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Sample records for adp-ribosyl cyclase cd38

  1. Thromboxane-induced renal vasoconstriction is mediated by the ADP-ribosyl cyclase CD38 and superoxide anion

    PubMed Central

    Vogel, Paul A.; Kopple, Tayler E.; Arendshorst, William J.

    2013-01-01

    The present renal hemodynamic study tested the hypothesis that CD38 and superoxide anion (O2·−) participate in the vasoconstriction produced by activation of thromboxane prostanoid (TP) receptors in the mouse kidney. CD38 is the major mammalian ADP-ribosyl cyclase contributing to vasomotor tone through the generation of cADP-ribose, a second messenger that activates ryanodine receptors to release Ca2+ from the sarcoplasmic reticulum in vascular smooth muscle cells. We evaluated whether the stable thromboxane mimetic U-46619 causes less pronounced renal vasoconstriction in CD38-deficient mice and the involvement of O2·− in U-46619-induced renal vasoconstriction. Our results indicate that U-46619 activation of TP receptors causes renal vasoconstriction in part by activating cADP-ribose signaling in renal resistance arterioles. Based on maximal renal blood flow and renal vascular resistance responses to bolus injections of U-46619, CD38 contributes 30–40% of the TP receptor-induced vasoconstriction. We also found that the antioxidant SOD mimetic tempol attenuated the magnitude of vasoconstriction by U-46619 in both groups of mice, suggesting mediation by O2·−. The degree of tempol blockage of U-46619-induced renal vasoconstriction was greater in wild-type mice, attenuating renal vasoconstriction by 40% compared with 30% in CD38-null mice. In other experiments, U-46619 rapidly stimulated O2·− production (dihydroethidium fluorescence) in isolated mouse afferent arterioles, an effect abolished by tempol. These observations provide the first in vivo demonstration of CD38 and O2·− involvement in the vasoconstrictor effects of TP receptor activation in the kidney and in vitro evidence for TP receptor stimulation of O2·− production by the afferent arteriole. PMID:23884143

  2. Disordered osteoclast formation and function in a CD38 (ADP-ribosyl cyclase)-deficient mouse establishes an essential role for CD38 in bone resorption.

    PubMed

    Sun, Li; Iqbal, Jameel; Dolgilevich, Svetlana; Yuen, Tony; Wu, Xue-Bin; Moonga, Baljit S; Adebanjo, Olugbenga A; Bevis, Peter J R; Lund, Frances; Huang, Christopher L-H; Blair, Harry C; Abe, Etsuko; Zaidi, Mone

    2003-03-01

    We have evaluated the role of the ADP-ribosyl cyclase, CD38, in bone remodeling, a process by which the skeleton is being renewed constantly through the coordinated activity of osteoclasts and osteoblasts. CD38 catalyzes the cyclization of its substrate, NAD+, to the Ca2+-releasing second messenger, cyclic ADP-ribose (cADPr). We have shown previously that CD38 is expressed both in osteoblasts and osteoclasts. Its activation in the osteoclast triggers Ca2+ release through ryanodine receptors (RyRs), stimulation of interleukin-6 (IL-6), and an inhibition of bone resorption. Here, we have examined the consequences of deleting the CD38 gene in mice on skeletal remodeling. We report that CD38-/- mice displayed a markedly reduced bone mineral density (BMD) at the femur, tibia, and lumbar spine at 3 months and at the lumbar spine at 4 months, with full normalization of the BMD at all sites at 5 months. The osteoporosis at 3 months was accompanied by a reduction in primary spongiosa and increased osteoclast surfaces on histomorphometric analysis. Hematopoetic stem cells isolated ex vivo from CD38-/- mice showed a dramatic approximately fourfold increase in osteoclast formation in response to incubation for 6 days with RANK-L and M-CSF. The osteoclasts so formed in these cultures showed a approximately 2.5-fold increase in resorptive activity compared with wild-type cells. However, when adherent bone marrow stromal cells were allowed to mature into alkaline phosphatase-positive colony-forming units (CFU-Fs), those derived from CD38-/- mice showed a significant reduction in differentiation compared with wild-type cells. Real-time RT-PCR on mRNA isolated from osteoclasts at day 6 showed a significant reduction in IL-6 and IL-6 receptor mRNA, together with significant decreases in the expression of all calcineurin A isoforms, alpha, beta, and gamma. These findings establish a critical role for CD38 in osteoclast formation and bone resorption. We speculate that CD38 functions

  3. Human CD38 is an authentic NAD(P)+ glycohydrolase.

    PubMed Central

    Berthelier, V; Tixier, J M; Muller-Steffner, H; Schuber, F; Deterre, P

    1998-01-01

    The leucoyte surface antigen CD38 has been shown to be an ecto-enzyme with multiple catalytic activities. It is principally a NAD+ glycohydrolase that transforms NAD+ into ADP-ribose and nicotinamide. CD38 is also able to produce small amounts of cyclic ADP-ribose (ADP-ribosyl cyclase activity) and to hydrolyse this cyclic metabolite into ADP-ribose (cyclic ADP-ribose hydrolase activity). To classify CD38 among the enzymes that transfer the ADP-ribosyl moiety of NAD+ to a variety of acceptors, we have investigated its substrate specificity and some characteristics of its kinetic and molecular mechanisms. We find that CD38-catalysed cleavage of the nicotinamide-ribose bond results in the formation of an E.ADP-ribosyl intermediary complex, which is common to all reaction pathways; this intermediate reacts (1) with acceptors such as water (hydrolysis), methanol (methanolysis) or pyridine (transglycosidation), and (2) intramolecularly, yielding cyclic ADP-ribose with a low efficiency. This reaction scheme is also followed when using nicotinamide guanine dinucleotide as an alternative substrate; in this case, however, the cyclization process is highly favoured. The results obtained here are not compatible with the prevailing model for the mode of action of CD38, according to which this enzyme produces first cyclic ADP-ribose which is then immediately hydrolysed into ADP-ribose (i.e. sequential ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities). We show instead that the cyclic metabolite was a reaction product of CD38 rather than an obligatory reaction intermediate during the glycohydrolase activity. Altogether our results lead to the conclusion that CD38 is an authentic 'classical' NAD(P)+ glycohydrolase (EC 3.2.2.6). PMID:9494110

  4. Human lymphocyte antigen CD38 catalyzes the production of cyclic ADP-ribose.

    PubMed

    Summerhill, R J; Jackson, D G; Galione, A

    1993-12-01

    The human lymphocyte antigen CD38 has been shown to share sequence homology with ADP-ribosyl cyclase, the enzyme that catalyzes the conversion of NAD+ to cyclic ADP-ribose (cADPR), a potent Ca(2+)-mobilizing agent. In this study COS1 cells from African Green Monkey kidney were transiently transfected with CD38 cDNA, inducing expression of authentic CD38 on the cell surface. We demonstrate that CD38 expressed in this manner can convert NAD+ to cADPR in the extracellular medium as assessed by Ca2+ release from sea-urchin egg microsomes. PMID:8253202

  5. Functional expression of soluble forms of human CD38 in Escherichia coli and Pichia pastoris.

    PubMed

    Fryxell, K B; O'Donoghue, K; Graeff, R M; Lee, H C; Branton, W D

    1995-06-01

    Cyclic adenosine diphosphate (ADP)-ribose (cADPR), a metabolite of nicotinamide adenine dinucleotide (NAD+), mobilizes calcium from intracellular stores in many cells. The synthesis of cADPR from NAD+ and its subsequent hydrolysis to ADPR is catalyzed by an ADP-ribosyl cyclase and a cADPR hydrolase, respectively. The ADP-ribosyl cyclase cloned from the ovotestis of the marine invertebrate Aplysia californica has amino acid sequence homology to the human lymphocyte surface antigen CD38. CD38 has been shown to catalyze both the formation and the hydrolysis of cADPR. In this study, we produced soluble, enzymatically active CD38 using recombinant expression techniques in bacteria and yeast. We engineered a gene coding for a soluble form of CD38 by excision of the region of the gene coding for the N-terminal amino acids representing the putative membrane spanning sequence and short putative intracellular sequence. For expression in bacteria (Escherichia coli), this construct was cloned into the pFlag-1 plasmid which allows induced, periplasmic expression and relatively simple purification of the soluble CD38. For expression in yeast (Pichia pastoris) the CD38 sequence was further modified to eliminate four putative N-linked glycosylation sites and the resulting construct was expressed as a secreted protein. Both systems produce soluble enzymes of approximately 30 kDa and both recombinant enzymes display similar cyclase and hydrolase activities. PMID:7663169

  6. ADP-ribosylation of membrane components by pertussis and cholera toxin

    SciTech Connect

    Ribeiro-Neto, F.A.P.; Mattera, F.; Hildebrandt, J.D.; Codina, J.; Field, J.B.; Birnbaumer, L.; Sekura, R.D.

    1985-01-01

    Pertussis and cholera toxins are important tools to investigate functional and structural aspects of the stimulatory (N/sub s/) and inhibitory (N/sub i/) regulatory components of adenylyl cyclase. Cholera toxin acts on N/sub s/ by ADP-ribosylating its ..cap alpha../sub s/ subunit; pertussis toxin acts on N/sub i/ by ADP-ribosylating its ..cap alpha..; subunit. By using (/sup 32/P)NAD/sup +/ and determining the transfer of its (/sup 32/P)ADP-ribose moiety to membrane components, it is possible to obtain information on N/sub s/ and N/sub i/. A set of protocols is presented that can be used to study simultaneously and comparatively the susceptibility of N/sub s/ and N/sub i/ to be ADP-ribosylated by cholera and pertussis toxin.

  7. Wnt pathway activation by ADP-ribosylation.

    PubMed

    Yang, Eungi; Tacchelly-Benites, Ofelia; Wang, Zhenghan; Randall, Michael P; Tian, Ai; Benchabane, Hassina; Freemantle, Sarah; Pikielny, Claudio; Tolwinski, Nicholas S; Lee, Ethan; Ahmed, Yashi

    2016-01-01

    Wnt/β-catenin signalling directs fundamental processes during metazoan development and can be aberrantly activated in cancer. Wnt stimulation induces the recruitment of the scaffold protein Axin from an inhibitory destruction complex to a stimulatory signalosome. Here we analyse the early effects of Wnt on Axin and find that the ADP-ribose polymerase Tankyrase (Tnks)--known to target Axin for proteolysis-regulates Axin's rapid transition following Wnt stimulation. We demonstrate that the pool of ADP-ribosylated Axin, which is degraded under basal conditions, increases immediately following Wnt stimulation in both Drosophila and human cells. ADP-ribosylation of Axin enhances its interaction with the Wnt co-receptor LRP6, an essential step in signalosome assembly. We suggest that in addition to controlling Axin levels, Tnks-dependent ADP-ribosylation promotes the reprogramming of Axin following Wnt stimulation; and propose that Tnks inhibition blocks Wnt signalling not only by increasing destruction complex activity, but also by impeding signalosome assembly. PMID:27138857

  8. Wnt pathway activation by ADP-ribosylation

    PubMed Central

    Yang, Eungi; Tacchelly-Benites, Ofelia; Wang, Zhenghan; Randall, Michael P.; Tian, Ai; Benchabane, Hassina; Freemantle, Sarah; Pikielny, Claudio; Tolwinski, Nicholas S.; Lee, Ethan; Ahmed, Yashi

    2016-01-01

    Wnt/β-catenin signalling directs fundamental processes during metazoan development and can be aberrantly activated in cancer. Wnt stimulation induces the recruitment of the scaffold protein Axin from an inhibitory destruction complex to a stimulatory signalosome. Here we analyse the early effects of Wnt on Axin and find that the ADP-ribose polymerase Tankyrase (Tnks)—known to target Axin for proteolysis—regulates Axin's rapid transition following Wnt stimulation. We demonstrate that the pool of ADP-ribosylated Axin, which is degraded under basal conditions, increases immediately following Wnt stimulation in both Drosophila and human cells. ADP-ribosylation of Axin enhances its interaction with the Wnt co-receptor LRP6, an essential step in signalosome assembly. We suggest that in addition to controlling Axin levels, Tnks-dependent ADP-ribosylation promotes the reprogramming of Axin following Wnt stimulation; and propose that Tnks inhibition blocks Wnt signalling not only by increasing destruction complex activity, but also by impeding signalosome assembly. PMID:27138857

  9. The natural history of ADP-ribosyltransferases and the ADP-ribosylation system.

    PubMed

    Aravind, L; Zhang, Dapeng; de Souza, Robson F; Anand, Swadha; Iyer, Lakshminarayan M

    2015-01-01

    Catalysis of NAD(+)-dependent ADP-ribosylation of proteins, nucleic acids, or small molecules has evolved in at least three structurally unrelated superfamilies of enzymes, namely ADP-ribosyltransferase (ART), the Sirtuins, and probably TM1506. Of these, the ART superfamily is the most diverse in terms of structure, active site residues, and targets that they modify. The primary diversification of the ART superfamily occurred in the context of diverse bacterial conflict systems, wherein ARTs play both offensive and defensive roles. These include toxin-antitoxin systems, virus-host interactions, intraspecific antagonism (polymorphic toxins), symbiont/parasite effectors/toxins, resistance to antibiotics, and repair of RNAs cleaved in conflicts. ARTs evolving in these systems have been repeatedly acquired by lateral transfer throughout eukaryotic evolution, starting from the PARP family, which was acquired prior to the last eukaryotic common ancestor. They were incorporated into eukaryotic regulatory/epigenetic control systems (e.g., PARP family and NEURL4), and also used as defensive (e.g., pierisin and CARP-1 families) or immunity-related proteins (e.g., Gig2-like ARTs). The ADP-ribosylation system also includes other domains, such as the Macro, ADP-ribosyl glycohydrolase, NADAR, and ADP-ribosyl cyclase, which appear to have initially diversified in bacterial conflict-related systems. Unlike ARTs, sirtuins appear to have a much smaller presence in conflict-related systems.

