-15

    cADPR (cADP-ribose), a metabolite of NAD+, is known to modulate intracellular calcium levels and to be involved in calcium-dependent processes, including synaptic transmission, plasticity and neuronal excitability. However, the enzyme that is responsible for producing cADPR in the cytoplasm of neural cells, and particularly at the synaptic terminals of neurons, remains unknown. In the present study, we show that endogenous concentrations of cADPR are much higher in embryonic and neonate mouse brain compared with the adult tissue. We also demonstrate, by comparing wild-type and Cd38-/- tissues, that brain cADPR content is independent of the presence of CD38 (the best characterized mammalian ADP-ribosyl cyclase) not only in adult but also in developing tissues. We show that Cd38-/- synaptosome preparations contain high ADP-ribosyl cyclase activities, which are more important in neonates than in adults, in line with the levels of endogenous cyclic nucleotide. By using an HPLC method and adapting the cycling assay developed initially to study endogenous cADPR, we accurately examined the properties of the synaptosomal ADP-ribosyl cyclase. This intracellular enzyme has an estimated K(m) for NAD+ of 21 microM, a broad optimal pH at 6.0-7.0, and the concentration of free calcium has no major effect on its cADPR production. It binds NGD+ (nicotinamide-guanine dinucleotide), which inhibits its NAD+-metabolizing activities (K(i)=24 microM), despite its incapacity to cyclize this analogue. Interestingly, it is fully inhibited by low (micromolar) concentrations of zinc. We propose that this novel mammalian ADP-ribosyl cyclase regulates the production of cADPR and therefore calcium levels within brain synaptic terminals. In addition, this enzyme might be a potential target of neurotoxic Zn2+.

  8. CelR, an Ortholog of the Diguanylate Cyclase PleD of Caulobacter, Regulates Cellulose Synthesis in Agrobacterium tumefaciens

    OpenAIRE

    Barnhart, D. Michael; Su, Shengchang; Baccaro, Brenna E.; Banta, Lois M.; Farrand, Stephen K.

    2013-01-01

    Cellulose fibrils play a role in attachment of Agrobacterium tumefaciens to its plant host. While the genes for cellulose biosynthesis in the bacterium have been identified, little is known concerning the regulation of the process. The signal molecule cyclic di-GMP (c-di-GMP) has been linked to the regulation of exopolysaccharide biosynthesis in many bacterial species, including A. tumefaciens. In this study, we identified two putative diguanylate cyclase genes, celR (atu1297) and atu1060, th...

  9. Protein-protein docking and analysis reveal that two homologous bacterial adenylyl cyclase toxins interact with calmodulin differently.

    Science.gov (United States)

    Guo, Qing; Jureller, Justin E; Warren, Julia T; Solomaha, Elena; Florián, Jan; Tang, Wei-Jen

    2008-08-29

    Calmodulin (CaM), a eukaryotic calcium sensor that regulates diverse biological activities, consists of N- and C-terminal globular domains (N-CaM and C-CaM, respectively). CaM serves as the activator of CyaA, a 188-kDa adenylyl cyclase toxin secreted by Bordetella pertussis, which is the etiologic agent for whooping cough. Upon insertion of the N-terminal adenylyl cyclase domain (ACD) of CyaA to its targeted eukaryotic cells, CaM binds to this domain tightly ( approximately 200 pm affinity). This interaction activates the adenylyl cyclase activity of CyaA, leading to a rise in intracellular cAMP levels to disrupt normal cellular signaling. We recently solved the structure of CyaA-ACD in complex with C-CaM to elucidate the mechanism of catalytic activation. However, the structure of the interface between N-CaM and CyaA, the formation of which contributes a 400-fold increase of binding affinity between CyaA and CaM, remains elusive. Here, we used site-directed mutations and molecular dynamic simulations to generate several working models of CaM-bound CyaA-ACD. The validity of these models was evaluated by disulfide bond cross-linking, point mutations, and fluorescence resonance energy transfer experiments. Our study reveals that a beta-hairpin region (amino acids 259-273) of CyaA-ACD likely makes contacts with the second calcium binding motif of the extended CaM. This mode of interaction differs from the interaction of N-CaM with anthrax edema factor, which binds N-CaM via its helical domain. Thus, two structurally conserved, bacterial adenylyl cyclase toxins have evolved to utilize distinct binding surfaces and modes of activation in their interaction with CaM, a highly conserved eukaryotic signaling protein.

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

    Directory of Open Access Journals (Sweden)

    Kabir Hassan Biswas

    2015-04-01

    Full Text Available 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.

  11. Molecular cloning and characterization of a rat homolog of CAP, the adenylyl cyclase-associated protein from Saccharomyces cerevisiae.

    Science.gov (United States)

    Zelicof, A; Gatica, J; Gerst, J E

    1993-06-25

    We have isolated a rat cDNA whose expression suppresses the physiological consequences of the chromosomal disruption of CAP, the gene encoding the adenylyl cyclase-associated protein of Saccharomyces cerevisiae. Yeast CAP is a bifunctional protein: the NH2 terminus is necessary and sufficient for cellular responsiveness to activated RAS proteins, while the COOH terminus is required for normal cellular morphology and growth control. The rat MCH1 cDNA encodes a protein of 474 amino acids that is 36% identical to S. cerevisiae CAP and is capable of suppressing the loss of the COOH-terminal functions of CAP when expressed in yeast. The MCH1 protein therefore appears to be a structural and functional homolog of the yeast cyclase-associated proteins. Northern analysis of MCH1 gene expression shows it to be constitutively expressed in all cell and tissue types examined. The cloning of a rat homolog of CAP, in addition to the cloning of a human CAP homolog by Matviw et al. (Matviw, H., Yu, G., and Young, D. (1992) Mol. Cell. Biol. 12, 5033-5040), demonstrates that both cyclase-associated proteins and their functions may have evolved with mammalian cells.

  12. Extracellular regulation of sperm transmembrane adenylyl cyclase by a forward motility stimulating protein.

    Directory of Open Access Journals (Sweden)

    Souvik Dey

    Full Text Available 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 tm

  13. Structural insights for activation of retinal guanylate cyclase by GCAP1.

    Directory of Open Access Journals (Sweden)

    Sunghyuk Lim

    Full Text Available Guanylyl cyclase activating protein 1 (GCAP1, a member of the neuronal calcium sensor (NCS subclass of the calmodulin superfamily, confers Ca(2+-sensitive activation of retinal guanylyl cyclase 1 (RetGC1 upon light activation of photoreceptor cells. Here we present NMR assignments and functional analysis to probe Ca(2+-dependent structural changes in GCAP1 that control activation of RetGC. NMR assignments were obtained for both the Ca(2+-saturated inhibitory state of GCAP1 versus a GCAP1 mutant (D144N/D148G, called EF4mut, which lacks Ca(2+ binding in EF-hand 4 and models the Ca(2+-free/Mg(2+-bound activator state of GCAP1. NMR chemical shifts of backbone resonances for Ca(2+-saturated wild type GCAP1 are overall similar to those of EF4mut, suggesting a similar main chain structure for assigned residues in both the Ca(2+-free activator and Ca(2+-bound inhibitor states. This contrasts with large Ca(2+-induced chemical shift differences and hence dramatic structural changes seen for other NCS proteins including recoverin and NCS-1. The largest chemical shift differences between GCAP1 and EF4mut are seen for residues in EF4 (S141, K142, V145, N146, G147, G149, E150, L153, E154, M157, E158, Q161, L166, but mutagenesis of EF4 residues (F140A, K142D, L153R, L166R had little effect on RetGC1 activation. A few GCAP1 residues in EF-hand 1 (K23, T27, G32 also show large chemical shift differences, and two of the mutations (K23D and G32N each decrease the activation of RetGC, consistent with a functional conformational change in EF1. GCAP1 residues at the domain interface (V77, A78, L82 have NMR resonances that are exchange broadened, suggesting these residues may be conformationally dynamic, consistent with previous studies showing these residues are in a region essential for activating RetGC1.

  14. Membrane Guanylate Cyclase catalytic Subdomain: Structure and Linkage with Calcium Sensors and Bicarbonate

    Directory of Open Access Journals (Sweden)

    Sarangan Ravichandran

    2017-06-01

    Full Text Available Membrane guanylate cyclase (MGC is a ubiquitous multi-switching cyclic GMP generating signaling machine linked with countless physiological processes. In mammals it is encoded by seven distinct homologous genes. It is a single transmembrane spanning multi-modular protein; composed of integrated blocks and existing in homo-dimeric form. Its core catalytic domain (CCD module is a common transduction center where all incoming signals are translated into the production of cyclic GMP, a cellular signal second messenger. Crystal structure of the MGC’s CCD does not exist and its precise identity is ill-defined. Here, we define it at a sub-molecular level for the phototransduction-linked MGC, the rod outer segment guanylate cyclase type 1, ROS-GC1. (1 The CCD is a conserved 145-residue structural unit, represented by the segment V820-P964. (2 It exists as a homo-dimer and contains seven conserved catalytic elements (CEs wedged into seven conserved motifs. (3 It also contains a conserved 21-residue neurocalcin δ-modulated structural domain, V836-L857. (4 Site-directed mutagenesis documents that each of the seven CEs governs the cyclase’s catalytic activity. (5 In contrast to the soluble and the bacterium MGC which use Mn2+-GTP substrate for catalysis, MGC CCD uses the natural Mg2+-GTP substrate. (6 Strikingly, the MGC CCD requires anchoring by the Transmembrane Domain (TMD to exhibit its major (∼92% catalytic activity; in isolated form the activity is only marginal. This feature is not linked with any unique sequence of the TMD; there is minimal conservation in TMD. Finally, (7 the seven CEs control each of four phototransduction pathways- -two Ca2+-sensor GCAPs-, one Ca2+-sensor, S100B-, and one bicarbonate-modulated. The findings disclose that the CCD of ROS-GC1 has built-in regulatory elements that control its signal translational activity. Due to conservation of these regulatory elements, it is proposed that these elements also control the

  15. NCBI nr-aa BLAST: CBRC-MDOM-11-0110 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MDOM-11-0110 sp|P30804|ADCY6_CANFA RecName: Full=Adenylate cyclase type 6; AltName: Full...=Adenylate cyclase type VI; AltName: Full=ATP pyrophosphate-lyase 6; AltName: Full=Adenylyl cyclase 6; AltName: Full=Ca(2+)-inhibitable adenylyl cyclase P30804 1e-123 75% ...

  16. Mechanism of oligomerisation of cyclase-associated protein from Dictyostelium discoideum in solution.

    Science.gov (United States)

    Yusof, Adlina Mohd; Jaenicke, Elmar; Pedersen, Jan Skov; Noegel, Angelika A; Schleicher, Michael; Hofmann, Andreas

    2006-10-06

    Cyclase-associated protein (CAP) is a highly conserved modular protein implicated in the regulation of actin filament dynamics and a variety of developmental and morphological processes. The protein exists as a high molecular weight complex in cell extracts and purified protein possesses a high tendency to aggregate, a major obstacle for crystallisation. Using a mutagenesis approach, we show that two structural features underlie the mechanism of oligomerisation in Dictyostelium discoideum CAP. Positively charged clusters on the surface of the N-terminal helix-barrel domain are involved in inter-molecular interactions with the N or C-terminal domains. Abolishing these interactions mainly renders dimers due to a domain swap feature in the extreme C-terminal region of the protein that was previously described. Based on earlier studies with yeast CAP, we also generated constructs with mutations in the extreme N-terminal region of Dictyostelium CAP that did not show significantly altered oligomerisation behaviour. Constructs with mutations in the earlier identified protein-protein interaction interface on the N-terminal domain of CAP could not be expressed as soluble protein. Assessment of the soluble proteins indicates that the mutations did not affect their overall fold. Further studies point to the correlation between stability of full-length CAP with its multimerisation behaviour, where oligomer formation leads to a more stable protein.