  10. The natural history of ADP-ribosyltransferases and the ADP-ribosylation system.

    PubMed

    Aravind, L; Zhang, Dapeng; de Souza, Robson F; Anand, Swadha; Iyer, Lakshminarayan M

    2015-01-01

    Catalysis of NAD(+)-dependent ADP-ribosylation of proteins, nucleic acids, or small molecules has evolved in at least three structurally unrelated superfamilies of enzymes, namely ADP-ribosyltransferase (ART), the Sirtuins, and probably TM1506. Of these, the ART superfamily is the most diverse in terms of structure, active site residues, and targets that they modify. The primary diversification of the ART superfamily occurred in the context of diverse bacterial conflict systems, wherein ARTs play both offensive and defensive roles. These include toxin-antitoxin systems, virus-host interactions, intraspecific antagonism (polymorphic toxins), symbiont/parasite effectors/toxins, resistance to antibiotics, and repair of RNAs cleaved in conflicts. ARTs evolving in these systems have been repeatedly acquired by lateral transfer throughout eukaryotic evolution, starting from the PARP family, which was acquired prior to the last eukaryotic common ancestor. They were incorporated into eukaryotic regulatory/epigenetic control systems (e.g., PARP family and NEURL4), and also used as defensive (e.g., pierisin and CARP-1 families) or immunity-related proteins (e.g., Gig2-like ARTs). The ADP-ribosylation system also includes other domains, such as the Macro, ADP-ribosyl glycohydrolase, NADAR, and ADP-ribosyl cyclase, which appear to have initially diversified in bacterial conflict-related systems. Unlike ARTs, sirtuins appear to have a much smaller presence in conflict-related systems. PMID:25027823

  11. Cholera toxin can catalyze ADP-ribosylation of cytoskeletal proteins

    SciTech Connect

    Kaslow, H.R.; Groppi, V.E.; Abood, M.E.; Bourne, H.R.

    1981-11-01

    Cholera toxin catalyzes transfer of radiolabel from (/sup 32/P)NAD/sup +/ to several peptides in particulate preparations of human foreskin fibroblasts. Resolution of these peptides by two-dimensional gel electrophoresis allowed identification of two peptides of M/sub r/ = 42,000 and 52,000 as peptide subunits of a regulatory component of adenylate cyclase. The radiolabeling of another group of peptides (M/sub r/ = 50,000 to 65,000) suggested that cholera toxin could catalyze ADP-ribosylation of cytoskeletal proteins. This suggestion was confirmed by showing that incubation with cholera toxin and (/sup 32/P)NAD/sup +/ caused radiolabeling of purified microtubule and intermediate filament proteins.

  12. NADP/sup +/ enhances cholera and pertussis toxin-catalyzed ADP-ribosylation of membrane proteins

    SciTech Connect

    Kawai, Y.; Whitsel, C.; Arinze, I.J.

    1986-05-01

    Cholera or pertussis toxin-catalyzed (/sup 32/P)ADP-ribosylation is frequently used to estimate the concentration of the stimulatory (Ns) or inhibitory (Ni) guanine nucleotide regulatory proteins which modulate the activity of adenylate cyclase. With this assay, however, the degradation of the substrate, NAD/sup +/, by endogenous enzymes such as NAD/sup +/-glycohydrolase (NADase) present in the test membranes can influence the results. In this study the authors show that both cholera and pertussis toxin-catalyzed (/sup 32/P)ADP-ribosylation of liver membrane proteins is markedly enhanced by NADP/sup +/. The effect is concentration dependent; with 20 ..mu..M (/sup 32/P)NAD/sup +/ as substrate maximal enhancement is obtained at 0.5-1.0 mM NADP/sup +/. The enhancement of (/sup 32/P)ADP-ribosylation by NADP/sup +/ was much greater than that by other known effectors such as Mg/sup 2 +/, phosphate or isoniazid. The effect of NADP/sup +/ on ADP-ribosylation may occur by inhibition of the degradation of NAD/sup +/ probably by acting as an alternate substrate for NADase. Among inhibitors tested (NADP/sup +/, isoniazid, imidazole, nicotinamide, L-Arg-methyl-ester and HgCl/sub 2/) to suppress NADase activity, NADP/sup +/ was the most effective and, 10 mM, inhibited activity of the enzyme by about 90%. In membranes which contain substantial activities of NADase the inclusion of NADP/sup +/ in the assay is necessary to obtain maximal ADP-ribosylation.

  13. Proteomics Approaches to Identify Mono(ADP-ribosyl)ated and Poly(ADP-ribosyl)ated proteins

    PubMed Central

    Vivelo, Christina A.; Leung, Anthony K. L.

    2015-01-01

    ADP-ribosylation refers to the addition of one or more ADP-ribose units onto protein substrates and this protein modification has been implicated in various cellular processes including DNA damage repair, RNA metabolism, transcription and cell cycle regulation. This review focuses on a compilation of large-scale proteomics studies that identify ADP-ribosylated proteins and their associated proteins by mass spectrometry using a variety of enrichment strategies. Some methods, such as the use of a poly(ADP-ribose)-specific antibody and boronate affinity chromatography and NAD+ analogues, have been employed for decades while others, such as the use of protein microarrays and recombinant proteins that bind ADP-ribose moieties (such as macrodomains), have only recently been developed. The advantages and disadvantages of each method and whether these methods are specific for identifying mono(ADP-ribosyl)ated and poly(ADP-ribosyl)ated proteins will be discussed. Lastly, since poly(ADP-ribose) is heterogeneous in length, it has been difficult to attain a mass signature associated with the modification sites. Several strategies on how to reduce polymer chain length heterogeneity for site identification will be reviewed. PMID:25263235

  14. Proteomics approaches to identify mono-(ADP-ribosyl)ated and poly(ADP-ribosyl)ated proteins.

    PubMed

    Vivelo, Christina A; Leung, Anthony K L

    2015-01-01

    ADP-ribosylation refers to the addition of one or more ADP-ribose units onto protein substrates and this protein modification has been implicated in various cellular processes including DNA damage repair, RNA metabolism, transcription, and cell cycle regulation. This review focuses on a compilation of large-scale proteomics studies that identify ADP-ribosylated proteins and their associated proteins by MS using a variety of enrichment strategies. Some methods, such as the use of a poly(ADP-ribose)-specific antibody and boronate affinity chromatography and NAD(+) analogues, have been employed for decades while others, such as the use of protein microarrays and recombinant proteins that bind ADP-ribose moieties (such as macrodomains), have only recently been developed. The advantages and disadvantages of each method and whether these methods are specific for identifying mono(ADP-ribosyl)ated and poly(ADP-ribosyl)ated proteins will be discussed. Lastly, since poly(ADP-ribose) is heterogeneous in length, it has been difficult to attain a mass signature associated with the modification sites. Several strategies on how to reduce polymer chain length heterogeneity for site identification will be reviewed. PMID:25263235

  15. A Clickable Aminooxy Probe for Monitoring Cellular ADP-Ribosylation

    PubMed Central

    Morgan, Rory K.; Cohen, Michael S.

    2015-01-01

    ADP-ribosylation is essential for cell function, yet there is a dearth of methods for detecting this post-translational modification in cells. Here, we describe a clickable aminooxy alkyne (AO-alkyne) probe that can detect cellular ADP-ribosylation on acidic amino acids following Cu-catalyzed conjugation to an azide-containing reporter. Using AO-alkyne, we show that PARP10 and PARP11 are auto-ADP-ribosylated in cells. We also demonstrate that AO-alkyne can be used to monitor stimulus-induced ADP-ribosylation in cells. Functional studies using AO-alkyne support a previously unknown mechanism for ADP-ribosylation on acidic amino acids, wherein a glutamate or aspartate at the initial C1′-position of ADP-ribose transfers to the C2′ position. This new mechanism for ADP-ribosylation has important implications for how glutamyl/aspartyl-ADP-ribose is recognized by proteins in cells. PMID:25978521

  16. ADP-ribosylation of proteins: Enzymology and biological significance

    SciTech Connect

    Althaus, F.R.; Richter, C.

    1987-01-01

    This book presents an overview of the molecular and biological consequences of the posttranslational modification of proteins with ADP-ribose monomers and polymers. Part one focuses on chromatin-associated poly ADP-ribosylation reactions which have evolved in higher eukaryotes as modulators of chromatin functions. The significance of poly ADP-ribosylation in DNA repair, carcinogenesis, and gene expression during terminal differentiation is discussed. Part two reviews mono ADP-ribosylation reactions which are catalyzed by prokaryotic and eukaryotic enzymes. Consideration is given to the action of bacterial toxins, such as cholera toxin, pertussis toxin, and diphtheria toxin. These toxins have emerged as tools for the molecular probing of proteins involved in signal transduction and protein biosynthesis.

  17. PARPs and ADP-Ribosylation: Fifty Years… and Counting

    PubMed Central

    Kraus, W. Lee

    2015-01-01

    Summary Over 50 years ago, the discovery of poly(ADP-ribose) (PAR) set a new field of science in motion - the field of poly(ADP-ribosyl) transferases (PARPs) and ADP-ribosylation. The field is still flourishing today. The diversity of biological processes now known to require PARPs and ADP-ribosylation was practically unimaginable even two decades ago. From an initial focus on DNA damage detection and repair in response to genotoxic stresses, the field has expanded to include the regulation of chromatin structure, gene expression, and RNA processing in a wide range of biological systems, including reproduction, development, aging, stem cells, inflammation, metabolism, and cancer. This special focus issue of Molecular Cell includes a collection of three Reviews, three Perspectives, and a SnapShot, which together summarize the current state of the field and suggest where it may be headed. PMID:26091339

  18. Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation*

    PubMed Central

    Watanabe, Yukihide; Papoutsoglou, Panagiotis; Maturi, Varun; Tsubakihara, Yutaro; Hottiger, Michael O.; Heldin, Carl-Henrik; Moustakas, Aristidis

    2016-01-01

    We previously established a mechanism of negative regulation of transforming growth factor β signaling mediated by the nuclear ADP-ribosylating enzyme poly-(ADP-ribose) polymerase 1 (PARP1) and the deribosylating enzyme poly-(ADP-ribose) glycohydrolase (PARG), which dynamically regulate ADP-ribosylation of Smad3 and Smad4, two central signaling proteins of the pathway. Here we demonstrate that the bone morphogenetic protein (BMP) pathway can also be regulated by the opposing actions of PARP1 and PARG. PARG positively contributes to BMP signaling and forms physical complexes with Smad5 and Smad4. The positive role PARG plays during BMP signaling can be neutralized by PARP1, as demonstrated by experiments where PARG and PARP1 are simultaneously silenced. In contrast to PARG, ectopic expression of PARP1 suppresses BMP signaling, whereas silencing of endogenous PARP1 enhances signaling and BMP-induced differentiation. The two major Smad proteins of the BMP pathway, Smad1 and Smad5, interact with PARP1 and can be ADP-ribosylated in vitro, whereas PARG causes deribosylation. The overall outcome of this mode of regulation of BMP signal transduction provides a fine-tuning mechanism based on the two major enzymes that control cellular ADP-ribosylation. PMID:27129221

  19. Nuclear CD38 in retinoic acid-induced HL-60 cells

    SciTech Connect

    Yalcintepe, Leman . E-mail: lemany@istanbul.edu.tr; Albeniz, Isil; Adin-Cinar, Suzan; Tiryaki, Demir; Bermek, Engin; Graeff, Richard M.; Lee, Hon Cheung

    2005-02-01

    The cell surface antigen, CD38, is a 45-kDa transmembrane protein which is predominantly expressed on hematopoietic cells during differentiation. As a bifunctional ectoenzyme, it catalyzes the synthesis of cyclic ADP-ribose (cADPR) from NAD{sup +} and hydrolysis of either NAD{sup +} or cADPR to ADP-ribose. All-trans-retinoic acid (RA) is a potent and specific inducer of CD38 in myeloid cells. In this report, we demonstrate that the nuclei of RA-treated human HL-60 myeloblastic cells reveal enzymatic activities inherent to CD38. Thus, GDP-ribosyl cyclase and NAD{sup +} glycohydrolase activities in the nuclear fraction increased very significantly in response to incubation with RA. With Western blotting, we detected in the nuclear protein fraction from RA-treated cells a {approx}43-kDa protein band which was reactive with the CD38-specific monoclonal antibody OKT10. The expression of CD38 in HL-60 nuclei was also shown with FACScan analysis. RA treatment gave rise to an increase in in vitro ADP ribosylation of the {approx}43-kDa nuclear protein. Moreover, nuclei isolated from RA-treated HL-60 cells revealed calcium release in response to cADPR, whereas a similar response was not observed in control nuclei. These results suggest that CD38 is expressed in HL-60 cell nuclei during RA-induced differentiation.

  20. Structure of Plasmodium falciparum ADP-ribosylation factor 1

    SciTech Connect

    Cook, William J.; Smith, Craig D.; Senkovich, Olga; Holder, Anthony A.; Chattopadhyay, Debasish

    2011-09-26

    Vesicular trafficking may play a crucial role in the pathogenesis and survival of the malaria parasite. ADP-ribosylation factors (ARFs) are among the major components of vesicular trafficking pathways in eukaryotes. The crystal structure of ARF1 GTPase from Plasmodium falciparum has been determined in the GDP-bound conformation at 2.5 {angstrom} resolution and is compared with the structures of mammalian ARF1s.

  1. ADP-ribosylation of histones by ARTD1: an additional module of the histone code?

    PubMed

    Hottiger, Michael O

    2011-06-01

    ADP-ribosylation is a covalent post-translational protein modification catalyzed by ADP-ribosyltransferases and is involved in important processes such as cell cycle regulation, DNA damage response, replication or transcription. Histones are ADP-ribosylated by ADP-ribosyltransferase diphtheria toxin-like 1 at specific amino acid residues, in particular lysines, of the histones tails. Specific ADP-ribosyl hydrolases and poly-ADP-ribose glucohydrolases degrade the ADP-ribose polymers. The ADP-ribose modification is read by zinc finger motifs or macrodomains, which then regulate chromatin structure and transcription. Thus, histone ADP-ribosylation may be considered an additional component of the histone code.

  2. The Promise of Proteomics for the Study of ADP-ribosylation

    PubMed Central

    Daniels, Casey M.; Ong, Shao-En; Leung, Anthony K. L.

    2015-01-01

    ADP-ribosylation is a post-translational modification where single units (mono-ADP-ribosylation) or polymeric chains (poly-ADP-ribosylation) of ADP-ribose are conjugated to proteins by ADP-ribosyltransferases. This post-translational modification and the ADP-ribosyltransferases (also known as PARPs) responsible for its synthesis have been found to play a role in nearly all major cellular processes, including DNA repair, transcription, translation, cell signaling and cell death. Furthermore, dysregulation of ADP-ribosylation has been linked to diseases including cancers, diabetes, neurodegenerative disorders and heart failure, leading to the development of therapeutic PARP inhibitors, many of which are currently in clinical trials. The study of this therapeutically important modification has recently been bolstered by the application of mass spectrometry-based proteomics, arguably the most powerful tool for the unbiased analysis of protein modifications. Unfortunately, progress has been hampered by the inherent challenges that stem from the physicochemical properties of ADP-ribose which as a post-translational modification is highly charged, heterogeneous (linear or branched polymers, as well as monomers), labile, and found on a wide range of amino acid acceptors. In this perspective, we discuss the progress that has been made in addressing these challenges, including the recent breakthroughs in proteomics techniques to identify ADP-ribosylation sites, and future developments to provide a proteome-wide view of the many cellular processes regulated by ADP-ribosylation. PMID:26091340

  3. Inhibiting poly(ADP-ribosylation) improves axon regeneration

    PubMed Central

    Byrne, Alexandra B; McWhirter, Rebecca D; Sekine, Yuichi; Strittmatter, Stephen M; Miller, David M; Hammarlund, Marc

    2016-01-01

    The ability of a neuron to regenerate its axon after injury depends in part on its intrinsic regenerative potential. Here, we identify novel intrinsic regulators of axon regeneration: poly(ADP-ribose) glycohodrolases (PARGs) and poly(ADP-ribose) polymerases (PARPs). PARGs, which remove poly(ADP-ribose) from proteins, act in injured C. elegans GABA motor neurons to enhance axon regeneration. PARG expression is regulated by DLK signaling, and PARGs mediate DLK function in enhancing axon regeneration. Conversely, PARPs, which add poly(ADP-ribose) to proteins, inhibit axon regeneration of both C. elegans GABA neurons and mammalian cortical neurons. Furthermore, chemical PARP inhibitors improve axon regeneration when administered after injury. Our results indicate that regulation of poly(ADP-ribose) levels is a critical function of the DLK regeneration pathway, that poly-(ADP ribosylation) inhibits axon regeneration across species, and that chemical inhibition of PARPs can elicit axon regeneration. DOI: http://dx.doi.org/10.7554/eLife.12734.001 PMID:27697151

  4. ADP-ribosylation of dinitrogenase reductase from Clostridium pasteurianum prevents its inhibition of nitrogenase from Azotobacter vinelandii.

    PubMed

    Murrell, S A; Lowery, R G; Ludden, P W

    1988-04-15

    The effect of ADP-ribosylation of dinitrogenase reductase on its binding to dinitrogenase was investigated. Dinitrogenase reductase from Clostridium pasteurianum (Cp2) was a substrate for the ADP-ribosyltransferase and the dinitrogenase-reductase-activating glycohydrolase from Rhodospirillum rubrum. ADP-ribosylation inactivated Cp2 and prevented its formation of a tight complex with dinitrogenase from Azotobacter vinelandii (Av1). The complex between Cp2 and Av1 could not be ADP-ribosylated once it formed.

  5. The role of ADP-ribosylation in regulating DNA interstrand crosslink repair

    PubMed Central

    Gunn, Alasdair R.; Banos-Pinero, Benito; Paschke, Peggy; Sanchez-Pulido, Luis; Ariza, Antonio; Day, Joseph; Emrich, Mehera; Leys, David; Ponting, Chris P.

    2016-01-01

    ABSTRACT ADP-ribosylation by ADP-ribosyltransferases (ARTs) has a well-established role in DNA strand break repair by promoting enrichment of repair factors at damage sites through ADP-ribose interaction domains. Here, we exploit the simple eukaryote Dictyostelium to uncover a role for ADP-ribosylation in regulating DNA interstrand crosslink repair and redundancy of this pathway with non-homologous end-joining (NHEJ). In silico searches were used to identify a protein that contains a permutated macrodomain (which we call aprataxin/APLF-and-PNKP-like protein; APL). Structural analysis reveals that this permutated macrodomain retains features associated with ADP-ribose interactions and that APL is capable of binding poly(ADP-ribose) through this macrodomain. APL is enriched in chromatin in response to cisplatin treatment, an agent that induces DNA interstrand crosslinks (ICLs). This is dependent on the macrodomain of APL and the ART Adprt2, indicating a role for ADP-ribosylation in the cellular response to cisplatin. Although adprt2− cells are sensitive to cisplatin, ADP-ribosylation is evident in these cells owing to redundant signalling by the double-strand break (DSB)-responsive ART Adprt1a, promoting NHEJ-mediated repair. These data implicate ADP-ribosylation in DNA ICL repair and identify that NHEJ can function to resolve this form of DNA damage in the absence of Adprt2. PMID:27587838

  6. State of the art of protein mono-ADP-ribosylation: biological role and therapeutic potential.

    PubMed

    Fabrizio, Gaia; Scarpa, Emanuele Salvatore; Di Girolamo, Maria

    2015-01-01

    Mono-ADP-ribosylation is a post-translational modification that was discovered more than five decades ago, and it consists of the enzymatic transfer of ADP-ribose from NAD⁺ to acceptor proteins. In viruses and prokaryotes, mono-ADP-ribosylation is mainly, but not exclusively, a mechanism used to take control of the host cell. In mammals, mono-ADP-ribosylation serves to regulate protein functions, and it is catalysed by two families of toxin-related cellular ADP-ribosyltransferases: ecto-enzymes that modify various cell-surface proteins, like integrins and receptors, and intracellular enzymes that act on a variety of nuclear and cytosolic proteins. These two families have been recently renamed the ARTCs (clostridia toxin like) and ARTDs (diphtheria toxin like), depending on their conserved structural features, and in terms of their relationships to the bacterial toxins. In addition, two members of the structurally non-related sirtuin family can also modify cellular proteins by mono-ADP-ribosylation. Recently, new examples of ADP-ribosylation of proteins involved in signal transduction and intracellular trafficking have been discovered, thus opening the route to the better molecular understanding of this reaction and of its role in human cell physiology and pathology.

  7. Identification of a Class of Protein ADP-Ribosylating Sirtuins in Microbial Pathogens

    PubMed Central

    Rack, Johannes Gregor Matthias; Morra, Rosa; Barkauskaite, Eva; Kraehenbuehl, Rolf; Ariza, Antonio; Qu, Yue; Ortmayer, Mary; Leidecker, Orsolya; Cameron, David R.; Matic, Ivan; Peleg, Anton Y.; Leys, David; Traven, Ana; Ahel, Ivan

    2015-01-01

    Summary Sirtuins are an ancient family of NAD+-dependent deacylases connected with the regulation of fundamental cellular processes including metabolic homeostasis and genome integrity. We show the existence of a hitherto unrecognized class of sirtuins, found predominantly in microbial pathogens. In contrast to earlier described classes, these sirtuins exhibit robust protein ADP-ribosylation activity. In our model organisms, Staphylococcus aureus and Streptococcus pyogenes, the activity is dependent on prior lipoylation of the target protein and can be reversed by a sirtuin-associated macrodomain protein. Together, our data describe a sirtuin-dependent reversible protein ADP-ribosylation system and establish a crosstalk between lipoylation and mono-ADP-ribosylation. We propose that these posttranslational modifications modulate microbial virulence by regulating the response to host-derived reactive oxygen species. PMID:26166706

  8. Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions.

    PubMed Central

    D'Amours, D; Desnoyers, S; D'Silva, I; Poirier, G G

    1999-01-01

    Poly(ADP-ribosyl)ation is a post-translational modification of proteins. During this process, molecules of ADP-ribose are added successively on to acceptor proteins to form branched polymers. This modification is transient but very extensive in vivo, as polymer chains can reach more than 200 units on protein acceptors. The existence of the poly(ADP-ribose) polymer was first reported nearly 40 years ago. Since then, the importance of poly(ADP-ribose) synthesis has been established in many cellular processes. However, a clear and unified picture of the physiological role of poly(ADP-ribosyl)ation still remains to be established. The total dependence of poly(ADP-ribose) synthesis on DNA strand breaks strongly suggests that this post-translational modification is involved in the metabolism of nucleic acids. This view is also supported by the identification of direct protein-protein interactions involving poly(ADP-ribose) polymerase (113 kDa PARP), an enzyme catalysing the formation of poly(ADP-ribose), and key effectors of DNA repair, replication and transcription reactions. The presence of PARP in these multiprotein complexes, in addition to the actual poly(ADP-ribosyl)ation of some components of these complexes, clearly supports an important role for poly(ADP-ribosyl)ation reactions in DNA transactions. Accordingly, inhibition of poly(ADP-ribose) synthesis by any of several approaches and the analysis of PARP-deficient cells has revealed that the absence of poly(ADP-ribosyl)ation strongly affects DNA metabolism, most notably DNA repair. The recent identification of new poly(ADP-ribosyl)ating enzymes with distinct (non-standard) structures in eukaryotes and archaea has revealed a novel level of complexity in the regulation of poly(ADP-ribose) metabolism. PMID:10455009

  9. Cholera toxin-induced ADP-ribosylation of a 46 kDa protein is decreased in brains of ethanol-fed mice

    SciTech Connect

    Nhamburo, P.T.; Hoffman, P.L.; Tabakoff, B.

    1988-01-01

    The acute in vitro effects of ethanol on cerebral cortical adenylate cyclase activity and beta-adrenergic receptor characteristics suggested a site of action of ethanol at Gs, the stimulatory guanine nucleotide binding protein. After chronic ethanol ingestion, the beta-adrenergic receptor appeared to be uncoupled (i.e., the form of the receptor with high affinity for agonist was undetectable), and stimulation of adenylate cyclase activity by isoproterenol or guanine nucleotides was reduced, suggesting an alteration in the properties of Gs. To further characterize this change, cholera and pertussis toxin-mediated /sup 32/P-ADP-ribosylation of mouse cortical membranes was assessed in mice that had chronically ingested ethanol in a liquid diet. /sup 32/P-labeled proteins were separated by SDS-PAGE and quantitated by autoradiography. There was a selective 30-50% decrease in cholera toxin-induced labeling of 46 kDa protein band in membranes of ethanol-fed mice, with no apparent change in pertussis toxin-induced labeling. The 46 kDa protein has a molecular weight similar to that of the alpha subunit of Gs, suggesting a reduced amount of this protein or a change in its characteristics as a substrate for cholera toxin-induced ADP-ribosylation in cortical membranes of ethanol-fed mice.