  17. Crystal structure of the actin binding domain of the cyclase-associated protein.

    Science.gov (United States)

    Dodatko, Tetyana; Fedorov, Alexander A; Grynberg, Marcin; Patskovsky, Yury; Rozwarski, Denise A; Jaroszewski, Lukasz; Aronoff-Spencer, Eliah; Kondraskina, Elena; Irving, Tom; Godzik, Adam; Almo, Steven C

    2004-08-24

    Cyclase-associated protein (CAP or Srv2p) is a modular actin monomer binding protein that directly regulates filament dynamics and has been implicated in a number of complex developmental and morphological processes, including mRNA localization and the establishment of cell polarity. The crystal structure of the C-terminal dimerization and actin monomer binding domain (C-CAP) reveals a highly unusual dimer, composed of monomers possessing six coils of right-handed beta-helix flanked by antiparallel beta-strands. Domain swapping, involving the last two strands of each monomer, results in the formation of an extended dimer with an extensive interface. This structural and biochemical characterization provides new insights into the organization and potential mechanistic properties of the multiprotein assemblies that integrate dynamic actin processes into the overall physiology of the cell. An unanticipated finding is that the unique tertiary structure of the C-CAP monomer provides a structural model for a wide range of molecules, including RP2 and cofactor C, proteins involved in X-linked retinitis pigmentosa and tubulin maturation, respectively, as well as several uncharacterized proteins that exhibit very diverse domain organizations. Thus, the unusual right-handed beta-helical fold present in C-CAP appears to support a wide range of biological functions.

  18. Ablation of cyclase-associated protein 2 (CAP2) leads to cardiomyopathy.

    Science.gov (United States)

    Peche, Vivek S; Holak, Tad A; Burgute, Bhagyashri D; Kosmas, Kosmas; Kale, Sushant P; Wunderlich, F Thomas; Elhamine, Fatiha; Stehle, Robert; Pfitzer, Gabriele; Nohroudi, Klaus; Addicks, Klaus; Stöckigt, Florian; Schrickel, Jan W; Gallinger, Julia; Schleicher, Michael; Noegel, Angelika A

    2013-02-01

    Cyclase-associated proteins are highly conserved proteins that have a role in the regulation of actin dynamics. Higher eukaryotes have two isoforms, CAP1 and CAP2. To study the in vivo function of CAP2, we generated mice in which the CAP2 gene was inactivated by a gene-trap approach. Mutant mice showed a decrease in body weight and had a decreased survival rate. Further, they developed a severe cardiac defect marked by dilated cardiomyopathy (DCM) associated with drastic reduction in basal heart rate and prolongations in atrial and ventricular conduction times. Moreover, CAP2-deficient myofibrils exhibited reduced cooperativity of calcium-regulated force development. At the microscopic level, we observed disarrayed sarcomeres with development of fibrosis. We analyzed CAP2's role in actin assembly and found that it sequesters G-actin and efficiently fragments filaments. This activity resides completely in its WASP homology domain. Thus CAP2 is an essential component of the myocardial sarcomere and is essential for physiological functioning of the cardiac system, and a deficiency leads to DCM and various cardiac defects.

  19. Molecular Cloning, and Characterization of an Adenylyl Cyclase-Associated Protein from Gossypium arboreum L.

    Institute of Scientific and Technical Information of China (English)

    WANG Sheng; ZHAO Guo-hong; JIA Yin-hua; DU Xiong-ming

    2009-01-01

    The aim of this study was to clone CAP (adenylyl cyclase-associated protein) gene from Gossypium arboreum L. and develop a platform for expressing and purifying CAP protein, which is a base for the construction and function researches of CAP. In this work, a CAP homolog from cotton (DPL971) ovule was identified and cloned. And the cDNA sequence consisted of an open reading frame of 1416 nucleotides encoding a protein of 471 amino acid residues with a calculated molecular weight of 50.6 kDa. To gain insight on the CAP role in cotton fiber development, the cloned CAP cDNA was expressed. A significant higher yield pure protein was obtained with the chromatographic method. Further experiments showed that the purified protein can bind with the actin in vitro indicating that the recombinant cotton CAP is functional. The procedure described here produced high yield pure protein through one chromatographic step, suitable for further structure-function studies.

  20. CAP2, cyclase-associated protein 2, is a dual compartment protein.

    Science.gov (United States)

    Peche, V; Shekar, S; Leichter, M; Korte, H; Schröder, R; Schleicher, M; Holak, T A; Clemen, C S; Ramanath-Y, B; Pfitzer, G; Karakesisoglou, I; Noegel, A A

    2007-10-01

    Cyclase-associated proteins (CAPs) are evolutionarily conserved proteins with roles in regulating the actin cytoskeleton and in signal transduction. Mammals have two CAP genes encoding the related CAP1 and CAP2. We studied the distribution and subcellular localization of CAP1 and CAP2 using specific antibodies. CAP1 shows a broad tissue distribution, whereas CAP2 is significantly expressed only in brain, heart and skeletal muscle, and skin. CAP2 is found in the nucleus in undifferentiated myoblasts and at the M-line of differentiated myotubes. In PAM212, a mouse keratinocyte cell line, CAP2 is enriched in the nucleus, and sparse in the cytosol. By contrast, CAP1 localizes to the cytoplasm in PAM212 cells. In human skin, CAP2 is present in all living layers of the epidermis localizing to the nuclei and the cell periphery. In in vitro studies, a C-terminal fragment of CAP2 interacts with actin, indicating that CAP2 has the capacity to bind to actin.

  1. Guanylyl cyclase C and guanylin reduce fat droplet accumulation in cattle mesenteric adipose tissue.

    Science.gov (United States)

    Yasuda, Masahiro; Kawabata, Jyunya; Akieda-Asai, Sayaka; Nasu, Tetsuo; Date, Yukari

    2017-09-30

    Guanylyl cyclase C (GC-C) is a member of a family of enzymes that metabolize GTP to cGMP and was first identified as a receptor for heat-stable enterotoxin. Guanylin (GNY) has since been identified as an endogenous ligand for GC-C in the intestine of several mammalian species. The GNY/GC-C system regulates ion transportation and pH in the mucosa. Recently, it was reported that GC-C and GNY are involved in lipid metabolism in rat mesenteric adipose tissue macrophages. To examine the role of GC-C and GNY in lipid metabolism in cattle, we used a bovine mesenteric adipocyte primary culture system and a coculture system for bovine adipocytes and GNY-/GC-C-expressing macrophages. Fat droplets were observed to accumulate in bovine mesenteric adipocytes cultured alone, whereas few fat droplets accumulated in adipocytes indirectly cocultured with macrophages. We also observed that GC-C was present in bovine mesenteric adipose tissue, and that fat droplet accumulation decreased after in vitro GNY administration. Expressions of mRNAs encoding lipogenic factors decreased significantly in adipocytes after either coculture or GNY administration. These results suggest that the GNY/GC-C system is part of the control system for lipid accumulation in bovine mesenteric adipose tissue.

  2. cNMP-AMs mimic and dissect bacterial nucleotidyl cyclase toxin effects.

    Science.gov (United States)

    Beckert, Ulrike; Grundmann, Manuel; Wolter, Sabine; Schwede, Frank; Rehmann, Holger; Kaever, Volkhard; Kostenis, Evi; Seifert, Roland

    2014-09-05

    In addition to the well-known second messengers cAMP and cGMP, mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. The Pseudomonas aeruginosa toxin ExoY massively increases cGMP and cUMP in cells, whereas the Bordetella pertussis toxin CyaA increases cAMP and, to a lesser extent, cCMP. To mimic and dissect toxin effects, we synthesized cNMP-acetoxymethylesters as prodrugs. cNMP-AMs rapidly and effectively released the corresponding cNMP in cells. The combination of cGMP-AM plus cUMP-AM mimicked cytotoxicity of ExoY. cUMP-AM and cGMP-AM differentially activated gene expression. Certain cCMP and cUMP effects were independent of the known cNMP effectors protein kinases A and G and guanine nucleotide exchange factor Epac. In conclusion, cNMP-AMs are useful tools to mimic and dissect bacterial nucleotidyl cyclase toxin effects.

  3. Activity Regulation by Heteromerization of Arabidopsis Allene Oxide Cyclase Family Members

    Science.gov (United States)

    Otto, Markus; Naumann, Christin; Brandt, Wolfgang; Wasternack, Claus; Hause, Bettina

    2016-01-01

    Jasmonates (JAs) are lipid-derived signals in plant stress responses and development. A crucial step in JA biosynthesis is catalyzed by allene oxide cyclase (AOC). Four genes encoding functional AOCs (AOC1, AOC2, AOC3 and AOC4) have been characterized for Arabidopsis thaliana in terms of organ- and tissue-specific expression, mutant phenotypes, promoter activities and initial in vivo protein interaction studies suggesting functional redundancy and diversification, including first hints at enzyme activity control by protein-protein interaction. Here, these analyses were extended by detailed analysis of recombinant proteins produced in Escherichia coli. Treatment of purified AOC2 with SDS at different temperatures, chemical cross-linking experiments and protein structure analysis by molecular modelling approaches were performed. Several salt bridges between monomers and a hydrophobic core within the AOC2 trimer were identified and functionally proven by site-directed mutagenesis. The data obtained showed that AOC2 acts as a trimer. Finally, AOC activity was determined in heteromers formed by pairwise combinations of the four AOC isoforms. The highest activities were found for heteromers containing AOC4 + AOC1 and AOC4 + AOC2, respectively. All data are in line with an enzyme activity control of all four AOCs by heteromerization, thereby supporting a putative fine-tuning in JA formation by various regulatory principles. PMID:27135223

  4. Structure of a diguanylate cyclase from Thermotoga maritima: insights into activation, feedback inhibition and thermostability.

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    Angeline Deepthi

    Full Text Available Large-scale production of bis-3'-5'-cyclic-di-GMP (c-di-GMP would facilitate biological studies of numerous bacterial signaling pathways and phenotypes controlled by this second messenger molecule, such as virulence and biofilm formation. C-di-GMP constitutes also a potentially interesting molecule as a vaccine adjuvant. Even though chemical synthesis of c-di-GMP can be done, the yields are incompatible with mass-production. tDGC, a stand-alone diguanylate cyclase (DGC or GGDEF domain from Thermotoga maritima, enables the robust enzymatic production of large quantities of c-di-GMP. To understand the structural correlates of tDGC thermostability, its catalytic mechanism and feedback inhibition, we determined structures of an active-like dimeric conformation with both active (A sites facing each other and of an inactive dimeric conformation, locked by c-di-GMP bound at the inhibitory (I site. We also report the structure of a single mutant of tDGC, with the R158A mutation at the I-site, abolishing product inhibition and unproductive dimerization. A comparison with structurally characterized DGC homologues from mesophiles reveals the presence of a higher number of salt bridges in the hyperthermophile enzyme tDGC. Denaturation experiments of mutants disrupting in turn each of the salt bridges unique to tDGC identified three salt-bridges critical to confer thermostability.

  5. Regulation of oxidative response and extracellular polysaccharide synthesis by a diadenylate cyclase in Streptococcus mutans.

    Science.gov (United States)

    Cheng, Xingqun; Zheng, Xin; Zhou, Xuedong; Zeng, Jumei; Ren, Zhi; Xu, Xin; Cheng, Lei; Li, Mingyun; Li, Jiyao; Li, Yuqing

    2016-03-01

    Cyclic diadenosine monophosphate (c-di-AMP) has been implicated in the control of many important bacterial activities. However, the function of this molecule in Streptococcus mutans, the primary aetiological agent of human dental caries, is unknown. In this study, we identified and characterized a diadenylate cyclase, named CdaA, in S. mutans. Furthermore, we showed that in-frame deletion of the cdaA gene in S. mutans causes decreased c-di-AMP levels, increased sensitivity to hydrogen peroxide and increased production of extracellular polysaccharides. Global gene expression profiling revealed that more than 200 genes were significantly upregulated or downregulated (> 2.0-fold) in the cdaA mutant. Interestingly, genes with increased or decreased expression were clustered in cellular polysaccharide biosynthetic processes and oxidoreductase activity respectively. Notably, the expression of several genomic islands, such as GTF-B/C, TnSmu, CRISPR1-Cas and CRISPR2-Cas, was found to be altered in the cdaA mutant, indicating a possible link between these genomic islands and c-di-AMP signalling. Collectively, the results reported here show that CdaA is an important global modulator in S. mutans and is required for optimal growth and environmental adaption. This report also paves the way to unveil further the roles of c-di-AMP signalling networks in the biology and pathogenicity of S. mutans.