  10. Guanine nucleotide-binding proteins that enhance choleragen ADP-ribosyltransferase activity: nucleotide and deduced amino acid sequence of an ADP-ribosylation factor cDNA.

    PubMed Central

    Price, S R; Nightingale, M; Tsai, S C; Williamson, K C; Adamik, R; Chen, H C; Moss, J; Vaughan, M

    1988-01-01

    Three (two soluble and one membrane) guanine nucleotide-binding proteins (G proteins) that enhance ADP-ribosylation of the Gs alpha stimulatory subunit of the adenylyl cyclase (EC 4.6.1.1) complex by choleragen have recently been purified from bovine brain. To further define the structure and function of these ADP-ribosylation factors (ARFs), we isolated a cDNA clone (lambda ARF2B) from a bovine retinal library by screening with a mixed heptadecanucleotide probe whose sequence was based on the partial amino acid sequence of one of the soluble ARFs from bovine brain. Comparison of the deduced amino acid sequence of lambda ARF2B with sequences of peptides from the ARF protein (total of 60 amino acids) revealed only two differences. Whether these are cloning artifacts or reflect the existence of more than one ARF protein remains to be determined. Deduced amino acid sequences of ARF, Go alpha (the alpha subunit of a G protein that may be involved in regulation of ion fluxes), and c-Ha-ras gene product p21 show similarities in regions believed to be involved in guanine nucleotide binding and GTP hydrolysis. ARF apparently lacks a site analogous to that ADP-ribosylated by choleragen in G-protein alpha subunits. Although both the ARF proteins and the alpha subunits bind guanine nucleotides and serve as choleragen substrates, they must interact with the toxin A1 peptide in different ways. In addition to serving as an ADP-ribose acceptor, ARF interacts with the toxin in a manner that modifies its catalytic properties. PMID:3135549

  11. Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization

    SciTech Connect

    Scaife, R.M. ); Wilson, L. ); Purich, D.L. )

    1992-01-14

    Bovine brain microtubule protein, containing both tubulin and microtubule-associated proteins, undergoes ADP-ribosylation in the presence of ({sup 14}C)NAD{sup +} and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the {alpha} and {beta} chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight microtubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated ({sup 14}C)ADP-ribose to an average extent of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state microtubules with ADP-ribosyltransferase and NAD{sup +} resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly.

  12. Agonist-induced ADP-ribosylation of a cytosolic protein in human platelets

    SciTech Connect

    Bruene, B.; Molina Y Vedia, L.; Lapetina, E.G. )

    1990-05-01

    {alpha}-Thrombin and phorbol 12,13-dibutyrate stimulated the mono(ADP-ribosyl)ation of a 42-kDa cytosolic protein of human platelets. This effect was mediated by protein kinase C activation and was inhibited by protein kinase C inhibitor staurosporine. It also was prevented by prostacyclin, which is known to inhibit the phospholipase C-induced formation of 1,2-diacylglycerol, which is one of the endogenous activators of protein kinase C. On sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the 42-kDa protein that is ADP-ribosylated by {alpha}-thrombin was clearly distinct from the {alpha} subunits of membrane-bound inhibitory and stimulatory guanine nucleotide-binding regulatory proteins, respectively G{sub i{alpha}} and G{sub s{alpha}}; the 47-kDa protein that is phosphorylated by protein kinase C in platelets; and the 39-kDa protein that has been shown to be endogenously ADP-ribosylated by agents that release nitric oxide. This information shows that agonist-induced activation of protein kinase leads to the ADP-ribosylation of a specific protein. This covalent modification might have a functional role in platelet activation.

  13. Radiolabelling of bovine myristoylated alanine-rich protein kinase C substrate (MARCKS) in an ADP-ribosylation reaction.

    PubMed

    Chao, D; Severson, D L; Zwiers, H; Hollenberg, M D

    1994-01-01

    In an ADP-ribosylation reaction, we have observed the radiolabelling of a protein in a crude bovine brain homogenate, which upon two-dimensional gel electrophoresis migrated with an acidic pI (< 4.5) and an apparent molecular mass (80-90 kDa) consistent with the properties of the myristoylated, alanine-rich, protein kinase C substrate protein termed MARCKS. To establish the identity of this radiolabelled constituent in brain homogenates, we first purified bovine brain MARCKS using calmodulin-Sepharose affinity chromatography and we then supplemented the crude ADP-ribosylation reaction mixture with this purified MARCKS fraction. Concordant increases in radiolabelling and silver staining of the same protein component from the MARCKS-supplemented ADP-ribosylation reaction, as compared with the ADP-ribosylated crude homogenate, established the identity of this constituent as MARCKS. The radiolabelling of MARCKS was lower in comparison with the ADP-ribosylation of the related neuronal protein B-50/GAP-43 under identical reaction conditions. The potential functional consequences of the ADP-ribosylation of MARCKS are discussed and the possibility is raised that other members of the MARCKS family, such as the F52/MacMARCKS/MRP protein, may also be subject to ADP-ribosylation. PMID:7605610

  14. Photorhabdus luminescens toxins ADP-ribosylate actin and RhoA to force actin clustering.

    PubMed

    Lang, Alexander E; Schmidt, Gudula; Schlosser, Andreas; Hey, Timothy D; Larrinua, Ignacio M; Sheets, Joel J; Mannherz, Hans G; Aktories, Klaus

    2010-02-26

    The bacterium Photorhabdus luminescens is mutualistically associated with entomopathogenetic nematodes. These nematodes invade insect larvae and release the bacteria from their intestine, which kills the insects through the action of toxin complexes. We elucidated the mode of action of two of these insecticidal toxins from P. luminescens. We identified the biologically active components TccC3 and TccC5 as adenosine diphosphate (ADP)-ribosyltransferases, which modify unusual amino acids. TccC3 ADP-ribosylated threonine-148 of actin, resulting in actin polymerization. TccC5 ADP-ribosylated Rho guanosine triphosphatase proteins at glutamine-61 and glutamine-63, inducing their activation. The concerted action of both toxins inhibited phagocytosis of target insect cells and induced extensive intracellular polymerization and clustering of actin. Several human pathogenic bacteria produce related toxins. PMID:20185726

  15. Bacillus cereus Certhrax ADP-ribosylates vinculin to disrupt focal adhesion complexes and cell adhesion.

    PubMed

    Simon, Nathan C; Barbieri, Joseph T

    2014-04-11

    Bacillus cereus is often associated with mild to moderate gastroenteritis; however, some recent isolates cause inhalational anthrax-like diseases and death. These potential emerging human pathogens express multiple virulence factors. B. cereus strain G9241 expresses anthrax toxin, several polysaccharide capsules, and the novel ADP-ribosyltransferase, Certhrax. In this study, we show that Certhrax ADP-ribosylates Arg-433 of vinculin, a protein that coordinates actin cytoskeleton and extracellular matrix interactions. ADP-ribosylation of vinculin disrupted focal adhesion complexes and redistributed vinculin to the cytoplasm. Exogenous vinculin rescued these phenotypes. This provides a mechanism for strain G9241 to breach host barrier defenses and promote bacterial growth and spread. Certhrax is the first bacterial toxin to add a post-translational modification to vinculin to disrupt the actin cytoskeleton.

  16. Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody

    PubMed Central

    Rohrbeck, Astrid; Fühner, Viola; Schröder, Anke; Hagemann, Sandra; Vu, Xuan-Khang; Berndt, Sarah; Hust, Michael; Pich, Andreas; Just, Ingo

    2016-01-01

    Clostridium botulinum exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry. PMID:27043630

  17. Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody.

    PubMed

    Rohrbeck, Astrid; Fühner, Viola; Schröder, Anke; Hagemann, Sandra; Vu, Xuan-Khang; Berndt, Sarah; Hust, Michael; Pich, Andreas; Just, Ingo

    2016-04-01

    Clostridium botulinum exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry. PMID:27043630

  18. Post-Transcriptional Regulation by Poly(ADP-ribosyl)ation of the RNA-Binding Proteins

    PubMed Central

    Ji, Yingbiao; Tulin, Alexei V.

    2013-01-01

    Gene expression is intricately regulated at the post-transcriptional level by RNA-binding proteins (RBPs) via their interactions with pre-messenger RNA (pre-mRNA) and mRNA during development. However, very little is known about the mechanism regulating RBP activities in RNA metabolism. During the past few years, a large body of evidence has suggested that many RBPs, such as heterogeneous nuclear ribonucleoproteins (hnRNPs), undergo post-translational modification through poly(ADP-ribosyl)ation to modulate RNA processing, including splicing, polyadenylation, translation, miRNA biogenesis and rRNA processing. Accordingly, RBP poly(ADP-ribosyl)ation has been shown to be involved in stress responses, stem cell differentiation and retinal morphogenesis. Here, we summarize recent advances in understanding the biological roles of RBP poly(ADP-ribosyl)ation, as controlled by Poly(ADP-ribose) Polymerases (PARPs) and Poly(ADP-ribose) Glycohydrolase (PARG). In addition, we discuss the potential of PARP and PARG inhibitors for the treatment of RBP-related human diseases, including cancer and neurodegenerative disorders. PMID:23921685

  19. ADP-ribosylation by cholera toxin: functional analysis of a cellular system that stimulates the enzymic activity of cholera toxin fragment A/sub 1/

    SciTech Connect

    Gill, D.M.; Coburn, J.

    1987-10-06

    The authors have clarified relationships between cholera toxin, cholera toxin substrates, a membrane protein S that is required for toxin activity, and a soluble protein CF that is needed for the function of S. The toxin has little intrinsic ability to catalyze ADP-ribosylations unless it encounters the active form of the S protein, which is S liganded to GTP or to a GTP analogue. In the presence of CF, S x GTP forms readily, though reversibly, but a more permanent active species, S-guanosine 5'-O-(3-thiotriphosphate) (S x GTP..gamma..S), forms over a period of 10-15 min at 37/sup 0/C. Both guanosine 5'-O-(2-thiodiphosphate) and GTP block this quasi-permanent activation. Some S x GTP..gamma..S forms in membranes that are exposed to CF alone and then to GTP..gamma..S, with a wash in between, and it is possible that CF facilitates a G nucleotide exchange. S x GTP..gamma..S dissolved by nonionic detergents persists in solution and can be used to support the ADP-ribosylation of nucleotide-free substrates. In this circumstance, added guanyl nucleotides have no further effect. This active form of S is unstable, especially when heated, but the thermal inactivation above 45/sup 0/C is decreased by GTP..gamma..S. Active S is required equally for the ADP-ribosylation of all of cholera toxin's protein substrates, regardless of whether they bind GTP or not. They suggest that active S interacts directly with the enzymic A/sub 1/ fragments of cholera toxin and not with any toxin substrate. The activation and activity of S are independent of the state, or even the presence, of adenylate cyclase and seem to be involved with the cyclase system only via cholera toxin. S is apparently not related by function to certain other GTP binding proteins, including p21/sup ras/, and appears to be a new GTP binding protein whose physiologic role remains to be identified.

  20. An Entamoeba histolytica ADP-ribosyl transferase from the diphtheria toxin family modifies the bacterial elongation factor Tu.

    PubMed

    Avila, Eva E; Rodriguez, Orlando I; Marquez, Jaqueline A; Berghuis, Albert M

    2016-06-01

    ADP-ribosyl transferases are enzymes involved in the post-translational modification of proteins; they participate in multiple physiological processes, pathogenesis and host-pathogen interactions. Several reports have characterized the functions of these enzymes in viruses, prokaryotes and higher eukaryotes, but few studies have reported ADP-ribosyl transferases in lower eukaryotes, such as parasites. The locus EHI_155600 from Entamoeba histolytica encodes a hypothetical protein that possesses a domain from the ADP-ribosylation superfamily; this protein belongs to the diphtheria toxin family according to a homology model using poly-ADP-ribosyl polymerase 12 (PARP12 or ARTD12) as a template. The recombinant protein expressed in Escherichia coli exhibited in vitro ADP-ribosylation activity that was dependent on the time and temperature. Unlabeled βNAD(+), but not ADP-ribose, competed in the enzymatic reaction using biotin-βNAD(+) as the ADP-ribose donor. The recombinant enzyme, denominated EhToxin-like, auto-ADP-ribosylated and modified an acceptor from E. coli that was identified by MS/MS as the elongation factor Tu (EF-Tu). To the best of our knowledge, this is the first report to identify an ADP-ribosyl transferase from the diphtheria toxin family in a protozoan parasite. The known toxins from this family (i.e., the diphtheria toxin, the Pseudomonas aeruginosa toxin Exo-A, and Cholix from Vibrio cholerae) modify eukaryotic elongation factor two (eEF-2), whereas the amoeba EhToxin-like modified EF-Tu, which is another elongation factor involved in protein synthesis in bacteria and mitochondria. PMID:27234208

  1. Distribution of cytotoxic and DNA ADP-ribosylating activity in crude extracts from butterflies among the family Pieridae

    PubMed Central

    Matsumoto, Yasuko; Nakano, Tsuyoshi; Yamamoto, Masafumi; Matsushima-Hibiya, Yuko; Odagiri, Ken-Ichi; Yata, Osamu; Koyama, Kotaro; Sugimura, Takashi; Wakabayashi, Keiji

    2008-01-01

    Cabbage butterflies, Pieris rapae and Pieris brassicae, contain strong cytotoxic proteins, designated as pierisin-1 and -2, against cancer cell lines. These proteins exhibit DNA ADP-ribosylating activity. To determine the distribution of substances with cytotoxicity and DNA ADP-ribosylating activity among other species, crude extracts from 20 species of the family Pieridae were examined for cytotoxicity in HeLa cells and DNA ADP-ribosylating activity. Both activities were detected in extracts from 13 species: subtribes Pierina (Pieris rapae, Pieris canidia, Pieris napi, Pieris melete, Pieris brassicae, Pontia daplidice, and Talbotia naganum), Aporiina (Aporia gigantea, Aporia crataegi, Aporia hippia, and Delias pasithoe), and Appiadina (Appias nero and Appias paulina). All of these extracts contained substances recognized by anti-pierisin-1 antibodies, with a molecular mass of ≈100 kDa established earlier for pierisin-1. Moreover, sequences containing NAD-binding sites, conserved in ADP-ribosyltransferases, were amplified from genomic DNA from 13 species of butterflies with cytotoxicity and DNA ADP-ribosylating activity by PCR. Extracts from seven species, Appias lyncida, Leptosia nina, Anthocharis scolymus, Eurema hecabe, Catopsilia pomona, Catopsilia scylla, and Colias erate, showed neither cytotoxicity nor DNA ADP-ribosylating activity, and did not contain substances recognized by anti-pierisin-1 antibodies. Sequences containing NAD-binding sites were not amplified from genomic DNA from these seven species. Thus, pierisin-like proteins, showing cytotoxicity and DNA ADP-ribosylating activity, are suggested to be present in the extracts from butterflies not only among the subtribe Pierina, but also among the subtribes Aporiina and Appiadina. These findings offer insight to understanding the nature of DNA ADP-ribosylating activity in the butterfly. PMID:18256183

  2. Purification and molecular cloning of a DNA ADP-ribosylating protein, CARP-1, from the edible clam Meretrix lamarckii

    PubMed Central

    Nakano, Tsuyoshi; Matsushima-Hibiya, Yuko; Yamamoto, Masafumi; Enomoto, Shigeki; Matsumoto, Yasuko; Totsuka, Yukari; Watanabe, Masahiko; Sugimura, Takashi; Wakabayashi, Keiji

    2006-01-01

    The cabbage butterflies Pieris rapae and Pieris brassicae have unique enzymes, named pierisin-1 and -2, respectively, that catalyze the ADP-ribosylation of guanine residues of DNA, which has been linked with induction of apoptosis and mutation in mammalian cell lines. In the present study, we identified ADP-ribosylation activity targeting DNA in six kinds of edible clam. Similar to our observations with pierisin-1 and -2, crude extracts from the clams Meretrix lamarckii, Ruditapes philippinarum, and Corbicula japonica incubated with calf thymus DNA and β-NAD resulted in production of N2-(ADP-ribos-1-yl)-2′-deoxyguanosine. The DNA ADP-ribosylating protein in the hard clam M. lamarckii, designated as CARP-1, was purified by column chromatography, and its cDNA was cloned. The cDNA encodes a 182-aa protein with a calculated molecular mass of 20,332. The protein synthesized in vitro from the cDNA in a reticulocyte lysate exhibited the same ADP-ribosylating activity as that of purified CARP-1. Neither the nucleotide nor the deduced amino acid sequence of CARP-1 showed homology with pierisin-1 or -2. However, a glutamic acid residue (E128) at the putative NAD-binding site, conserved in all ADP-ribosyltransferases, was found in CARP-1, and replacement of aspartic acid for this glutamic acid resulted in loss of almost all ADP-ribosylating activity. CARP-1 in the culture medium showed no cytotoxicity against HeLa and TMK-1 cells; however, introduction of this protein by electroporation induced apoptosis in these cells. The finding of clam ADP-ribosylating protein targeting guanine residues in DNA could offer new insights into the biological significance of ADP-ribosylation of DNA. PMID:16945908

  3. Stimulation of mono-ADP ribosylation in rat liver plasma membranes after long-term alcohol intake.

    PubMed

    Nomura, F; Noda, M

    1993-10-01

    ADP ribosylation is considered one of the important covalent modifications of cellular proteins catalyzed by ADP ribosyltransferase, which transfers ADP ribose moiety of NAD to an acceptor protein. Because a growing body of evidence has suggested significant biological roles for mono-ADP ribosylations in transmembrane signal transduction and other cell metabolism, how alcohol intake alters them is of interest. Cholera toxin and pertussis toxin have been widely used as probes to investigate the roles of GTP-binding proteins (G-proteins) in the transduction of hormonal and sensory signals. We first tested effects of long-term alcohol intake on these toxin-catalyzed ADP ribosylations of G-proteins in rat liver plasma membranes. Treatment of rat liver plasma membrane with [32P]NAD and thiol-preactivated cholera toxin resulted in the labeling of a 44-kD band, most likely an alpha-subunit of the stimulatory GTP-binding protein, the extent of which was much greater in alcohol-fed rats than in pair-fed controls. Analogous experiments with pertussis toxin also demonstrated enhancement of toxin-catalyzed ADP ribosylation of the inhibitory GTP-binding protein after long-term alcohol intake. More interesting was that long-term alcohol intake remarkably stimulated endogenous mono-ADP ribosylation of a 58-kD protein in a GTP-dependent manner. In vitro, ethanol (50 mmol/L) or a single load of ethanol (3 gm/kg) did not stimulate the reaction. Thus long-term alcohol intake stimulated both toxin-catalyzed and endogenous mono-ADP ribosylations of proteins in rat liver plasma membranes. Pursuit of alcohol interaction with mono-ADP ribosylation may provide an interesting approach to the study of alcohol's effects on the liver.

  4. HPF1/C4orf27 Is a PARP-1-Interacting Protein that Regulates PARP-1 ADP-Ribosylation Activity

    PubMed Central

    Gibbs-Seymour, Ian; Fontana, Pietro; Rack, Johannes Gregor Matthias; Ahel, Ivan

    2016-01-01

    Summary We report the identification of histone PARylation factor 1 (HPF1; also known as C4orf27) as a regulator of ADP-ribosylation signaling in the DNA damage response. HPF1/C4orf27 forms a robust protein complex with PARP-1 in cells and is recruited to DNA lesions in a PARP-1-dependent manner, but independently of PARP-1 catalytic ADP-ribosylation activity. Functionally, HPF1 promotes PARP-1-dependent in trans ADP-ribosylation of histones and limits DNA damage-induced hyper-automodification of PARP-1. Human cells lacking HPF1 exhibit sensitivity to DNA damaging agents and PARP inhibition, thereby suggesting an important role for HPF1 in genome maintenance and regulating the efficacy of PARP inhibitors. Collectively, our results demonstrate how a fundamental step in PARP-1-dependent ADP-ribosylation signaling is regulated and suggest that HPF1 functions at the crossroads of histone ADP-ribosylation and PARP-1 automodification. PMID:27067600

  5. Roles of Asp179 and Glu270 in ADP-Ribosylation of Actin by Clostridium perfringens Iota Toxin

    PubMed Central

    Belyy, Alexander; Tabakova, Irina; Lang, Alexander E.; Jank, Thomas; Belyi, Yury; Aktories, Klaus

    2015-01-01

    Clostridium perfringens iota toxin is a binary toxin composed of the enzymatically active component Ia and receptor binding component Ib. Ia is an ADP-ribosyltransferase, which modifies Arg177 of actin. The previously determined crystal structure of the actin-Ia complex suggested involvement of Asp179 of actin in the ADP-ribosylation reaction. To gain more insights into the structural requirements of actin to serve as a substrate for toxin-catalyzed ADP-ribosylation, we engineered Saccharomyces cerevisiae strains, in which wild type actin was replaced by actin variants with substitutions in residues located on the Ia-actin interface. Expression of the actin mutant Arg177Lys resulted in complete resistance towards Ia. Actin mutation of Asp179 did not change Ia-induced ADP-ribosylation and growth inhibition of S. cerevisiae. By contrast, substitution of Glu270 of actin inhibited the toxic action of Ia and the ADP-ribosylation of actin. In vitro transcribed/translated human β-actin confirmed the crucial role of Glu270 in ADP-ribosylation of actin by Ia. PMID:26713879

  6. Distribution of protein poly(ADP-ribosyl)ation systems across all domains of life

    PubMed Central

    Perina, Dragutin; Mikoč, Andreja; Ahel, Josip; Ćetković, Helena; Žaja, Roko; Ahel, Ivan

    2014-01-01

    Poly(ADP-ribosyl)ation is a post-translational modification of proteins involved in regulation of many cellular pathways. Poly(ADP-ribose) (PAR) consists of chains of repeating ADP-ribose nucleotide units and is synthesized by the family of enzymes called poly(ADP-ribose) polymerases (PARPs). This modification can be removed by the hydrolytic action of poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3). Hydrolytic activity of macrodomain proteins (MacroD1, MacroD2 and TARG1) is responsible for the removal of terminal ADP-ribose unit and for complete reversion of protein ADP-ribosylation. Poly(ADP-ribosyl)ation is widely utilized in eukaryotes and PARPs are present in representatives from all six major eukaryotic supergroups, with only a small number of eukaryotic species that do not possess PARP genes. The last common ancestor of all eukaryotes possessed at least five types of PARP proteins that include both mono and poly(ADP-ribosyl) transferases. Distribution of PARGs strictly follows the distribution of PARP proteins in eukaryotic species. At least one of the macrodomain proteins that hydrolyse terminal ADP-ribose is also always present. Therefore, we can presume that the last common ancestor of all eukaryotes possessed a fully functional and reversible PAR metabolism and that PAR signalling provided the conditions essential for survival of the ancestral eukaryote in its ancient environment. PARP proteins are far less prevalent in bacteria and were probably gained through horizontal gene transfer. Only eleven bacterial species possess all proteins essential for a functional PAR metabolism, although it is not known whether PAR metabolism is truly functional in bacteria. Several dsDNA viruses also possess PARP homologues, while no PARP proteins have been identified in any archaeal genome. Our analysis of the distribution of enzymes involved in PAR metabolism provides insight into the evolution of these important signalling systems, as well as

  7. Poly(ADP-ribosyl)ation is recognized by ECT2 during mitosis.

    PubMed

    Li, Mo; Bian, Chunjing; Yu, Xiaochun

    2014-01-01

    Poly(ADP-ribosyl)ation is an unique posttranslational modification and required for spindle assembly and function during mitosis. However, the molecular mechanism of poly(ADP-ribose) (PAR) in mitosis remains elusive. Here, we show the evidence that PAR is recognized by ECT2, a key guanine nucleotide exchange factor in mitosis. The BRCT domain of ECT2 directly binds to PAR both in vitro and in vivo. We further found that α-tubulin is PARylated during mitosis. PARylation of α-tubulin is recognized by ECT2 and recruits ECT2 to mitotic spindle for completing mitosis. Taken together, our study reveals a novel mechanism by which PAR regulates mitosis.

  8. Mechanism of activation of cholera toxin by ADP-ribosylation factor (ARF): both low- and high-affinity interactions of ARF with guanine nucleotides promote toxin activation.

    PubMed

    Bobak, D A; Bliziotes, M M; Noda, M; Tsai, S C; Adamik, R; Moss, J

    1990-01-30

    Activation of adenylyl cyclase by cholera toxin A subunit (CT-A) results from the ADP-ribosylation of the stimulatory guanine nucleotide binding protein (GS alpha). This process requires GTP and an endogenous guanine nucleotide binding protein known as ADP-ribosylation factor (ARF). One membrane (mARF) and two soluble forms (sARF I and sARF II) of ARF have been purified from bovine brain. Because the conditions reported to enhance the binding of guanine nucleotides by ARF differ from those observed to promote optimal activity, we sought to characterize the determinants influencing the functional interaction of guanine nucleotides with ARF. High-affinity GTP binding by sARF II (apparent KD of approximately 70 nM) required Mg2+, DMPC, and sodium cholate. sARF II, in DMPC/cholate, also enhanced CT-A ADP-ribosyltransferase activity (apparent EC50 for GTP of approximately 50 nM), although there was a delay before achievement of a maximal rate of sARF II stimulated toxin activity. The delay was abolished by incubation of sARF II with GTP at 30 degrees C before initiation of the assay. In contrast, a maximal rate of activation of toxin by sARF II, in 0.003% SDS, occurred without delay (apparent EC50 for GTP of approximately 5 microM). High-affinity GTP binding by sARF II was not detectable in SDS. Enhancement of CT-A ADP-ribosyltransferase activity by sARF II, therefore, can occur under conditions in which sARF II exhibits either a relatively low affinity or a relatively high affinity for GTP. The interaction of GTP with ARF under these conditions may reflect ways in which intracellular membrane and cytosolic environments modulate GTP-mediated activation of ARF.