  6. Soluble Guanylate Cyclase Stimulators: a Novel Treatment Option for Heart Failure Associated with Cardiorenal Syndromes?

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  8. Characterization and expression of soluble guanylate cyclase in skins and melanocytes of sheep.

    Science.gov (United States)

    Yang, Shanshan; Zhang, Junzhen; Ji, Kaiyuan; Jiao, Dingxing; Fan, Ruiwen; Dong, Changsheng

    2016-04-01

    The study reported the characterization of soluble guanylate cyclase (sGC) with the size of CDS of 1860bp, encoding a protein of 620 amino acids and containing several conserved functional domains including HNOB, HNOBA, and CHD. Quantitative real time PCR analysis of sGC showed that the expression of sGC mRNA is higher (∼5 fold) in white sheep skin relative to black sheep skin with significant difference (Pmelanocytes in vitro of sheep skin. Over expression of sGC in melanocytes resulted in decreased expression of key melanogenic genes including microphthalmia transcription factor (MITF), tyrosinase (TYR), tyrosinase related protein 1(TYRTP1), and tyrosinase related protein 2(TYRP2) both at mRNA and protein level. Moreover, the melanocytes was capable of producing cGMP and cAMP. The observed differential expression and localization of sGC in sheep skins and melanocytes and the capability of producing cGMP and cAMP, which suggested a potential role for this gene in hair color regulation.

  9. Role of signal transduction crosstalk between adenylyl cyclase and MAP kinase in hippocampus-dependent memory.

    Science.gov (United States)

    Xia, Zhengui; Storm, Daniel R

    2012-08-16

    One of the intriguing questions in neurobiology is how long-term memory (LTM) traces are established and maintained in the brain. Memory can be divided into at least two temporally and mechanistically distinct forms. Short-term memory (STM) lasts no longer than several hours, while LTM persists for days or longer. A crucial step in the generation of LTM is consolidation, a process in which STM is converted to LTM. Hippocampus-dependent LTM depends on activation of Ca(2+), Erk/MAP kinase (MAPK), and cAMP signaling pathways, as well as de novo gene expression and translation. One of the transcriptional pathways strongly implicated in LTM is the CREB/CRE (calcium, cAMP response element) transcriptional pathway. Interestingly, this transcriptional pathway may also contribute to other forms of neuroplasticity including adaptive responses to drugs. Evidence discussed in this review indicates that activation of the Erk1/2 MAP Kinase (MAPK)/CRE transcriptional pathway during the formation of hippocampus-dependent memory depends on calmodulin (CaM)-stimulated adenylyl cyclases.

  10. The Functional State of Hormone-Sensitive Adenylyl Cyclase Signaling System in Diabetes Mellitus

    Directory of Open Access Journals (Sweden)

    Alexander O. Shpakov

    2013-01-01

    Full Text Available Diabetes mellitus (DM induces a large number of diseases of the nervous, cardiovascular, and some other systems of the organism. One of the main causes of the diseases is the changes in the functional activity of hormonal signaling systems which lead to the alterations and abnormalities of the cellular processes and contribute to triggering and developing many DM complications. The key role in the control of physiological and biochemical processes belongs to the adenylyl cyclase (AC signaling system, sensitive to biogenic amines and polypeptide hormones. The review is devoted to the changes in the GPCR-G protein-AC system in the brain, heart, skeletal muscles, liver, and the adipose tissue in experimental and human DM of the types 1 and 2 and also to the role of the changes in AC signaling in the pathogenesis and etiology of DM and its complications. It is shown that the changes of the functional state of hormone-sensitive AC system are dependent to a large extent on the type and duration of DM and in experimental DM on the model of the disease. The degree of alterations and abnormalities of AC signaling pathways correlates very well with the severity of DM and its complications.

  11. Adenylyl cyclase plays a regulatory role in development, stress resistance and secondary metabolism in Fusarium fujikuroi.

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    Jorge García-Martínez

    Full Text Available The ascomycete fungus Fusarium fujikuroi (Gibberella fujikuroi MP-C produces secondary metabolites of biotechnological interest, such as gibberellins, bikaverin, and carotenoids. Production of these metabolites is regulated by nitrogen availability and, in a specific manner, by other environmental signals, such as light in the case of the carotenoid pathway. A complex regulatory network controlling these processes is recently emerging from the alterations of metabolite production found through the mutation of different regulatory genes. Here we show the effect of the targeted mutation of the acyA gene of F. fujikuroi, coding for adenylyl cyclase. Mutants lacking the catalytic domain of the AcyA protein showed different phenotypic alterations, including reduced growth, enhanced production of unidentified red pigments, reduced production of gibberellins and partially derepressed carotenoid biosynthesis in the dark. The phenotype differs in some aspects from that of similar mutants of the close relatives F. proliferatum and F. verticillioides: contrary to what was observed in these species, ΔacyA mutants of F. fujikuroi showed enhanced sensitivity to oxidative stress (H(2O(2, but no change in heavy metal resistance or in the ability to colonize tomato tissue, indicating a high versatility in the regulatory roles played by cAMP in this fungal group.

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

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    Erin J Heckler

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  14. Functional analysis of the sporulation-specific diadenylate cyclase CdaS in Bacillus thuringiensis

    Science.gov (United States)

    Zheng, Cao; Ma, Yang; Wang, Xun; Xie, Yuqun; Ali, Maria K.; He, Jin

    2015-01-01

    Cyclic di-AMP (c-di-AMP) is a recently discovered bacterial secondary messenger molecule, which is associated with various physiological functions. In the genus Bacillus, the intracellular level and turnover of c-di-AMP are mainly regulated by three diadenylate cyclases (DACs), including DisA, CdaA and CdaS, and two c-di-AMP-specific phosphodiesterases (GdpP and PgpH). In this study, we demonstrated that CdaS protein from B. thuringiensis is a hexameric DAC protein that can convert ATP or ADP to c-di-AMP in vitro and the N-terminal YojJ domain is essential for the DAC activity. Based on the markerless gene knock-out method, we demonstrated that the transcription of cdaS was initiated by the sporulation-specific sigma factor σH and the deletion of cdaS significantly delayed sporulation and parasporal crystal formation. These findings contrast with similar experiments conducted using B. subtilis, wherein transcription of its cdaS was initiated by the sigma factor σG. Deletion of all the three DAC genes from a single strain was unsuccessful, suggesting that c-di-AMP is an indispensable molecule in B. thuringiensis. Phylogenetic analysis indicated increased diversity of CdaS in the B. cereus and B. subtilis Bacillus subgroups. In summary, this study identifies important aspects in the regulation of c-di-AMP in the genus Bacillus. PMID:26441857

  15. The kiwifruit lycopene beta-cyclase plays a significant role in carotenoid accumulation in fruit.

    Science.gov (United States)

    Ampomah-Dwamena, Charles; McGhie, Tony; Wibisono, Reginald; Montefiori, Mirco; Hellens, Roger P; Allan, Andrew C

    2009-01-01

    The composition of carotenoids, along with anthocyanins and chlorophyll, accounts for the distinctive range of colour found in the Actinidia (kiwifruit) species. Lutein and beta-carotene are the most abundant carotenoids found during fruit development, with beta-carotene concentration increasing rapidly during fruit maturation and ripening. In addition, the accumulation of beta-carotene and lutein is influenced by the temperature at which harvested fruit are stored. Expression analysis of carotenoid biosynthetic genes among different genotypes and fruit developmental stages identified Actinidia lycopene beta-cyclase (LCY-beta) as the gene whose expression pattern appeared to be associated with both total carotenoid and beta-carotene accumulation. Phytoene desaturase (PDS) expression was the least variable among the different genotypes, while zeta carotene desaturase (ZDS), beta-carotene hydroxylase (CRH-beta), and epsilon carotene hydroxylase (CRH-epsilon) showed some variation in gene expression. The LCY-beta gene was functionally tested in bacteria and shown to convert lycopene and delta-carotene to beta-carotene and alpha-carotene respectively. This indicates that the accumulation of beta-carotene, the major carotenoid in these kiwifruit species, appears to be controlled by the level of expression of LCY-beta gene.

  16. Overexpression of Gs proteins and adenylyl cyclase in normal and diabetic islets.

    Science.gov (United States)

    Portela-Gomes, Guida M; Abdel-Halim, Samy M

    2002-08-01

    Knowledge about the relation between G proteins and adenylyl cyclases (ACs) is important for the construction of signaling paradigms to increase our understanding of signal transduction in the normal state and its alterations in pathologic states, such as type-2 diabetes. The immunocytochemical expression patterns of the stimulatory Gs proteins (G alpha-s and G alpha-olf) and the in vitro Ca2+-stimulated ACs (AC1, 3, and 8) were studied in normal and spontaneously diabetic Goto-Kakizaki (GK) rat pancreatic islets with use of well-characterized antibodies. The expressions of G alpha-11 and AC2, abundant in pancreatic islets, were also studied. G alpha-s and G alpha-olf were mainly expressed in insulin cells, and G alpha-11 in glucagon cells. The immunoreactivity to G alpha-s and G alpha-olf and to AC1 and AC3 was higher in the GK islets than in the controls, whereas AC8 was found only in the diabetic islets. Strong G alpha-11 and AC2 immunoreactivity was seen equally in both animal groups. G alpha-s was colocalized with all ACs, whereas G alpha-olf was mainly colocalized with AC3, and G alpha-11 with AC1. The current findings may help in drawing a more specific signaling paradigm coupling Gs proteins to ACs.

  17. Exogenous irritant-induced airway hyperreactivity and inhibition of soluble guanylyl cyclase.

    Science.gov (United States)

    Antosová, Martina; Strapková, Anna; Turcan, Tomás

    2008-10-01

    The majority of nitric oxide (NO) effects in the respiratory system are caused by stimulation of soluble guanylyl cyclase (sGC) with subsequent increase of cyclic guanosine monophosphate (cGMP) production. The importance of this mechanism of NO action in airway hyperreactivity (AHR) pathogenesis is unknown. Therefore, the aim of our experiment was to examine the changes of airway reactivity enhanced by toluene vapor exposure in the presence or inhibition of sGC activity in guinea pigs. Animals were treated with a nonspecific sGC inhibitor, methylene blue, in a dose of 50 or 100 mg/kg body weight, administered by intraperitoneal injection 30 min before or after exposure to toluene vapors. The toluene exposure lasted 2 hr in each of 3 consecutive days under in vivo conditions. Thereafter, the tracheal and lung tissue smooth muscle response to cumulative doses of mediators (histamine or acetylcholine) was recorded under in vitro conditions. The exposure to toluene vapors significantly increased the airway reactivity to both mediators in comparison with the healthy animal group. The administration of methylene blue decreased the amplitude of airway smooth muscle contraction in toluene-induced hyperreactivity. The decreases were dependent on the inhibitor doses, on a regimen of administration (before or after toluene inhalation), the level of the respiratory system (trachea, lung), and the bronchoconstrictor mediators. Our results suggest that the interaction between NO and sGC may be important for airway reactivity changes, but other mechanisms of NO action are important in AHR pathogenesis, too.

  18. Functional analysis of the sporulation-specific diadenylate cyclase CdaS in Bacillus thuringiensis

    Directory of Open Access Journals (Sweden)

    Cao eZheng

    2015-09-01

    Full Text Available Cyclic di-AMP (c-di-AMP is a recently discovered bacterial secondary messenger molecule, which is associated with various physiological functions. In Bacillus, the intracellular level and turnover of c-di-AMP is mainly regulated by three diadenylate cyclases (DACs, including DisA, CdaA and CdaS, and one c-di-AMP-specific phosphodiesterase. In this study, we demonstrated that CdaS protein from B. thuringiensisis is a hexameric DAC protein that can convert ATP or ADP to c-di-AMP in vitro and the N-terminal YojJ domain was essential for the DAC activity. Based on the markerless gene knock-out method, we demonstrated that the transcription of cdaS was initiated by the sporulation-specific sigma factor σH and the deletion of cdaS significantly delayed sporulation and parasporal crystal formation. These findings contrast with similar experiments conducted using B. subtilis, wherein transcription of its cdaS was initiated by the sigma factor σG. Deletion of all the three DAC genes from a single strain was unsuccessful, suggesting that c-di-AMP is an indispensable molecule in B. thuringiensis. Phylogenetic analysis indicated increased diversity of CdaS in the B. cereus and B. subtilis Bacillus subgroups. In summary, this study identifies important aspects in the regulation of c-di-AMP in Bacillus.