  9. Analysis of Chromatin ADP-Ribosylation at the Genome-wide Level and at Specific Loci by ADPr-ChAP.

    PubMed

    Bartolomei, Giody; Leutert, Mario; Manzo, Massimiliano; Baubec, Tuncay; Hottiger, Michael O

    2016-02-01

    Chromatin ADP-ribosylation regulates important cellular processes. However, the exact location and magnitude of chromatin ADP-ribosylation are largely unknown. A robust and versatile method for assessing chromatin ADP-ribosylation is therefore crucial for further understanding its function. Here, we present a chromatin affinity precipitation method based on the high specificity and avidity of two well-characterized ADP-ribose binding domains to map chromatin ADP-ribosylation at the genome-wide scale and at specific loci. Our ADPr-ChAP method revealed that in cells exposed to oxidative stress, ADP-ribosylation of chromatin scales with histone density, with highest levels at heterochromatic sites and depletion at active promoters. Furthermore, in growth factor-induced adipocyte differentiation, increased chromatin ADP-ribosylation was observed at PPARγ target genes, whose expression is ADP-ribosylation dependent. In combination with deep-sequencing and conventional chromatin immunoprecipitation, the established ADPr-ChAP provides a valuable resource for the bioinformatic comparison of ADP-ribosylation with other chromatin modifications and for addressing its role in other biologically important processes. PMID:26833088

  10. Tuning IL-2 signaling by ADP-ribosylation of CD25

    PubMed Central

    Teege, Sophie; Hann, Alexander; Miksiewicz, Maria; MacMillan, Cary; Rissiek, Björn; Buck, Friedrich; Menzel, Stephan; Nissen, Marion; Bannas, Peter; Haag, Friedrich; Boyer, Olivier; Seman, Michel; Adriouch, Sahil; Koch-Nolte, Friedrich

    2015-01-01

    Control of immunologic tolerance and homeostasis rely on Foxp3+CD4+CD25+ regulatory T cells (Tregs) that constitutively express the high affinity receptor for Interleukin-2, CD25. Tregs proliferate in response to injections of IL-2/anti-IL-2 antibody complexes or low doses of IL-2. However, little is known about endogenous mechanisms that regulate the sensitivity of CD25 to signaling by IL-2. Here we demonstrate that CD25 is ADP-ribosylated at Arg35 in the IL-2 binding site by ecto-ADP-ribosyltransferase ARTC2.2, a toxin-related GPI-anchored ecto-enzyme. ADP-ribosylation inhibits binding of IL-2 by CD25, IL-2- induced phosphorylation of STAT5, and IL-2-dependent cell proliferation. Our study elucidates an as-yet-unrecognized mechanism to tune IL-2 signaling. This newly found mechanism might thwart Tregs at sites of inflammation and thereby permit a more potent response of activated effector T cells. PMID:25753532

  11. Poly(ADP-ribosyl)ation-dependent Transient Chromatin Decondensation and Histone Displacement following Laser Microirradiation.

    PubMed

    Strickfaden, Hilmar; McDonald, Darin; Kruhlak, Michael J; Haince, Jean-Francois; Th'ng, John P H; Rouleau, Michele; Ishibashi, Toytaka; Corry, Gareth N; Ausio, Juan; Underhill, D Alan; Poirier, Guy G; Hendzel, Michael J

    2016-01-22

    Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.

  12. Pierisins and CARP-1: ADP-ribosylation of DNA by ARTCs in butterflies and shellfish.

    PubMed

    Nakano, Tsuyoshi; Takahashi-Nakaguchi, Azusa; Yamamoto, Masafumi; Watanabe, Masahiko

    2015-01-01

    The cabbage butterfly, Pieris rapae, and related species possess a previously unknown ADP-ribosylating toxin, guanine specific ADP-ribosyltransferase. This enzyme toxin, known as pierisin, consists of enzymatic N-terminal domain and receptor-binding C-terminal domain, or typical AB-toxin structure. Pierisin efficiently transfers an ADP-ribosyl moiety to the N(2) position of the guanine base of dsDNA. Receptors for pierisin are suggested to be the neutral glycosphingolipids, globotriaosylceramide (Gb3), and globotetraosylceramide (Gb4). This DNA-modifying toxin exhibits strong cytotoxicity and induces apoptosis in various human cell lines, which can be blocked by Bcl-2. Pierisin also produces detrimental effects on the eggs and larvae of the non-habitual parasitoids. In contrast, a natural parasitoid of the cabbage butterfly, Cotesia glomerata, was resistant to this toxin. The physiological role of pierisin in the butterfly is suggested to be a defense factor against parasitization by wasps. Other type of DNA ADP-ribosyltransferase is present in certain kinds of edible clams. For example, the CARP-1 protein found in Meretrix lamarckii consists of an enzymatic domain without a possible receptor-binding domain. Pierisin and CARP-1 are almost fully non-homologous at the amino acid sequence level, but other ADP-ribosyltransferases homologous to pierisin are present in different biological species such as eubacterium Streptomyces. Possible diverse physiological roles of the DNA ADP-ribosyltransferases are discussed.

  13. Poly(ADP-ribosyl)ation-dependent Transient Chromatin Decondensation and Histone Displacement following Laser Microirradiation.

    PubMed

    Strickfaden, Hilmar; McDonald, Darin; Kruhlak, Michael J; Haince, Jean-Francois; Th'ng, John P H; Rouleau, Michele; Ishibashi, Toytaka; Corry, Gareth N; Ausio, Juan; Underhill, D Alan; Poirier, Guy G; Hendzel, Michael J

    2016-01-22

    Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement. PMID:26559976

  14. Poly(ADP-ribosyl)ation of p53 Contributes to TPEN-Induced Neuronal Apoptosis

    PubMed Central

    Kim, Hyun-Lim; Ra, Hana; Kim, Ki-Ryeong; Lee, Jeong-Min; Im, Hana; Kim, Yang-Hee

    2015-01-01

    Depletion of intracellular zinc by N,N,N′,N′-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces p53-mediated protein synthesis-dependent apoptosis of mouse cortical neurons. Here, we examined the requirement for poly(ADP-ribose) polymerase (PARP)-1 as an upstream regulator of p53 in zinc depletion-induced neuronal apoptosis. First, we found that chemical inhibition or genetic deletion of PARP-1 markedly attenuated TPEN-induced apoptosis of cultured mouse cortical neurons. Poly(ADP-ribosyl)ation of p53 occurred starting 1 h after TPEN treatment. Suggesting the critical role of PARP-1, the TPEN-induced increase of stability and activity of p53 as well as poly(ADP-ribosyl)ation of p53 was almost completely blocked by PARP inhibition. Consistent with this, the induction of downstream proapoptotic proteins PUMA and NOXA was noticeably reduced by chemical inhibitors or genetic deletion of PARP-1. TPEN-induced cytochrome C release into the cytosol and caspase-3 activation were also blocked by inhibition of PARP-1. Taken together, these findings indicate that PARP-1 is essential for TPEN-induced neuronal apoptosis. PMID:25813624

  15. Cholera toxin partially inhibits the T-cell response to phytohaemagglutinin through the ADP-ribosylation of a 45 kDa membrane protein.

    PubMed Central

    Nel, A E; Vandenplas, M; Wooten, M M; Cooper, R; Vandenplas, S; Rheeder, A; Daniels, J

    1988-01-01

    This study examines the influence of cholera toxin (CT) on T lymphocyte activation by the mitogenic lectin phytohaemagglutinin (PHA). CT suppressed lectin-induced [3H]thymidine uptake in a dose-dependent fashion and acted synergistically with PHA in the generation of intracellular cyclic AMP. The toxin was assumed to act on Gs, because it also stimulated ADP-ribosylation of a 45 kDa membrane protein in vitro; no additional substrates were seen. The inhibitory effect of the adenylate cyclase/cyclic AMP pathway was shown to be directed at a concomitant stimulatory pathway, namely inositol phospholipid turnover. Lectin-stimulated 32P incorporation into both phosphatidylinositol as well as its 4,5-biphosphate derivative was depressed in the presence of CT or exogenous dibutyryl cyclic AMP. This, in turn, was associated with reduced activation of C-kinase as determined by decreased lectin-induced translocation from the cytosol to the surface membrane. These results indicate that Gs probably acts as a transducer between the PHA receptor and adenylate cyclase and may give rise to an exaggerated adenylate cyclase response in the presence of CT. It would seem as if reduction in inositol phospholipid turnover is related to the elevation of cyclic AMP rather than a CT effect on a putative transducer which acts directly on phospholipase C. Our study does not exclude the existence of non-CT-sensitive transducers in this capacity. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. PMID:2851989

  16. Social memory, amnesia, and autism: brain oxytocin secretion is regulated by NAD+ metabolites and single nucleotide polymorphisms of CD38.

    PubMed

    Higashida, Haruhiro; Yokoyama, Shigeru; Huang, Jian-Jun; Liu, Li; Ma, Wen-Jie; Akther, Shirin; Higashida, Chiharu; Kikuchi, Mitsuru; Minabe, Yoshio; Munesue, Toshio

    2012-11-01

    Previously, we demonstrated that CD38, a transmembrane protein with ADP-ribosyl cyclase activity, plays a critical role in mouse social behavior by regulating the release of oxytocin (OXT), which is essential for mutual recognition. When CD38 was disrupted, social amnesia was observed in Cd38 knockout mice. The autism spectrum disorders (ASDs), characterized by defects in reciprocal social interaction and communication, occur either sporadically or in a familial pattern. However, the etiology of ASDs remains largely unknown. Therefore, the theoretical basis for pharmacological treatments has not been established. Hence, there is a rationale for investigating single nucleotide polymorphisms (SNPs) in the human CD38 gene in ASD subjects. We found several SNPs in this gene. The SNP rs3796863 (C>A) was associated with high-functioning autism (HFA) in American samples from the Autism Gene Resource Exchange. Although this finding was partially confirmed in low-functioning autism subjects in Israel, it has not been replicated in Japanese HFA subjects. The second SNP of interest, rs1800561 (4693C>T), leads to the substitution of an arginine (R) at codon 140 by tryptophan (W; R140W) in CD38. This mutation was found in four probands of ASD and in family members of three pedigrees with variable levels of ASD or ASD traits. The plasma levels of OXT in ASD subjects with the R140W allele were lower than those in ASD subjects lacking this allele. The OXT levels were unchanged in healthy subjects with or without this mutation. One proband with the R140W allele receiving intranasal OXT for approximately 3years showed improvement in areas of social approach, eye contact and communication behaviors, emotion, irritability, and aggression. Five other ASD subjects with mental deficits received nasal OXT for various periods; three subjects showed improved symptoms, while two showed little or no effect. These results suggest that SNPs in CD38 may be possible risk factors for ASD by

  17. Regulation of E2F1-induced apoptosis by poly(ADP-ribosyl)ation

    PubMed Central

    Kumari, A; Iwasaki, T; Pyndiah, S; Cassimere, E K; Palani, C D; Sakamuro, D

    2015-01-01

    The transcription factor adenovirus E2 promoter-binding factor (E2F)-1 normally enhances cell-cycle progression, but it also induces apoptosis under certain conditions, including DNA damage and serum deprivation. Although DNA damage facilitates the phosphorylation and stabilization of E2F1 to trigger apoptosis, how serum starvation renders cells vulnerable to E2F1-induced apoptosis remains unclear. Because poly(ADP-ribose) polymerase 1 (PARP1), a nuclear enzyme essential for genomic stability and chromatin remodeling, interacts directly with E2F1, we investigated the effects of PARP1 on E2F1-mediated functions in the presence and absence of serum. PARP1 attenuation, which increased E2F1 transactivation, induced G2/M cell-cycle arrest under normal growth conditions, but enhanced E2F1-induced apoptosis in serum-starved cells. Interestingly, basal PARP1 activity was sufficient to modify E2F1 by poly(ADP-ribosyl)ation, which stabilized the interaction between E2F1 and the BIN1 tumor suppressor in the nucleus. Accordingly, BIN1 acted as an RB1-independent E2F1 corepressor. Because E2F1 directly activates the BIN1 gene promoter, BIN1 curbed E2F1 activity through a negative-feedback mechanism. Conversely, when the BIN1–E2F1 interaction was abolished by PARP1 suppression, E2F1 continuously increased BIN1 levels. This is functionally germane, as PARP1-depletion-associated G2/M arrest was reversed by the transfection of BIN1 siRNA. Moreover, PARP-inhibitor-associated anti-transformation activity was compromised by the coexpression of dominant-negative BIN1. Because serum starvation massively reduced the E2F1 poly(ADP-ribosyl)ation, we conclude that the release of BIN1 from hypo-poly(ADP-ribosyl)ated E2F1 is a mechanism by which serum starvation promotes E2F1-induced apoptosis. PMID:25257171

  18. Poly(ADP-ribosylation) regulates chromatin organization through histone H3 modification and DNA methylation of the first cell cycle of mouse embryos

    SciTech Connect

    Osada, Tomoharu; Rydén, Anna-Margareta; Masutani, Mitsuko

    2013-04-26

    Highlights: •Histone modification of the mouse pronuclei is regulated by poly(ADP-ribosylation). •Hypermethylation of the mouse female pronuclei is maintained by poly(ADP-ribosylation). •Parp1 is physically interacted with Suz12, which may function in the pronuclei. •Poly(ADP-ribosylation) affects ultrastructure of chromatin of the mouse pronucleus. -- Abstract: We examined the roles of poly(ADP-ribosylation) in chromatin remodeling during the first cell cycle of mouse embryos. Drug-based inhibition of poly(ADP-ribosylation) by a PARP inhibitor, PJ-34, revealed up-regulation of dimethylation of histone H3 at lysine 4 in male pronuclei and down-regulation of dimethylation of histone H3 at lysine 9 (H3K9) and lysine 27 (H3K27). Association of poly(ADP-ribosylation) with histone modification was suggested to be supported by the interaction of Suz12, a histone methyltransferase in the polycomb complex, with Parp1. PARP activity was suggested to be required for a proper localization and maintenance of Suz12 on chromosomes. Notably, DNA methylation level of female pronuclei in one-cell embryos was robustly decreased by PJ-34. Electron microscopic analysis showed a frequent appearance of unusual electron-dense areas within the female pronuclei, implying the disorganized and hypercondensed chromatin ultrastructure. These results show that poly(ADP-ribosylation) is important for the integrity of non-equivalent epigenetic dynamics of pronuclei during the first cell cycle of mouse embryos.

  19. Localization and characterization of the human ADP-ribosylation factor 5 (ARF5) gene

    SciTech Connect

    McGuire, R.E. |; Daiger, S.P.; Green, E.D.

    1997-05-01

    ADP-ribosylation factor 5 (ARF5) is a member of the ARF gene family. The ARF proteins stimulate the in vitro ADP-ribosyltransferase activity of cholera toxin and appear to play a role in vesicular trafficking in vivo. We have mapped ARF5, one of the six known mammalian ARF genes, to a well-defined yeast artificial chromosome contig on human chromosome 7q31.3. In addition, we have isolated and sequenced an {approximately}3.2-kb genomic segment that contains the entire ARF5 coding region, revealing the complete intron-exon structure of the gene. With six coding exons and five introns, the genomic structure of ARF5 is unique among the mammalian ARF genes and provides insight about the evolutionary history of this ancient gene family. 20 refs., 2 figs., 1 tab.

  20. Chemical reporters for exploring ADP-ribosylation and AMPylation at the host-pathogen interface

    PubMed Central

    Westcott, Nathan P.; Hang, Howard C.

    2014-01-01

    Bacterial pathogens secrete protein toxins and effectors that hijack metabolites to covalently modify key host proteins and interfere with their function during infection. Adenosine metabolites, such as nicotinamide adenine dinucleotide (NAD) and adenosine triphosphate (ATP), have in particular been co-opted by these secreted virulence factors to reprogram host pathways. While some host targets for secreted virulence factors have been identified, other toxin and effector substrates have been elusive, which require new methods for their characterization. In this review, we focus on chemical reporters based on NAD and ATP that should facilitate the discovery and characterization of adenosine diphosphate (ADP)-ribosylation and adenylylation/AMPylation in bacterial pathogenesis and cell biology. PMID:25461386

  1. Two novel human members of an emerging mammalian gene family related to mono-ADP-ribosylating bacterial toxins

    SciTech Connect

    Koch-Nolte, F.; Haag, F.; Braren, R.

    1997-02-01

    Mono-ADP-ribosylation is one of the posttranslational protein modifications regulating cellular metabolism, e.g., nitrogen fixation, in prokaryotes. Several bacterial toxins mono-ADP-ribosylate and inactivate specific proteins in their animal hosts. Recently, two mammalian GPI-anchored cell surface enzymes with similar activities were cloned (designated ART1 and ART2). We have now identified six related expressed sequence tags (ESTs) in the public database and cloned the two novel human genes from which these are derived (designated ART3 and ART4). The deduced amino acid sequences of the predicted gene products show 28% sequence identity to one another and 32-41% identity vs the muscle and T cell enzymes. They contain signal peptide sequences characteristic of GPI anchorage. Southern Zoo blot analyses suggest the presence of related genes in other mammalian species. By PCR screening of somatic cell hybrids and by in situ hybridization, we have mapped the two genes to human chromosomes 4p14-p15.l and 12q13.2- q13.3. Northern blot analyses show that these genes are specifically expressed in testis and spleen, respectively. Comparison of genomic and cDNA sequences reveals a conserved exon/intron structure, with an unusually large exon encoding the predicted mature membrane proteins. Secondary structure prediction analyses indicate conserved motifs and amino acid residues consistent with a common ancestry of this emerging mammalian enzyme family and bacterial mono(ADP-ribosyl)transferases. It is possible that the four human gene family members identified so far represent the {open_quotes}tip of an iceberg,{close_quote} i.e., a larger family of enzymes that influences the function of target proteins via mono-ADP-ribosylation. 35 refs., 4 figs.

  2. Poly(ADP-ribosyl)ation of Methyl CpG Binding Domain Protein 2 Regulates Chromatin Structure.

    PubMed

    Becker, Annette; Zhang, Peng; Allmann, Lena; Meilinger, Daniela; Bertulat, Bianca; Eck, Daniel; Hofstaetter, Maria; Bartolomei, Giody; Hottiger, Michael O; Schreiber, Valérie; Leonhardt, Heinrich; Cardoso, M Cristina

    2016-03-01

    The epigenetic information encoded in the genomic DNA methylation pattern is translated by methylcytosine binding proteins like MeCP2 into chromatin topology and structure and gene activity states. We have shown previously that the MeCP2 level increases during differentiation and that it causes large-scale chromatin reorganization, which is disturbed by MeCP2 Rett syndrome mutations. Phosphorylation and other posttranslational modifications of MeCP2 have been described recently to modulate its function. Here we show poly(ADP-ribosyl)ation of endogenous MeCP2 in mouse brain tissue. Consequently, we found that MeCP2 induced aggregation of pericentric heterochromatin and that its chromatin accumulation was enhanced in poly(ADP-ribose) polymerase (PARP) 1(-/-) compared with wild-type cells. We mapped the poly(ADP-ribosyl)ation domains and engineered MeCP2 mutation constructs to further analyze potential effects on DNA binding affinity and large-scale chromatin remodeling. Single or double deletion of the poly(ADP-ribosyl)ated regions and PARP inhibition increased the heterochromatin clustering ability of MeCP2. Increased chromatin clustering may reflect increased binding affinity. In agreement with this hypothesis, we found that PARP-1 deficiency significantly increased the chromatin binding affinity of MeCP2 in vivo. These data provide novel mechanistic insights into the regulation of MeCP2-mediated, higher-order chromatin architecture and suggest therapeutic opportunities to manipulate MeCP2 function.

  3. Molecular recognition of an ADP-ribosylating Clostridium botulinum C3 exoenzyme by RalA GTPase

    PubMed Central

    Holbourn, Kenneth P.; Sutton, J. Mark; Evans, Hazel R.; Shone, Clifford C.; Acharya, K. Ravi

    2005-01-01

    C3 exoenzymes (members of the ADP-ribosyltranferase family) are produced by Clostridium botulinum (C3bot1 and -2), Clostridium limosum (C3lim), Bacillus cereus (C3cer), and Staphylococcus aureus (C3stau1–3). These exoenzymes lack a translocation domain but are known to specifically inactivate Rho GTPases in host target cells. Here, we report the crystal structure of C3bot1 in complex with RalA (a GTPase of the Ras subfamily) and GDP at a resolution of 2.66 Å. RalA is not ADP-ribosylated by C3 exoenzymes but inhibits ADP-ribosylation of RhoA by C3bot1, C3lim, and C3cer to different extents. The structure provides an insight into the molecular interactions between C3bot1 and RalA involving the catalytic ADP-ribosylating turn–turn (ARTT) loop from C3bot1 and helix α4 and strand β6 (which are not part of the GDP-binding pocket) from RalA. The structure also suggests a molecular explanation for the different levels of C3-exoenzyme inhibition by RalA and why RhoA does not bind C3bot1 in this manner. PMID:15809419

  4. Modes of Action of ADP-Ribosylated Elongation Factor 2 in Inhibiting the Polypeptide Elongation Cycle: A Modeling Study

    PubMed Central

    Chen, Kevin C.; Xie, Honglin; Cai, Yujie

    2013-01-01

    Despite the fact that ADP-ribosylation of eukaryotic elongation factor 2 (EF2) leads to inhibition of protein synthesis, the mechanism by which ADP-ribosylated EF2 (ADPR•EF2) causes this inhibition remains controversial. Here, we applied modeling approaches to investigate the consequences of various modes of ADPR•EF2 inhibitory actions on the two coupled processes, the polypeptide chain elongation and ADP-ribosylation of EF2. Modeling of experimental data indicates that ADPR•EF2 fully blocks the late-phase translocation of tRNAs; but the impairment in the translocation upstream process, mainly the GTP-dependent factor binding with the pretranslocation ribosome and/or the guanine nucleotide exchange in EF2, is responsible for the overall inhibition kinetics. The reduced ADPR•EF2-ribosome association spares the ribosome to bind and shield native EF2 against toxin attack, thereby deferring the inhibition of protein synthesis inhibition and inactivation of EF2. Minimum association with the ribosome also keeps ADPR•EF2 in an accessible state for toxins to catalyze the reverse reaction when nicotinamide becomes available. Our work underscores the importance of unveiling the interactions between ADPR•EF2 and the ribosome, and argues against that toxins inhibit protein synthesis through converting native EF2 to a competitive inhibitor to actively disable the ribosome. PMID:23861744

  5. The Escherichia coli effector EspJ blocks Src kinase activity via amidation and ADP ribosylation

    PubMed Central

    Young, Joanna C.; Clements, Abigail; Lang, Alexander E.; Garnett, James A.; Munera, Diana; Arbeloa, Ana; Pearson, Jaclyn; Hartland, Elizabeth L.; Matthews, Stephen J.; Mousnier, Aurelie; Barry, David J.; Way, Michael; Schlosser, Andreas; Aktories, Klaus; Frankel, Gad

    2014-01-01

    The hallmark of enteropathogenic Escherichia coli (EPEC) infection is the formation of actin-rich pedestal-like structures, which are generated following phosphorylation of the bacterial effector Tir by cellular Src and Abl family tyrosine kinases. This leads to recruitment of the Nck–WIP–N-WASP complex that triggers Arp2/3-dependent actin polymerization in the host cell. The same phosphorylation-mediated signalling network is also assembled downstream of the Vaccinia virus protein A36 and the phagocytic Fc-gamma receptor FcγRIIa. Here we report that the EPEC type-III secretion system effector EspJ inhibits autophosphorylation of Src and phosphorylation of the Src substrates Tir and FcγRIIa. Consistent with this, EspJ inhibits actin polymerization downstream of EPEC, Vaccinia virus and opsonized red blood cells. We identify EspJ as a unique adenosine diphosphate (ADP) ribosyltransferase that directly inhibits Src kinase by simultaneous amidation and ADP ribosylation of the conserved kinase-domain residue, Src E310, resulting in glutamine-ADP ribose. PMID:25523213

  6. The Escherichia coli effector EspJ blocks Src kinase activity via amidation and ADP ribosylation.

    PubMed

    Young, Joanna C; Clements, Abigail; Lang, Alexander E; Garnett, James A; Munera, Diana; Arbeloa, Ana; Pearson, Jaclyn; Hartland, Elizabeth L; Matthews, Stephen J; Mousnier, Aurelie; Barry, David J; Way, Michael; Schlosser, Andreas; Aktories, Klaus; Frankel, Gad

    2014-01-01

    The hallmark of enteropathogenic Escherichia coli (EPEC) infection is the formation of actin-rich pedestal-like structures, which are generated following phosphorylation of the bacterial effector Tir by cellular Src and Abl family tyrosine kinases. This leads to recruitment of the Nck-WIP-N-WASP complex that triggers Arp2/3-dependent actin polymerization in the host cell. The same phosphorylation-mediated signalling network is also assembled downstream of the Vaccinia virus protein A36 and the phagocytic Fc-gamma receptor FcγRIIa. Here we report that the EPEC type-III secretion system effector EspJ inhibits autophosphorylation of Src and phosphorylation of the Src substrates Tir and FcγRIIa. Consistent with this, EspJ inhibits actin polymerization downstream of EPEC, Vaccinia virus and opsonized red blood cells. We identify EspJ as a unique adenosine diphosphate (ADP) ribosyltransferase that directly inhibits Src kinase by simultaneous amidation and ADP ribosylation of the conserved kinase-domain residue, Src E310, resulting in glutamine-ADP ribose.