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

    Science.gov (United States)

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

    2015-01-01

    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

  20. Bioinformatics analysis of the oxidosqualene cyclase gene and the amino acid sequence in mangrove plants

    Science.gov (United States)

    Basyuni, M.; Wati, R.

    2017-01-01

    This study described the bioinformatics methods to analyze seven oxidosqualene cyclase (OSC) genes from mangrove plants on DDBJ/EMBL/GenBank as well as predicted the structure, composition, similarity, subcellular localization and phylogenetic. The physical and chemical properties of seven mangrove OSC showed variation among the genes. The percentage of the secondary structure of seven mangrove OSC genes followed the order of a helix > random coil > extended chain structure. The values of chloroplast or signal peptide were too low, indicated that no chloroplast transit peptide or signal peptide of secretion pathway in mangrove OSC genes. The target peptide value of mitochondria varied from 0.163 to 0.430, indicated it was possible to exist. These results suggested the importance of understanding the diversity and functional of properties of the different amino acids in mangrove OSC genes. To clarify the relationship among the mangrove OSC gene, a phylogenetic tree was constructed. The phylogenetic tree shows that there are three clusters, Kandelia KcMS join with Bruguiera BgLUS, Rhizophora RsM1 was close to Bruguiera BgbAS, and Rhizophora RcCAS join with Kandelia KcCAS. The present study, therefore, supported the previous results that plant OSC genes form distinct clusters in the tree.

  1. Soluble guanylate cyclase stimulators increase sensitivity to cisplatin in head and neck squamous cell carcinoma cells.

    Science.gov (United States)

    Tuttle, Traci R; Takiar, Vinita; Kumar, Bhavna; Kumar, Pawan; Ben-Jonathan, Nira

    2017-03-28

    Head and neck squamous cell carcinoma (HNSCC) is an aggressive and often fatal disease. Cisplatin is the most common chemotherapeutic drug in the treatment of HNSCC, but intrinsic and acquired resistance are frequent, and severe side effects occur at high doses. The second messenger cyclic GMP (cGMP) is produced by soluble guanylate cyclase (sGC). We previously reported that activation of the cGMP signaling cascade caused apoptosis in HNSCC cells, while others found that this pathway enhances cisplatin efficacy in some cell types. Here we found that sGC stimulators reduced HNSCC cell viability synergistically with cisplatin, and enhanced apoptosis by cisplatin. Moreover, the sGC stimulators effectively reduced viability in cells with acquired cisplatin resistance, and were synergistic with cisplatin. The sGC stimulator BAY 41-2272 reduced expression of the survival proteins EGFR and β-catenin, and increased pro-apoptotic Bax, suggesting a potential mechanism for the anti-tumorigenic effects of these drugs. The sGC stimulator Riociguat is FDA-approved to treat pulmonary hypertension, and others are being studied for therapeutic use in several diseases. These drugs could provide valuable addition or alternative to cisplatin in the treatment of HNSCC.

  2. Adenylyl cyclase 4 does not regulate collecting duct water and sodium handling.

    Science.gov (United States)

    Kittikulsuth, Wararat; Stuart, Deborah; Kohan, Donald E

    2014-01-01

    Abstract Adenylyl cyclase (AC)-stimulated cAMP is a key mediator of collecting duct (CD) Na and water transport. AC isoforms 3, 4, and 6 are expressed in the CD. Our group demonstrated that AC6, but not AC3, is involved in regulating CD Na and water transport. However, the role of AC4 in such regulation remains unknown. Therefore, we generated mice with loxP-flanked critical exons in the Adcy4 gene and bred with mice expressing the aquaporin-2/Cre recombinase transgene to yield CD principal cell-specific knockout of AC4 (CD AC4 KO). Isolated inner medullary CD showed 100% genomic target gene recombination in CD AC4 KO mice, while microdissected cortical CD and renal papillary AC4 mRNA was significantly reduced in CD AC4 KO mice. CD AC4 KO had no effect on vasopressin (AVP)-stimulated cAMP generation in the inner medulla. Water intake, urine volume, and urine osmolality were similar between CD AC4 KO and control mice during normal or restricted water intake. Sodium intake, urinary Na excretion, and blood pressure on a normal-, high-, or low-Na diet were not affected by CD AC4 KO. Moreover, there were no differences in plasma AVP or plasma renin concentration between CD AC4 KO and control mice. In summary, these data suggest that CD AC4 does not play a role in the physiologic regulation of CD Na and water handling.

  3. Two branches of the lupeol synthase gene in the molecular evolution of plant oxidosqualene cyclases.

    Science.gov (United States)

    Shibuya, M; Zhang, H; Endo, A; Shishikura, K; Kushiro, T; Ebizuka, Y

    1999-11-01

    Two new triterpene synthase cDNAs, named as OEW and TRW, were cloned from olive leaves (Olea europaea) and from dandelion roots (Taraxacum officinale), respectively, by the PCR method with primers designed from the conserved sequences found in the known oxidosqualene cyclases. Their ORFs consisted of 2274 bp nucleotides and coded for 758 amino acid long polypeptides. They shared high sequence identity (78%) to each other, while they showed only about 60% identities to the known triterpene synthases LUPI (lupeol synthase clone from Arabidopsis thaliana) and PNY (beta-amyrin synthase clone from Panax ginseng) at amino acid level. To determine the enzyme functions of the translates, they were expressed in an ERG7 deficient yeast mutant. Accumulation of lupeol in the cells of yeast transformants proved both of these clones code for lupeol synthase proteins. An EST (expression sequence tag) clone isolated from Medicago truncatula roots as a homologue of cycloartenol synthase gene, exhibits high sequence identity (75-77%) to these two lupeol synthase cDNAs, suggesting it to be another lupeol synthase clone. Comparatively low identity (approximately 57%) of LUP1 from Arabidopsis thaliana to either one of these clones leaves LUP1 as a distinct clone among lupeol synthases. From these sequence comparisons, now we propose that two branches of lupeol synthase gene have been generated in higher plants during the course of evolution.

  4. Metabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase

    Directory of Open Access Journals (Sweden)

    Papacchioli Velia

    2006-06-01

    Full Text Available Abstract Background Potato is a major staple food, and modification of its provitamin content is a possible means for alleviating nutritional deficiencies. beta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein, antheraxanthin, violaxanthin, and of xanthophyll esters. None of these carotenoids have provitamin A activity. Results We silenced the first dedicated step in the beta-epsilon- branch of carotenoid biosynthesis, lycopene epsilon cyclase (LCY-e, by introducing, via Agrobacterium-mediated transformation, an antisense fragment of this gene under the control of the patatin promoter. Real Time measurements confirmed the tuber-specific silencing of Lcy-e. Antisense tubers showed significant increases in beta-beta-carotenoid levels, with beta-carotene showing the maximum increase (up to 14-fold. Total carotenoids increased up to 2.5-fold. These changes were not accompanied by a decrease in lutein, suggesting that LCY-e is not rate-limiting for lutein accumulation. Tuber-specific changes in expression of several genes in the pathway were observed. Conclusion The data suggest that epsilon-cyclization of lycopene is a key regulatory step in potato tuber carotenogenesis. Upon tuber-specific silencing of the corresponding gene, beta-beta-carotenoid and total carotenoid levels are increased, and expression of several other genes in the pathway is modified.

  5. Metabolic communication between astrocytes and neurons via bicarbonate-responsive soluble adenylyl cyclase.

    Science.gov (United States)

    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

    2012-09-20

    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. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Expression of nitric oxide synthase and guanylate cyclase in the human ciliary body and trabecular meshwork

    Institute of Scientific and Technical Information of China (English)

    WU Ren-yi; MA Ning

    2012-01-01

    Background The role played by the nitric oxide (NO) signaling pathway in the aqueous humor dynamics is still unclear.This study was designed to investigate the expression and distribution of NO synthase (NOS) isoforms and guanylate cyclase (GC) in human ciliary body,trabecular meshwork and the Schlemm's canal.Methods Twelve eyes after corneal transplantation were used.Expression of three NOS isoforms (i.e.neuronal NOS (nNOS),inducible NOS (iNOS) and endothelial NOS (eNOS)) and GC were assessed in 10 eyes by immunohistochemical staining using monoclonal or polyclonal antibody of NOS and GC.Ciliary bodies were dissected free and the total proteins were extracted.Western blotting was performed to confirm the protein expression of 3 NOS isoforms and GC.Results Expression of 3 NOS isoforms and GC were observed in the ciliary epithelium,ciliary muscle,trabecular meshwork and the endothelium of the Schlemm's canal.Immunoreactivity of nNOS was detected mainly along the apical cytoplasmic junction of the non-pigmented epithelium (NPE) and pigmented epithelial (PE) cells.Protein expressions of 3 NOS isoforms and GC were confirmed in isolated human ciliary body by Western blotting.Conclusions The expression of NOS isoforms and GC in human ciliary body suggest the possible involvement of NO and cyclic guanosine monophosphate (cyclic GMP,cGMP) signaling pathway in the ciliary body,and may play a role in both processes of aqueous humor formation and drainage.

  7. ATP-binding site of adenylate kinase: mechanistic implications of its homology with ras-encoded p21, F1-ATPase, and other nucleotide-binding proteins.

    Science.gov (United States)

    Fry, D C; Kuby, S A; Mildvan, A S

    1986-02-01

    The MgATP binding site of adenylate kinase, located by a combination of NMR and x-ray diffraction, is near three protein segments, five to seven amino acids in length, that are homologous in sequence to segments found in other nucleotide-binding phosphotransferases, such as myosin and F1-ATPase, ras p21 and transducin GTPases, and cAMP-dependent and src protein kinases, suggesting equivalent mechanistic roles of these segments in all of these proteins. Segment 1 is a glycine-rich flexible loop that, on adenylate kinase, may control access to the ATP-binding site by changing its conformation. Segment 2 is an alpha-helix containing two hydrophobic residues that interact with the adenine-ribose moiety of ATP, and a lysine that may bind to the beta- and gamma-phosphates of ATP. Segment 3 is a hydrophobic strand of parallel beta-pleated sheet, terminated by a carboxylate, that flanks the triphosphate binding site. The various reported mutations of ras p21 that convert it to a transforming agent all appear to involve segment 1, and such substitutions may alter the properties of p21 by hindering a conformational change at this segment. In F1-ATPase, the flexible loop may, by its position, control both the accessibility and the ATP/ADP equilibrium constant on the enzyme.

  8. In vitro mutagenesis studies at the arginine residues of adenylate kinase. A revised binding site for AMP in the X-ray-deduced model

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyo Joon; Nishikawa, Satoshi; Tokutomi, Yuiko; Uesugi, Seiichi (Osaka Univ. (Japan)); Takenaka, Hitoshi; Hamada, Minoru (Miyazaki Medical College (Japan)); Kuby, S.A. (Univ. of Utah, Salt Lake City (USA))

    1990-02-06

    Although X-ray crystallographic and NMR studies have been made on the adenylate kinases, the substrate-binding sites are not unequivocally established. In an attempt to shed light on the binding sites for MgATP{sup 2{minus}} and for AMP{sup 2{minus}} in human cytosolic adenylate kinase, the authors have investigated the enzymic effects of replacement of the arginine residues, which had been assumed by Pai et al. to interact with the phosphoryl groups of AMP{sup 2{minus}} and MgATP{sup 2{minus}}. With use of the site-directed mutagenesis method, point mutations were made in the artificial gene for hAK1 to replace these arginine residues with alanyl residues and yield the mutants R44A hAK1, R132A hAK1, R138A hAK1, and R149A hAK1. The resulting large increases in the K{sub m,app} values for AMP{sup 2{minus}} of the mutant enzymes, the relatively small increases in the K{sub m,app} values for MgATP{sup 2{minus}}, and the fact that the R132A, R138A, and R149A mutant enzymes proved to be very poor catalysts are consistent with the idea that the assigned substrate binding sites of Pai et al. have been reversed and that their ATP-binding site may be assigned as the AMP site.

  9. Structure of the D-alanylgriseoluteic acid biosynthetic protein EhpF, an atypical member of the ANL superfamily of adenylating enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Bera, A.K.; Robinson, H.; Atanasova, V.; Gamage, S.; Parsons, J. F.

    2010-06-01

    The structure of EhpF, a 41 kDa protein that functions in the biosynthetic pathway leading to the broad-spectrum antimicrobial compound D-alanylgriseoluteic acid (AGA), is reported. A cluster of approximately 16 genes, including ehpF, located on a 200 kbp plasmid native to certain strains of Pantoea agglomerans encodes the proteins that are required for the conversion of chorismic acid to AGA. Phenazine-1,6-dicarboxylate has been identified as an intermediate in AGA biosynthesis and deletion of ehpF results in accumulation of this compound in vivo. The crystallographic data presented here reveal that EhpF is an atypical member of the acyl-CoA synthase or ANL superfamily of adenylating enzymes. These enzymes typically catalyze two-step reactions involving adenylation of a carboxylate substrate followed by transfer of the substrate from AMP to coenzyme A or another phosphopantetheine. EhpF is distinguished by the absence of the C-terminal domain that is characteristic of enzymes from this family and is involved in phosphopantetheine binding and in the second half of the canonical two-step reaction that is typically observed. Based on the structure of EhpF and a bioinformatic analysis, it is proposed that EhpF and EhpG convert phenazine-1,6-dicarboxylate to 6-formylphenazine-1-carboxylate via an adenylyl intermediate.

  10. Mutation in Mg-Protoporphyrin IX Monomethyl Ester Cyclase Decreases Photosynthesis Capacity in Rice

    Science.gov (United States)

    Wang, Xuexia; Huang, Rongfeng; Quan, Ruidang

    2017-01-01

    In photosynthesis, the pigments chlorophyll a/b absorb light energy to convert to chemical energy in chloroplasts. Though most enzymes of chlorophyll biosynthesis from glutamyl-tRNA to chlorophyll a/b have been identified, the exact composition and regulation of the multimeric enzyme Mg-protoporphyrin IX monomethyl ester cyclase (MPEC) is largely unknown. In this study, we isolated a rice pale-green leaf mutant m167 with yellow-green leaf phenotype across the whole lifespan. Chlorophyll content decreases 43–51% and the granal stacks of chloroplasts becomes thinner in m167. Chlorophyll fluorescence parameters, including Fv/Fm (the maximum quantum efficiency of PSII) and quantum yield of PSII (Y(II)), were lower in m167 than those in wild type plants (WT), and photosynthesis rate decreases 40% in leaves of m167 mutant compared with WT plants, which lead to yield reduction in m167. Genetic analysis revealed that yellow-green leaf phenotype of m167 is controlled by a single recessive genetic locus. By positional cloning, a single mutated locus, G286A (Alanine 96 to Threonine in protein), was found in the coding sequence of LOC_Os01g17170 (Rice Copper Response Defect 1, OsCRD1), encoding a putative subunit of MPEC. Expression profile analysis demonstrated that OsCRD1 is mainly expressed in green tissues of rice. Sequence alignment analysis of CRD1 indicated that Alanine 96 is very conserved in all green plants and photosynthetic bacteria. OsCRD1 protein mainly locates in chloroplast and the point mutation A96T in OsCRD1 does not change its location. Therefore, Alanine96 of OsCRD1 might be fundamental for MPEC activity, mutation of which leads to deficiency in chlorophyll biosynthesis and chloroplast development and decreases photosynthetic capacity in rice. PMID:28129387

  11. A peptide against soluble guanylyl cyclase α1: a new approach to treating prostate cancer.

    Directory of Open Access Journals (Sweden)

    Shuai Gao

    Full Text Available Among the many identified androgen-regulated genes, sGCα1 (soluble guanylyl cyclase α1 appears to play a pivotal role in mediating the pro-cancer effects of androgens and androgen receptor. The classical role for sGCα1 is to heterodimerize with the sGCβ1 subunit, forming sGC, the enzyme that mediates nitric oxide signaling by catalyzing the synthesis of cyclic guanosine monophosphate. Our published data show that sGCα1 can drive prostate cancer cell proliferation independent of hormone and provide cancer cells a pro-survival function, via a novel mechanism for p53 inhibition, both of which are independent of sGCβ1, NO, and cGMP. All of these properties make sGCα1 an important novel target for prostate cancer therapy. Thus, peptides were designed targeting sGCα1 with the aim of disrupting this protein's pro-cancer activities. One peptide (A-8R was determined to be strongly cytotoxic to prostate cancer cells, rapidly inducing apoptosis. Cytotoxicity was observed in both hormone-dependent and, significantly, hormone-refractory prostate cancer cells, opening the possibility that this peptide can be used to treat the usually lethal castration-resistant prostate cancer. In mouse xenograft studies, Peptide A-8R was able to stop tumor growth of not only hormone-dependent cells, but most importantly from hormone-independent cells. In addition, the mechanism of Peptide A cytotoxicity is generation of reactive oxygen species, which recently have been recognized as a major mode of action of important cancer drugs. Thus, this paper provides strong evidence that targeting an important AR-regulated gene is a new paradigm for effective prostate cancer therapy.

  12. The Diguanylate Cyclase GcbA Facilitates Pseudomonas aeruginosa Biofilm Dispersion by Activating BdlA

    Science.gov (United States)

    Petrova, Olga E.; Cherny, Kathryn E.

    2014-01-01

    Biofilm dispersion is a highly regulated process that allows biofilm bacteria to respond to changing environmental conditions and to disseminate to new locations. The dispersion of biofilms formed by the opportunistic pathogen Pseudomonas aeruginosa is known to require a number of cyclic di-GMP (c-di-GMP)-degrading phosphodiesterases (PDEs) and the chemosensory protein BdlA, with BdlA playing a pivotal role in regulating PDE activity and enabling dispersion in response to a wide array of cues. BdlA is activated during biofilm growth via posttranslational modifications and nonprocessive cleavage in a manner that is dependent on elevated c-di-GMP levels. Here, we provide evidence that the diguanylate cyclase (DGC) GcbA contributes to the regulation of BdlA cleavage shortly after initial cellular attachment to surfaces and, thus, plays an essential role in allowing biofilm cells to disperse in response to increasing concentrations of a variety of substances, including carbohydrates, heavy metals, and nitric oxide. DGC activity of GcbA was required for its function, as a catalytically inactive variant could not rescue impaired BdlA processing or the dispersion-deficient phenotype of gcbA mutant biofilms to wild-type levels. While modulating BdlA cleavage during biofilm growth, GcbA itself was found to be subject to c-di-GMP-dependent and growth-mode-specific regulation. GcbA production was suppressed in mature wild-type biofilms and could be induced by reducing c-di-GMP levels via overexpression of genes encoding PDEs. Taken together, the present findings demonstrate that the regulatory functions of c-di-GMP-synthesizing DGCs expand beyond surface attachment and biofilm formation and illustrate a novel role for DGCs in the regulation of the reverse sessile-motile transition of dispersion. PMID:25331436

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

    Science.gov (United States)

    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.

  14. The plant natriuretic peptide receptor is a guanylyl cyclase and enables cGMP-dependent signaling

    KAUST Repository

    Turek, Ilona

    2016-03-05

    The functional homologues of vertebrate natriuretic peptides (NPs), the plant natriuretic peptides (PNPs), are a novel class of peptidic hormones that signal via guanosine 3′,5′-cyclic monophosphate (cGMP) and systemically affect plant salt and water balance and responses to biotrophic plant pathogens. Although there is increasing understanding of the complex roles of PNPs in plant responses at the systems level, little is known about the underlying signaling mechanisms. Here we report isolation and identification of a novel Leucine-Rich Repeat (LRR) protein that directly interacts with A. thaliana PNP, AtPNP-A. In vitro binding studies revealed that the Arabidopsis AtPNP-A binds specifically to the LRR protein, termed AtPNP-R1, and the active region of AtPNP-A is sufficient for the interaction to occur. Importantly, the cytosolic part of the AtPNP-R1, much like in some vertebrate NP receptors, harbors a catalytic center diagnostic for guanylyl cyclases and the recombinant AtPNP-R1 is capable of catalyzing the conversion of guanosine triphosphate to cGMP. In addition, we show that AtPNP-A causes rapid increases of cGMP levels in wild type (WT) leaf tissue while this response is significantly reduced in the atpnp-r1 mutants. AtPNP-A also causes cGMP-dependent net water uptake into WT protoplasts, and hence volume increases, whereas responses of the protoplasts from the receptor mutant are impaired. Taken together, our results suggest that the identified LRR protein is an AtPNP-A receptor essential for the PNP-dependent regulation of ion and water homeostasis in plants and that PNP- and vertebrate NP-receptors and their signaling mechanisms share surprising similarities. © 2016 Springer Science+Business Media Dordrecht

  15. Loss of guanylyl cyclase C (GCC signaling leads to dysfunctional intestinal barrier.

    Directory of Open Access Journals (Sweden)

    Xiaonan Han

    Full Text Available BACKGROUND: Guanylyl Cyclase C (GCC signaling via uroguanylin (UGN and guanylin activation is a critical mediator of intestinal fluid homeostasis, intestinal cell proliferation/apoptosis, and tumorigenesis. As a mechanism for some of these effects, we hypothesized that GCC signaling mediates regulation of intestinal barrier function. METHODOLOGY/PRINCIPAL FINDINGS: Paracellular permeability of intestinal segments was assessed in wild type (WT and GCC deficient (GCC-/- mice with and without lipopolysaccharide (LPS challenge, as well as in UGN deficient (UGN-/- mice. IFNγ and myosin light chain kinase (MLCK levels were determined by real time PCR. Expression of tight junction proteins (TJPs, phosphorylation of myosin II regulatory light chain (MLC, and STAT1 activation were examined in intestinal epithelial cells (IECs and intestinal mucosa. The permeability of Caco-2 and HT-29 IEC monolayers, grown on Transwell filters was determined in the absence and presence of GCC RNA interference (RNAi. We found that intestinal permeability was increased in GCC-/- and UGN-/- mice compared to WT, accompanied by increased IFNγ levels, MLCK and STAT1 activation in IECs. LPS challenge promotes greater IFNγ and STAT1 activation in IECs of GCC-/- mice compared to WT mice. Claudin-2 and JAM-A expression were reduced in GCC deficient intestine; the level of phosphorylated MLC in IECs was significantly increased in GCC-/- and UGN-/- mice compared to WT. GCC knockdown induced MLC phosphorylation, increased permeability in IEC monolayers under basal conditions, and enhanced TNFα and IFNγ-induced monolayer hyperpermeability. CONCLUSIONS/SIGNIFICANCE: GCC signaling plays a protective role in the integrity of the intestinal mucosal barrier by regulating MLCK activation and TJ disassembly. GCC signaling activation may therefore represent a novel mechanism in maintaining the small bowel barrier in response to injury.

  16. Chaperone-dependent E3 ligase CHIP ubiquitinates and mediates proteasomal degradation of soluble guanylyl cyclase.

    Science.gov (United States)

    Xia, Tian; Dimitropoulou, Christiana; Zeng, Jingmin; Antonova, Galina N; Snead, Connie; Venema, Richard C; Fulton, David; Qian, Shuibing; Patterson, Cam; Papapetropoulos, Andreas; Catravas, John D

    2007-11-01

    The nitric oxide receptor soluble guanylyl cyclase (sGC) exists in multimeric protein complexes, including heat shock protein (HSP) 90 and endothelial nitric oxide synthase. Inhibition of HSP90 by geldanamycin causes proteasomal degradation of sGC protein. In this study, we have investigated whether COOH terminus of heat shock protein 70-interacting protein (CHIP), a co-chaperone molecule that is involved in protein folding but is also a chaperone-dependent ubiquitin E3 ligase, could play a role in the process of degradation of sGC. Transient overexpression of CHIP in COS-7 cells degraded heterologous sGC in a concentration-related manner; this downregulation of sGC was abrogated by the proteasome inhibitor MG-132. Transfection of tetratricopeptide repeats and U-box domain CHIP mutants attenuated sGC degradation, suggesting that both domains are indispensable for CHIP function. Results from immunoprecipitation and indirect immunofluorescent microscopy experiments demonstrated that CHIP is associated with sGC, HSP90, and HSP70 in COS-7 cells. Furthermore, CHIP increased the association of HSP70 with sGC. In in vitro ubiquitination assays using purified proteins and ubiquitin enzymes, E3 ligase CHIP directly ubiquitinated sGC; this ubiquitination was potentiated by geldanamycin in COS-7 cells, followed by proteasomal degradation. In rat aortic smooth muscle cells, endogenous sGC was also degraded by adenovirus-infected wild-type CHIP but not by the chaperone interaction-deficient K30A CHIP, whereas CHIP, but not K30A, attenuated sGC expression in, and nitric oxide donor-induced relaxation of, rat aortic rings, suggesting that CHIP plays a regulatory role under physiological conditions. This study reveals a new mechanism for the regulation of sGC, an important mediator of cellular and vascular function.

  17. Isolation and functional characterization of a lycopene β-cyclase gene promoter from citrus

    Directory of Open Access Journals (Sweden)

    Suwen Lu

    2016-09-01

    Full Text Available Lycopene β-cyclases are key enzymes located at the branch point of the carotenoid biosynthesis pathway. However, the transcriptional regulatory mechanisms of LCYb1 in citrus with abundant carotenoid accumulation are still unclear. To understand the molecular basis of CsLCYb1 expression, we isolated and functionally characterized the 5’ upstream sequences of CsLCYb1 from citrus. The full-length CsLCYb1 promoter and a series of its 5’ deletions were fused to the β-glucuronidase (GUS reporter gene and transferred into different plants (tomato, Arabidopsis and citrus callus to test the promoter activities. The results of all transgenic species showed that the 1584 bp upstream region from the translational start site displayed maximal promoter activity, and the minimal promoter containing 746 bp upstream sequences was sufficient for strong basal promoter activity. Furthermore, the CsLCYb1 promoter activity was developmentally and tissue-specially regulated in transgenic Arabidopsis, and it was affected by multiple hormones and environmental cues in transgenic citrus callus under various treatments. Finer deletion analysis identified an enhancer element existing as a tandem repeat in the promoter region between -574 to -513 bp and conferring strong promoter activity. The copy numbers of the enhancer element differed among various citrus species, leading to the development of a derived simple sequence repeat (SSR marker to distinguish different species. In conclusion, this study elucidates the expression characteristics of the LCYb1 promoter from citrus and further identifies a novel enhancer element required for the promoter activity. The characterized promoter fragment would be an ideal candidate for genetic engineering and seeking of upstream trans-acting elements.

  18. Characterization and phylogenetic epitope mapping of CD38 ADPR cyclase in the cynomolgus macaque

    Directory of Open Access Journals (Sweden)

    Titti Fausto

    2004-09-01

    Full Text Available Abstract Background The CD38 transmembrane glycoprotein is an ADP-ribosyl cyclase that moonlights as a receptor in cells of the immune system. Both functions are independently implicated in numerous areas related to human health. This study originated from an inherent interest in studying CD38 in the cynomolgus monkey (Macaca fascicularis, a species closely related to humans that also represents a cogent animal model for the biomedical analysis of CD38. Results A cDNA was isolated from cynomolgus macaque peripheral blood leukocytes and is predicted to encode a type II membrane protein of 301 amino acids with 92% identity to human CD38. Both RT-PCR-mediated cDNA cloning and genomic DNA PCR surveying were possible with heterologous human CD38 primers, demonstrating the striking conservation of CD38 in these primates. Transfection of the cDNA coincided with: (i surface expression of cynomolgus macaque CD38 by immunofluorescence; (ii detection of ~42 and 84 kDa proteins by Western blot and (iii the appearance of ecto-enzymatic activity. Monoclonal antibodies were raised against the cynomolgus CD38 ectodomain and were either species-specific or cross-reactive with human CD38, in which case they were directed against a common disulfide-requiring conformational epitope that was mapped to the C-terminal disulfide loop. Conclusion This multi-faceted characterization of CD38 from cynomolgus macaque demonstrates its high genetic and biochemical similarities with human CD38 while the immunological comparison adds new insights into the dominant epitopes of the primate CD38 ectodomain. These results open new prospects for the biomedical and pharmacological investigations of this receptor-enzyme.

  19. Corruption of homeostatic mechanisms in the guanylyl cyclase C signaling pathway underlying colorectal tumorigenesis

    Science.gov (United States)

    Waldman, Scott A

    2010-01-01

    Colon cancer, the second leading cause of cancer-related mortality worldwide, originates from the malignant transformation of intestinal epithelial cells. The intestinal epithelium undergoes a highly organized process of rapid regeneration along the crypt-villus axis, characterized by proliferation, migration, differentiation and apoptosis, whose coordination is essential to maintaining the mucosal barrier. Disruption of these homeostatic processes predisposes cells to mutations in tumor suppressors or oncogenes, whose dysfunction provides transformed cells an evolutionary growth advantage. While sequences of genetic mutations at different stages along the neoplastic continuum have been established, little is known of the events initiating tumorigenesis prior to adenomatous polyposis coli (APC) mutations. Here, we examine a role for the corruption of homeostasis induced by silencing novel tumor suppressors, including the intestine-specific transcription factor CDX2 and its gene target guanylyl cyclase C (GCC), as early events predisposing cells to mutations in APC and other sequential genes that initiate colorectal cancer. CDX2 and GCC maintain homeostatic regeneration in the intestine by restricting cell proliferation, promoting cell maturation and adhesion, regulating cell migration and defending the intestinal barrier and genomic integrity. Elimination of CDX2 or GCC promotes intestinal tumor initiation and growth in aged mice, mice carrying APC mutations or mice exposed to carcinogens. The roles of CDX2 and GCC in suppressing intestinal tumorigenesis, universal disruption in their signaling through silencing of hormones driving GCC, and the uniform overexpression of GCC by tumors underscore the potential value of oral replacement with GCC ligands as targeted prevention and therapy for colorectal cancer. PMID:20592492

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  1. Altered collecting duct adenylyl cyclase content in collecting duct endothelin-1 knockout mice

    Directory of Open Access Journals (Sweden)

    Kohan Donald E

    2007-05-01

    Full Text Available Abstract Background Endothelin-1 (ET-1 inhibition of vasopressin (AVP-stimulated water reabsorption by the inner medullary collecting duct (IMCD is associated with reduced cAMP accumulation. To determine the effect of ET-1 deficiency, AVP-stimulated cAMP responsiveness was assessed in IMCD from mice with collecting duct-specific deletion of ET-1 (CD ET-1 KO and from control animals. Methods Cyclic AMP production, adenylyl cyclase (AC mRNA, and AC protein were measured in acutely isolated IMCD. Results CD ET-1 KO IMCD had enhanced AVP-stimulated cAMP accumulation. Inhibition of calcium-stimulated AC using BAPTA did not prevent enhanced AVP responsiveness in CD ET-1 KO IMCD. Factors known to be modified by ET-1, including nitric oxide, cyclooxygenase metabolites, and superoxide did not affect the increased AVP responsiveness of CD ET-1 KO IMCD. Differential V2 receptor or G-protein activity was not involved since CD ET-1 KO IMCD had increased cAMP accumulation in response to forskolin and/or cholera toxin. CD ET-1 KO did not affect mRNA or protein levels of AC3, one of the major known collecting duct AC isoforms. However, the other known major collecting duct AC isoform (AC5/6 did have increased protein levels in CD ET-1 KO IMCD, although AC5 (weak signal and 6 mRNA levels were unchanged. Conclusion ET-1 deficiency increases IMCD AC5/6 content, an effect that may synergize with acute ET-1 inhibition of AVP-stimulated cAMP accumulation.

  2. Calcium influx through L-type channels attenuates skeletal muscle contraction via inhibition of adenylyl cyclases.

    Science.gov (United States)

    Menezes-Rodrigues, Francisco Sandro; Pires-Oliveira, Marcelo; Duarte, Thiago; Paredes-Gamero, Edgar Julian; Chiavegatti, Tiago; Godinho, Rosely Oliveira

    2013-11-15

    Skeletal muscle contraction is triggered by acetylcholine induced release of Ca(2+) from sarcoplasmic reticulum. Although this signaling pathway is independent of extracellular Ca(2+), L-type voltage-gated calcium channel (Cav) blockers have inotropic effects on frog skeletal muscles which occur by an unknown mechanism. Taking into account that skeletal muscle fiber expresses Ca(+2)-sensitive adenylyl cyclase (AC) isoforms and that cAMP is able to increase skeletal muscle contraction force, we investigated the role of Ca(2+) influx on mouse skeletal muscle contraction and the putative crosstalk between extracellular Ca(2+) and intracellular cAMP signaling pathways. The effects of Cav blockers (verapamil and nifedipine) and extracellular Ca(2+) chelator EGTA were evaluated on isometric contractility of mouse diaphragm muscle under direct electrical stimulus (supramaximal voltage, 2 ms, 0.1 Hz). Production of cAMP was evaluated by radiometric assay while Ca(2+) transients were assessed by confocal microscopy using L6 cells loaded with fluo-4/AM. Ca(2+) channel blockers verapamil and nifedipine had positive inotropic effect, which was mimicked by removal of extracellular Ca(+2) with EGTA or Ca(2+)-free Tyrode. While phosphodiesterase inhibitor IBMX potentiates verapamil positive inotropic effect, it was abolished by AC inhibitors SQ22536 and NYK80. Finally, the inotropic effect of verapamil was associated with increased intracellular cAMP content and mobilization of intracellular Ca(2+), indicating that positive inotropic effects of Ca(2+) blockers depend on cAMP formation. Together, our results show that extracellular Ca(2+) modulates skeletal muscle contraction, through inhibition of Ca(2+)-sensitive AC. The cross-talk between extracellular calcium and cAMP-dependent signaling pathways appears to regulate the extent of skeletal muscle contraction responses.

  3. Isolation and Functional Characterization of a Lycopene β-cyclase Gene Promoter from Citrus.

    Science.gov (United States)

    Lu, Suwen; Zhang, Yin; Zheng, Xiongjie; Zhu, Kaijie; Xu, Qiang; Deng, Xiuxin

    2016-01-01

    Lycopene β-cyclases are key enzymes located at the branch point of the carotenoid biosynthesis pathway. However, the transcriptional regulatory mechanisms of LCYb1 in citrus with abundant carotenoid accumulation are still unclear. To understand the molecular basis of CsLCYb1 expression, we isolated and functionally characterized the 5' upstream sequences of CsLCYb1 from citrus. The full-length CsLCYb1 promoter and a series of its 5' deletions were fused to the β-glucuronidase (GUS) reporter gene and transferred into different plants (tomato, Arabidopsis and citrus callus) to test the promoter activities. The results of all transgenic species showed that the 1584 bp upstream region from the translational start site displayed maximal promoter activity, and the minimal promoter containing 746 bp upstream sequences was sufficient for strong basal promoter activity. Furthermore, the CsLCYb1 promoter activity was developmentally and tissue-specially regulated in transgenic Arabidopsis, and it was affected by multiple hormones and environmental cues in transgenic citrus callus under various treatments. Finer deletion analysis identified an enhancer element existing as a tandem repeat in the promoter region between -574 to -513 bp and conferring strong promoter activity. The copy numbers of the enhancer element differed among various citrus species, leading to the development of a derived simple sequence repeat marker to distinguish different species. In conclusion, this study elucidates the expression characteristics of the LCYb1 promoter from citrus and further identifies a novel enhancer element required for the promoter activity. The characterized promoter fragment would be an ideal candidate for genetic engineering and seeking of upstream trans-acting elements.

  4. Nitroxyl (HNO stimulates soluble guanylyl cyclase to suppress cardiomyocyte hypertrophy and superoxide generation.

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    Eliane Q Lin

    Full Text Available BACKGROUND: New therapeutic targets for cardiac hypertrophy, an independent risk factor for heart failure and death, are essential. HNO is a novel redox sibling of NO• attracting considerable attention for the treatment of cardiovascular disorders, eliciting cGMP-dependent vasodilatation yet cGMP-independent positive inotropy. The impact of HNO on cardiac hypertrophy (which is negatively regulated by cGMP however has not been investigated. METHODS: Neonatal rat cardiomyocytes were incubated with angiotensin II (Ang II in the presence and absence of the HNO donor Angeli's salt (sodium trioxodinitrate or B-type natriuretic peptide, BNP (all 1 µmol/L. Hypertrophic responses and its triggers, as well as cGMP signaling, were determined. RESULTS: We now demonstrate that Angeli's salt inhibits Ang II-induced hypertrophic responses in cardiomyocytes, including increases in cardiomyocyte size, de novo protein synthesis and β-myosin heavy chain expression. Angeli's salt also suppresses Ang II induction of key triggers of the cardiomyocyte hypertrophic response, including NADPH oxidase (on both Nox2 expression and superoxide generation, as well as p38 mitogen-activated protein kinase (p38MAPK. The antihypertrophic, superoxide-suppressing and cGMP-elevating effects of Angeli's salt were mimicked by BNP. We also demonstrate that the effects of Angeli's salt are specifically mediated by HNO (with no role for NO• or nitrite, with subsequent activation of cardiomyocyte soluble guanylyl cyclase (sGC and cGMP signaling (on both cGMP-dependent protein kinase, cGK-I and phosphorylation of vasodilator-stimulated phosphoprotein, VASP. CONCLUSIONS: Our results demonstrate that HNO prevents cardiomyocyte hypertrophy, and that cGMP-dependent NADPH oxidase suppression contributes to these antihypertrophic actions. HNO donors may thus represent innovative pharmacotherapy for cardiac hypertrophy.

  5. Therapeutically targeting guanylate cyclase-C: computational modeling of plecanatide, a uroguanylin analog.

    Science.gov (United States)

    Brancale, Andrea; Shailubhai, Kunwar; Ferla, Salvatore; Ricci, Antonio; Bassetto, Marcella; Jacob, Gary S

    2017-04-01

    Plecanatide is a recently developed guanylate cyclase-C (GC-C) agonist and the first uroguanylin analog designed to treat chronic idiopathic constipation (CIC) and irritable bowel syndrome with constipation (IBS-C). GC-C receptors are found across the length of the intestines and are thought to play a key role in fluid regulation and electrolyte balance. Ligands of the GC-C receptor include endogenous agonists, uroguanylin and guanylin, as well as diarrheagenic, Escherichia coli heat-stable enterotoxins (ST). Plecanatide mimics uroguanylin in its 2 disulfide-bond structure and in its ability to activate GC-Cs in a pH-dependent manner, a feature associated with the presence of acid-sensing residues (Asp2 and Glu3). Linaclotide, a synthetic analog of STh (a 19 amino acid member of ST family), contains the enterotoxin's key structural elements, including the presence of three disulfide bonds. Linaclotide, like STh, activates GC-Cs in a pH-independent manner due to the absence of pH-sensing residues. In this study, molecular dynamics simulations compared the stability of plecanatide and linaclotide to STh. Three-dimensional structures of plecanatide at various protonation states (pH 2.0, 5.0, and 7.0) were simulated with GROMACS software. Deviations from ideal binding conformations were quantified using root mean square deviation values. Simulations of linaclotide revealed a rigid conformer most similar to STh. Plecanatide simulations retained the flexible, pH-dependent structure of uroguanylin. The most active conformers of plecanatide were found at pH 5.0, which is the pH found in the proximal small intestine. GC-C receptor activation in this region would stimulate intraluminal fluid secretion, potentially relieving symptoms associated with CIC and IBS-C.

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

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    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. Diversity of squalene-hopene cyclases in a tropical carbonate-rich environment

    Science.gov (United States)

    Leavitt, W. D.; Pearson, A.

    2007-12-01

    Hopanoids are isoprenoid lipids which derive primarily from bacteria and are ubiquitous in contemporary Earth surface environments. In the geologic record, hopanes found in sedimentary rocks are used as proxies to help decipher ancient biological communities. However, in contrast to the ubiquity of these lipid products, biosynthesis of hopanoids appears to be a relatively rare physiological trait among bacteria in complex environmental communities. We have recently estimated that fewer than one in ten bacterial cells in soils and fewer than one in twenty bacterial cells in the ocean contains the gene squalene-hopene cyclase (sqhC) [1]. Biosynthesis of hopanoids is rarer in natural communities than it is among species that have been propagated in pure culture [2]. Here we continue our previous work to survey the phylogeny and diversity of hopanoid producers using culture-independent methods. In particular, genes affiliated with known cyanobacterial sequences were not detected in the contemporary environments analyzed previously [1]. One possible explanation is that hopanoid-producing strains of cyanobacteria are regionally localized. It has been suggested that throughout the long-term sedimentary record there is a correlation between 2-methylhopanoid index (a putative indicator of cyanobacterial biomass) and the global prevalence of shallow carbonate platform environments [3], and in previous work we did not analyze any such environments. To address this question we surveyed a land-sea gradient across the Bahamian island of San Salvador. Samples were taken from upland soil, a hypersaline lake, a tidal creek, and the shallow open ocean. The data are remarkably similar to our previous results: environmental sqhCs average triterpenes in Prokaryotes. J. Gen. Microbiol. 130, 1137-1150. [3] Summons, RE (personal communication).

  8. Characterization and Inhibition of a Class II Diterpene Cyclase from Mycobacterium tuberculosis

    Science.gov (United States)

    Mann, Francis M.; Prisic, Sladjana; Hu, Huayou; Xu, Meimei; Coates, Robert M.; Peters, Reuben J.

    2009-01-01

    Mycobacterium tuberculosis remains a widespread and devastating human pathogen, whose ability to infiltrate macrophage host cells from the human immune system is an active area of investigation. We have recently reported the discovery of a novel diterpene from M. tuberculosis, edaxadiene, whose ability to arrest phagosomal maturation in isolation presumably contributes to this critical process in M. tuberculosis infections. (Mann, F. M., Xu, M., Chen, X., Fulton, D. B., Russell, D. G., and Peters, R. J. (2009) J. Am. Chem. Soc., in press). Here, we present characterization of the class II diterpene cyclase that catalyzes the committed step in edaxadiene biosynthesis, i.e. the previously identified halimadienyl-diphosphate synthase (HPS; EC 5.5.1.16). Intriguingly, our kinetic analysis suggests a potential biochemical regulatory mechanism that triggers edaxadiene production upon phagosomal engulfment. Furthermore, we report characterization of potential HPS inhibitors: specifically, two related transition state analogs (15-aza-14,15-dihydrogeranylgeranyl diphosphate (7a) and 15-aza-14,15-dihydrogeranylgeranyl thiolodiphosphate (7b)) that exhibit very tight binding. Although arguably not suitable for clinical use, these nevertheless provide a basis for pharmaceutical design against this intriguing biosynthetic pathway. Finally, we provide evidence indicating that this pathway exists only in M. tuberculosis and is not functional in the closely related Mycobacterium bovis because of an inactivating frameshift in the HPS-encoding gene. Thus, we hypothesize that the inability to produce edaxadiene may be a contributing factor in the decreased infectivity and/or virulence of M. bovis relative to M. tuberculosis in humans. PMID:19574210

  9. Cloning and functional characterization of three branch point oxidosqualene cyclases from Withania somnifera (L.) dunal.

    Science.gov (United States)

    Dhar, Niha; Rana, Satiander; Razdan, Sumeer; Bhat, Wajid Waheed; Hussain, Aashiq; Dhar, Rekha S; Vaishnavi, Samantha; Hamid, Abid; Vishwakarma, Ram; Lattoo, Surrinder K

    2014-06-13

    Oxidosqualene cyclases (OSCs) positioned at a key metabolic subdividing junction execute indispensable enzymatic cyclization of 2,3-oxidosqualene for varied triterpenoid biosynthesis. Such branch points present favorable gene targets for redirecting metabolic flux toward specific secondary metabolites. However, detailed information regarding the candidate OSCs covering different branches and their regulation is necessary for the desired genetic manipulation. The aim of the present study, therefore, was to characterize members of OSC superfamily from Withania somnifera (Ws), a medicinal plant of immense repute known to synthesize a large array of biologically active steroidal lactone triterpenoids called withanolides. Three full-length OSC cDNAs, β-amyrin synthase (WsOSC/BS), lupeol synthase (WsOSC/LS), and cycloartenol synthase (WsOSC/CS), having open reading frames of 2289, 2268, and 2277 bp, were isolated. Heterologous expression in Schizosaccharomyces pombe, LC-MS analyses, and kinetic studies confirmed their monofunctionality. The three WsOSCs were found to be spatially regulated at transcriptional level with WsOSC/CS being maximally expressed in leaf tissue. Promoter analysis of three WsOSCs genes resulted in identification of distinct cis-regulatory elements. Further, transcript profiling under methyl jasmonate, gibberellic acid, and yeast extract elicitations displayed differential transcriptional regulation of each of the OSCs. Changes were also observed in mRNA levels under elicitations and further substantiated with protein expression levels by Western blotting. Negative regulation by yeast extract resulted in significant increase in withanolide content. Empirical evidence suggests that repression of competitive branch OSCs like WsOSC/BS and WsOSC/LS possibly leads to diversion of substrate pool toward WsOSC/CS for increased withanolide production.

  10. Regulation of anterior chamber drainage by bicarbonate-sensitive soluble adenylyl cyclase in the ciliary body.

    Science.gov (United States)

    Lee, Yong S; Tresguerres, Martin; Hess, Kenneth; Marmorstein, Lihua Y; Levin, Lonny R; Buck, Jochen; Marmorstein, Alan D

    2011-12-02

    Glaucoma is a leading cause of blindness affecting as many as 2.2 million Americans. All current glaucoma treatment strategies aim to reduce intraocular pressure (IOP). IOP results from the resistance to drainage of aqueous humor (AH) produced by the ciliary body in a process requiring bicarbonate. Once secreted into the anterior chamber, AH drains from the eye via two pathways: uveoscleral and pressure-dependent or conventional outflow (C(t)). Modulation of "inflow" and "outflow" pathways is thought to occur via distinct, local mechanisms. Mice deficient in the bicarbonate channel bestrophin-2 (Best2), however, exhibit a lower IOP despite an increase in AH production. Best2 is expressed uniquely in nonpigmented ciliary epithelial (NPE) cells providing evidence for a bicarbonate-dependent communicative pathway linking inflow and outflow. Here, we show that bicarbonate-sensitive soluble adenylyl cyclase (sAC) is highly expressed in the ciliary body in NPE cells, but appears to be absent from drainage tissues. Pharmacologic inhibition of sAC in mice causes a significant increase in IOP due to a decrease in C(t) with no effect on inflow. In mice deficient in sAC IOP is elevated, and C(t) is decreased relative to wild-type mice. Pharmacologic inhibition of sAC did not alter IOP or C(t) in sAC-deficient mice. Based on these data we propose that the ciliary body can regulate C(t) and that sAC serves as a critical sensor of bicarbonate in the ciliary body regulating the secretion of substances into the AH that govern outflow facility independent of pressure.

  11. Regulation of Anterior Chamber Drainage by Bicarbonate-sensitive Soluble Adenylyl Cyclase in the Ciliary Body*

    Science.gov (United States)

    Lee, Yong S.; Tresguerres, Martin; Hess, Kenneth; Marmorstein, Lihua Y.; Levin, Lonny R.; Buck, Jochen; Marmorstein, Alan D.

    2011-01-01

    Glaucoma is a leading cause of blindness affecting as many as 2.2 million Americans. All current glaucoma treatment strategies aim to reduce intraocular pressure (IOP). IOP results from the resistance to drainage of aqueous humor (AH) produced by the ciliary body in a process requiring bicarbonate. Once secreted into the anterior chamber, AH drains from the eye via two pathways: uveoscleral and pressure-dependent or conventional outflow (Ct). Modulation of “inflow” and “outflow” pathways is thought to occur via distinct, local mechanisms. Mice deficient in the bicarbonate channel bestrophin-2 (Best2), however, exhibit a lower IOP despite an increase in AH production. Best2 is expressed uniquely in nonpigmented ciliary epithelial (NPE) cells providing evidence for a bicarbonate-dependent communicative pathway linking inflow and outflow. Here, we show that bicarbonate-sensitive soluble adenylyl cyclase (sAC) is highly expressed in the ciliary body in NPE cells, but appears to be absent from drainage tissues. Pharmacologic inhibition of sAC in mice causes a significant increase in IOP due to a decrease in Ct with no effect on inflow. In mice deficient in sAC IOP is elevated, and Ct is decreased relative to wild-type mice. Pharmacologic inhibition of sAC did not alter IOP or Ct in sAC-deficient mice. Based on these data we propose that the ciliary body can regulate Ct and that sAC serves as a critical sensor of bicarbonate in the ciliary body regulating the secretion of substances into the AH that govern outflow facility independent of pressure. PMID:21994938

  12. Solution structure of the 45-residue MgATP-binding peptide of adenylate kinase as examined by 2-D NMR, FTIR, and CD spectroscopy.

    Science.gov (United States)

    Fry, D C; Byler, D M; Susi, H; Brown, E M; Kuby, S A; Mildvan, A S

    1988-05-17

    The structure of a synthetic peptide corresponding to residues 1-45 of rabbit muscle adenylate kinase has been studied in aqueous solution by two-dimensional NMR, FTIR, and CD spectroscopy. This peptide, which binds MgATP and is believed to represent most of the MgATP-binding site of the enzyme [Fry, D.C., Kuby, S.A., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694], appears to maintain a conformation similar to that of residues 1-45 in the X-ray structure of intact porcine adenylate kinase [Sachsenheimer, W., & Schulz, G.E. (1977) J. Mol. Biol. 114, 23-26], with 42% of the residues of the peptide showing NOEs indicative of phi and psi angles corresponding to those found in the protein. The NMR studies suggest that the peptide is composed of two helical regions of residues 4-7 and 23-29, and three stretches of beta-strand at residues 8-15, 30-32, and 35-40, yielding an overall secondary structure consisting of 24% alpha-helix, 38% beta-structure, and 38% aperiodic. Although the resolution-enhanced amide I band of the peptide FTIR spectrum is broad and rather featureless, possibly due to disorder, it can be fit by using methods developed on well-characterized globular proteins. On this basis, the peptide consists of 35 +/- 10% beta-structure, 60 +/- 12% turns and aperiodic structure, and not more than 10% alpha-helix. The CD spectrum is best fit by assuming the presence of at most 13% alpha-helix in the peptide, 24 +/- 2% beta-structure, and 66 +/- 4% aperiodic. The inability of the high-frequency FTIR and CD methods to detect helices in the amount found by NMR may result from the short helical lengths as well as from static and dynamic disorder in the peptide. Upon binding of MgATP, numerous conformational changes in the backbone of the peptide are detected by NMR, with smaller alterations in the overall secondary structure as assessed by CD. Detailed assignments of resonances in the peptide spectrum and intermolecular NOEs between protons of bound MgATP and

  13. The Adenylate-Forming Enzymes AfeA and TmpB Are Involved in Aspergillus nidulans Self-Communication during Asexual Development

    Science.gov (United States)

    Soid-Raggi, Gabriela; Sánchez, Olivia; Ramos-Balderas, Jose L.; Aguirre, Jesús

    2016-01-01

    Aspergillus nidulans asexual sporulation (conidiation) is triggered by different environmental signals and involves the differentiation of specialized structures called conidiophores. The elimination of genes flbA-E, fluG, and tmpA results in a fluffy phenotype characterized by delayed conidiophore development and decreased expression of the conidiation essential gene brlA. While flbA-E encode regulatory proteins, fluG and tmpA encode enzymes involved in the biosynthesis of independent signals needed for normal conidiation. Here we identify afeA and tmpB as new genes encoding members the adenylate-forming enzyme superfamily, whose inactivation cause different fluffy phenotypes and decreased conidiation and brlA expression. AfeA is most similar to unknown function coumarate ligase-like (4CL-Lk) enzymes and consistent with this, a K544N active site modification eliminates AfeA function. TmpB, identified previously as a larger homolog of the oxidoreductase TmpA, contains a NRPS-type adenylation domain. A high degree of synteny in the afeA-tmpA and tmpB regions in the Aspergilli suggests that these genes are part of conserved gene clusters. afeA, tmpA, and tmpB double and triple mutant analysis as well as afeA overexpression experiments indicate that TmpA and AfeA act in the same conidiation pathway, with TmpB acting in a different pathway. Fluorescent protein tagging shows that functional versions of AfeA are localized in lipid bodies and the plasma membrane, while TmpA and TmpB are localized at the plasma membrane. We propose that AfeA participates in the biosynthesis of an acylated compound, either a p-cuomaryl type or a fatty acid compound, which might be oxidized by TmpA and/or TmpB, while TmpB adenylation domain would be involved in the activation of a hydrophobic amino acid, which in turn would be oxidized by the TmpB oxidoreductase domain. Both, AfeA-TmpA and TmpB signals are involved in self-communication and reproduction in A. nidulans. PMID:27047469

  14. The Adenylate-Forming Enzymes AfeA And TmpB Are Involved In Aspergillus nidulans Self-Communication During Asexual Development

    Directory of Open Access Journals (Sweden)

    Jesus eAguirre

    2016-03-01

    Full Text Available Aspergillus nidulans asexual sporulation (conidiation is triggered by different environmental signals and involves the differentiation of specialized structures called conidiophores. The elimination of genes flbA-E, fluG and tmpA results in a fluffy phenotype characterized by delayed conidiophore development and decreased expression of the conidiation essential gene brlA. While flbA-E encode regulatory proteins, fluG and tmpA encode enzymes involved in the biosynthesis of independent signals needed for normal conidiation. Here we identify afeA and tmpB as new genes encoding members the adenylate-forming enzyme superfamily, whose inactivation cause different fluffy phenotypes and decreased conidiation and brlA expression. AfeA is most similar to unknown function coumarate ligase-like (4CL-Lk enzymes and consistent with this, a K544N active site modification eliminates AfeA function. TmpB, identified previously as a larger homolog of the oxidoreductase TmpA, contains a NRPS-type adenylation domain. A high degree of synteny in the afeA-tmpA and tmpB regions in the Aspergilli suggests that these genes are part of conserved gene clusters. afeA, tmpA and tmpB double and triple mutant analysis as well as afeA overexpression experiments indicate that TmpA and AfeA act in the same conidiation pathway, with TmpB acting in a different pathway. Fluorescent protein tagging shows that functional versions of AfeA are localized in organelle-type lipid bodies and the plasma membrane, while TmpA and TmpB are localized at the plasma membrane. We propose that AfeA participates in the biosynthesis of an acylated compound, either a p-cuomaryl type or a fatty acid compound, which might be oxidized by TmpA and/or TmpB, while TmpB adenylation domain would be involved in the activation of a hydrophobic amino acid, which in turn would be oxidized by the TmpB oxidoreductase domain. Both, AfeA-TmpA and TmpB signals are involved in self-communication and reproduction in A

  15. Functional analysis of the Phycomyces carRA gene encoding the enzymes phytoene synthase and lycopene cyclase.

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    Catalina Sanz

    Full Text Available Phycomyces carRA gene encodes a protein with two domains. Domain R is characterized by red carR mutants that accumulate lycopene. Domain A is characterized by white carA mutants that do not accumulate significant amounts of carotenoids. The carRA-encoded protein was identified as the lycopene cyclase and phytoene synthase enzyme by sequence homology with other proteins. However, no direct data showing the function of this protein have been reported so far. Different Mucor circinelloides mutants altered at the phytoene synthase, the lycopene cyclase or both activities were transformed with the Phycomyces carRA gene. Fully transcribed carRA mRNA molecules were detected by Northern assays in the transformants and the correct processing of the carRA messenger was verified by RT-PCR. These results showed that Phycomyces carRA gene was correctly expressed in Mucor. Carotenoids analysis in these transformants showed the presence of ß-carotene, absent in the untransformed strains, providing functional evidence that the Phycomyces carRA gene complements the M. circinelloides mutations. Co-transformation of the carRA cDNA in E. coli with different combinations of the carotenoid structural genes from Erwinia uredovora was also performed. Newly formed carotenoids were accumulated showing that the Phycomyces CarRA protein does contain lycopene cyclase and phytoene synthase activities. The heterologous expression of the carRA gene and the functional complementation of the mentioned activities are not very efficient in E. coli. However, the simultaneous presence of both carRA and carB gene products from Phycomyces increases the efficiency of these enzymes, presumably due to an interaction mechanism.

  16. A guanylyl cyclase-like gene is associated with Gibberella ear rot resistance in maize (Zea mays L.).

    Science.gov (United States)

    Yuan, J; Liakat Ali, M; Taylor, J; Liu, J; Sun, G; Liu, W; Masilimany, P; Gulati-Sakhuja, A; Pauls, K P

    2008-02-01

    Gibberella ear rot, caused by the fungal pathogen Fusarium graminearum Schwabe, is a serious disease of maize (Zea mays L.) grown in northern climates. The infected maize grain contains toxins that are very harmful to livestock and humans. A maize gene that encodes a putative 267-amino acid guanylyl cyclase-like protein (ZmGC1) was characterized and shown to be associated with resistance to this disease. The putative ZmGC1 amino acid sequence is 53% identical and 65% similar to AtGC1, an Arabidopsis guanylyl cyclase. The Zmgc1 coding sequence is nearly identical in a Gibberella ear rot-resistant line (CO387) and a susceptible line (CG62) but several nucleotide sequence differences were observed in the UTRs and introns of the two alleles. Using a 463 bp probe derived from the CG62 allele of Zmgc1 and a recombinant inbred (RI) mapping population developed from a CG62 x CO387 cross, six Zmgc1 restriction fragment length polymorphism (RFLP) fragments (ER1_1, ER1_2, ER1_3, ER1_4, ER1_5, and ER5_1) were mapped on maize chromosomes 2, 3, 7, and 8. Markers ER1_1 and ER5_1 on chromosomes 7 and 8, respectively, were significantly associated with Gibberella ear rot resistance, each in three different environments. The amount of Zmgc1 transcript in ear tissues increased more quickly and to a greater extent in the resistant genotype compared to the susceptible genotype after inoculation with F. graminearum. Zmgc1 is the first guanylyl cyclase gene characterized in maize and the first gene found to be associated with Gibberella ear rot resistance in this plant.

  17. Induction of sesquiterpene cyclase and suppression of squalene synthetase activities in plant cell cultures treated with fungal elicitor.

    Science.gov (United States)

    Vögeli, U; Chappell, J

    1988-12-01

    Addition of elicitor, cell wall fragments of the fungus Phytophthora parasitica, to tobacco cell suspension cultures (Nicotiana tabacum) resulted in the rapid synthesis and secretion of large amounts of antibiotic sesquiterpenoids. Pulse-labeling experiments with [(14)C]acetate and [(3)H] mevalonate demonstrated that the induction of sesquiterpenoid biosynthesis, maximal by 6 to 9 hours after elicitor addition to the cell cultures, was paralleled by a rapid and large decline in the incorporation rate of radioactivity into sterols. Consequently, sterol accumulation was also inhibited upon addition of elicitor to the cell cultures. Sesquiterpene cyclase activity was absent from control cell cultures but induced to a maximum within 10 hours of elicitor addition to the cell cultures. The cyclase activity remained elevated for an additional 30 hours before declining. In contrast, squalene synthetase activity was suppressed to less than 15% of that found in control cells within 7 hours of elicitor addition. Our results suggest that the channeling of isoprenoid intermediates, and especially farnesyl diphosphate, into sesquiterpenoids occurred by a coordinated increase in the sesquiterpene cyclase and a decrease in the squalene synthetase enzyme activities. A reexamination of the data pertaining to the transient induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity (EC 1.1.1.34) in elicitor-treated cells suggested that, while the reductase activity was necessary for sesquiterpenoid biosynthesis, it functioned more to maintain a sufficient level of intermediates between mevalonate and farnesyl diphosphate rather than as a rate limiting step controlling the synthesis rate of any one class of isoprenoids.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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 s