  7. ADP-Ribosylation Factor 1 Regulates Proliferation, Migration, and Fusion in Early Stage of Osteoclast Differentiation

    PubMed Central

    Kim, Min Jae; Kim, Hyunsoo; Lee, Seoung Hoon; Gu, Dong Ryun; Lee, Soo Young; Lee, Kyunghee; Jeong, Daewon

    2015-01-01

    Small G-protein adenosine diphosphate (ADP)-ribosylation factors (ARFs) regulate a variety of cellular functions, including actin cytoskeleton remodeling, plasma membrane reorganization, and vesicular transport. Here, we propose the functional roles of ARF1 in multiple stages of osteoclast differentiation. ARF1 was upregulated during receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation and transiently activated in an initial stage of their differentiation. Differentiation of ARF1-deficient osteoclast precursors into mature osteoclasts temporarily increased in pre-maturation stage of osteoclasts followed by reduced formation of mature osteoclasts, indicating that ARF1 regulates the osteoclastogenic process. ARF1 deficiency resulted in reduced osteoclast precursor proliferation and migration as well as increasing cell-cell fusion. In addition, ARF1 silencing downregulated c-Jun N-terminal kinase (JNK), Akt, osteopontin, and macrophage colony-stimulating factor (M-CSF)-receptor c-Fms as well as upregulating several fusion-related genes including CD44, CD47, E-cadherin, and meltrin-α. Collectively, we showed that ARF1 stimulated proliferation and migration of osteoclast precursors while suppressing their fusion, suggesting that ARF1 may be a plausible inter-player that mediates the transition to osteoclast fusion at multiple steps during osteoclast differentiation PMID:26690137

  8. Poly-ADP-ribosylation of HMGB1 regulates TNFSF10/TRAIL resistance through autophagy.

    PubMed

    Yang, Minghua; Liu, Liying; Xie, Min; Sun, Xiaofang; Yu, Yan; Kang, Rui; Yang, Liangchun; Zhu, Shan; Cao, Lizhi; Tang, Daolin

    2015-01-01

    Both apoptosis ("self-killing") and autophagy ("self-eating") are evolutionarily conserved processes, and their crosstalk influences anticancer drug sensitivity and cell death. However, the underlying mechanism remains unclear. Here, we demonstrated that HMGB1 (high mobility group box 1), normally a nuclear protein, is a crucial regulator of TNFSF10/TRAIL (tumor necrosis factor [ligand] superfamily, member 10)-induced cancer cell death. Activation of PARP1 (poly [ADP-ribose] polymerase 1) was required for TNFSF10-induced ADP-ribosylation of HMGB1 in cancer cells. Moreover, pharmacological inhibition of PARP1 activity or knockdown of PARP1 gene expression significantly inhibited TNFSF10-induced HMGB1 cytoplasmic translocation and subsequent HMGB1-BECN1 complex formation. Furthermore, suppression of the PARP1-HMGB1 pathway diminished autophagy, increased apoptosis, and enhanced the anticancer activity of TNFSF10 in vitro and in a subcutaneous tumor model. These results indicate that PARP1 acts as a prominent upstream regulator of HMGB1-mediated autophagy and maintains a homeostatic balance between apoptosis and autophagy, which provides new insight into the mechanism of TNFSF10 resistance.

  9. MARTX effector cross kingdom activation by Golgi-associated ADP-ribosylation factors.

    PubMed

    Kim, Byoung Sik; Satchell, Karla J F

    2016-08-01

    Vibrio vulnificus infects humans and causes lethal septicemia. The primary virulence factor is a multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin consisting of conserved repeats-containing regions and various effector domains. Recent genomic analyses for the newly emerged V. vulnificus biotype 3 strain revealed that its MARTX toxin has two previously unknown effector domains. Herein, we characterized one of these domains, Domain X (DmXVv ). A structure-based homology search revealed that DmXVv belongs to the C58B cysteine peptidase subfamily. When ectopically expressed in cells, DmXVv was autoprocessed and induced cytopathicity including Golgi dispersion. When the catalytic cysteine or the region flanking the scissile bond was mutated, both autoprocessing and cytopathicity were significantly reduced indicating that DmXVv cytopathicity is activated by amino-terminal autoprocessing. Consistent with this, host cell protein export was affected by Vibrio cells producing a toxin with wild-type, but not catalytically inactive, DmXVv . DmXVv was found to localize to Golgi and to directly interact with Golgi-associated ADP-ribosylation factors ARF1, ARF3 and ARF4, although ARF binding was not necessary for the subcellular localization. Rather, this interaction was found to induce autoprocessing of DmXVv . These data demonstrate that the V. vulnificus hijacks the host ARF proteins to activate the cytopathic DmXVv effector domain of MARTX toxin. PMID:26780191

  10. ADP ribosylation factor like 2 (Arl2) protein influences microtubule dynamics in breast cancer cells

    SciTech Connect

    Beghin, Anne . E-mail: anne.beghin@recherche.univ-lyon1.fr; Honore, Stephane; Messana, Celine; Matera, Eva-Laure; Aim, Jennifer; Burlinchon, Sandrine; Braguer, Diane; Dumontet, Charles

    2007-02-01

    ADP ribosylation factor like 2 (Arl2) protein is involved in the folding of tubulin peptides. Variants of the human adenocarcinoma line MCF7 cells with increased or reduced content of Arl2 protein were produced and characterized. Western blot analysis performed after separation of the different fractions of tubulins showed that the content in polymerizable soluble heterodimers was significantly increased in cells with the highest Arl2 expression level (MA+) and reduced in cells with the lowest Arl2 expression level (MA-) in comparison to control cells (MP). Microtubule dynamic instability, measured after microinjection of rhodamine-labelled tubulin in living cells, was significantly enhanced in MA+ cells and reduced in MA- cells. These alterations involved modifications of the microtubule growth and shortening rates, duration of attenuation phases, percentage of time spent in each phase (growth, shortening and attenuation) and catastrophe frequency. We also observed modifications in the expression level of the tumor suppressor protein phosphatase 2Ac, which has been shown to form a complex with Arl2. Finally, cell cycle progression was modified in these cells, particularly in regard to duration of telophase. In summary, alterations in Arl2 protein content were found to be associated with modifications in tubulin pools, microtubule dynamics as well as cell cycle progression.

  11. Rifamycin Antibiotic Resistance by ADP-Ribosylation: Structure and Diversity of Arr

    SciTech Connect

    Baysarowich, J.; Koteva, K; Hughes, D; Ejim, L; Griffiths, E; Zhang, K; Junop, M; Wright, G

    2008-01-01

    The rifamycin antibiotic rifampin is important for the treatment of tuberculosis and infections caused by multidrug-resistant Staphylococcus aureus. Recent iterations of the rifampin core structure have resulted in new drugs and drug candidates for the treatment of a much broader range of infectious diseases. This expanded use of rifamycin antibiotics has the potential to select for increased resistance. One poorly characterized mechanism of resistance is through Arr enzymes that catalyze ADP-ribosylation of rifamycins. We find that genes encoding predicted Arr enzymes are widely distributed in the genomes of pathogenic and nonpathogenic bacteria. Biochemical analysis of three representative Arr enzymes from environmental and pathogenic bacterial sources shows that these have equally efficient drug resistance capacity in vitro and in vivo. The 3D structure of one of these orthologues from Mycobacterium smegmatis was determined and reveals structural homology with ADP-ribosyltransferases important in eukaryotic biology, including poly(ADP-ribose) polymerases (PARPs) and bacterial toxins, despite no significant amino acid sequence homology with these proteins. This work highlights the extent of the rifamycin resistome in microbial genera with the potential to negatively impact the expanded use of this class of antibiotic.

  12. ADP-Ribosylation Factor 1 Regulates Proliferation, Migration, and Fusion in Early Stage of Osteoclast Differentiation.

    PubMed

    Kim, Min Jae; Kim, Hyunsoo; Lee, Seoung Hoon; Gu, Dong Ryun; Lee, Soo Young; Lee, Kyunghee; Jeong, Daewon

    2015-12-09

    Small G-protein adenosine diphosphate (ADP)-ribosylation factors (ARFs) regulate a variety of cellular functions, including actin cytoskeleton remodeling, plasma membrane reorganization, and vesicular transport. Here, we propose the functional roles of ARF1 in multiple stages of osteoclast differentiation. ARF1 was upregulated during receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation and transiently activated in an initial stage of their differentiation. Differentiation of ARF1-deficient osteoclast precursors into mature osteoclasts temporarily increased in pre-maturation stage of osteoclasts followed by reduced formation of mature osteoclasts, indicating that ARF1 regulates the osteoclastogenic process. ARF1 deficiency resulted in reduced osteoclast precursor proliferation and migration as well as increasing cell-cell fusion. In addition, ARF1 silencing downregulated c-Jun N-terminal kinase (JNK), Akt, osteopontin, and macrophage colony-stimulating factor (M-CSF)-receptor c-Fms as well as upregulating several fusion-related genes including CD44, CD47, E-cadherin, and meltrin-α. Collectively, we showed that ARF1 stimulated proliferation and migration of osteoclast precursors while suppressing their fusion, suggesting that ARF1 may be a plausible inter-player that mediates the transition to osteoclast fusion at multiple steps during osteoclast differentiation.

  13. Small G proteins in peroxisome biogenesis: the potential involvement of ADP-ribosylation factor 6

    PubMed Central

    2009-01-01

    Background Peroxisomes execute diverse and vital functions in virtually every eukaryote. New peroxisomes form by budding from pre-existing organelles or de novo by vesiculation of the ER. It has been suggested that ADP-ribosylation factors and COPI coatomer complexes are involved in these processes. Results Here we show that all viable Saccharomyces cerevisiae strains deficient in one of the small GTPases which have an important role in the regulation of vesicular transport contain functional peroxisomes, and that the number of these organelles in oleate-grown cells is significantly upregulated in the arf1 and arf3 null strains compared to the wild-type strain. In addition, we provide evidence that a portion of endogenous Arf6, the mammalian orthologue of yeast Arf3, is associated with the cytoplasmic face of rat liver peroxisomes. Despite this, ablation of Arf6 did neither influence the regulation of peroxisome abundance nor affect the localization of peroxisomal proteins in cultured fetal hepatocytes. However, co-overexpression of wild-type, GTP hydrolysis-defective or (dominant-negative) GTP binding-defective forms of Arf1 and Arf6 caused mislocalization of newly-synthesized peroxisomal proteins and resulted in an alteration of peroxisome morphology. Conclusion These observations suggest that Arf6 is a key player in mammalian peroxisome biogenesis. In addition, they also lend strong support to and extend the concept that specific Arf isoform pairs may act in tandem to regulate exclusive trafficking pathways. PMID:19686593

  14. Differential interaction of ADP-ribosylation factors 1, 3, and 5 with rat brain Golgi membranes.

    PubMed

    Tsai, S C; Adamik, R; Haun, R S; Moss, J; Vaughan, M

    1992-10-01

    Six mammalian ADP-ribosylation factors (ARFs) identified by cDNA cloning were expressed as recombinant proteins (rARFs) that stimulated cholera toxin ADP-ribosyltransferase activity. Microsequencing of soluble ARFs I and II (sARFs I and II), purified from bovine brain, established that they are ARFs 1 and 3, respectively. Rabbit antibodies (IgG) against sARF II reacted similarly with ARFs 1, 2, and 3 (class I) on Western blots. ARFs 1 and 3 were distinguished by their electrophoretic mobilities. Antiserum against rARF 5 cross-reacted partially with rARF 4 but not detectably with rARF 6 and minimally with class I ARFs. Guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma S]) increased recovery of ARF activity and immunoreactivity in organelle fractions separated by density gradient centrifugation, after incubation of rat brain homogenate with ATP and a regenerating system. ARF 1 accumulated in microsomes plus Golgi and Golgi fractions, whereas ARF 5 seemed to localize more specifically in Golgi; the smaller increment in ARF 3 was distributed more evenly among fractions. On incubation of Golgi with a crude ARF fraction, GTP[gamma S], and an ATP-regenerating system, association of ARF activity with Golgi increased with increasing ATP concentration paralleled by increases in immunoreactive ARFs 1 and 5 and, to a lesser degree, ARF 3. Golgi incubated with GTP[gamma S] and purified ARF 1 or 3 bound more ARF 1 than ARF 3. Based on immunoreactivity and assay of ARF activity, individual ARFs 1, 3, and 5 appeared to behave independently and selectively in their GTP-dependent association with Golgi in vitro.

  15. Differential interaction of ADP-ribosylation factors 1, 3, and 5 with rat brain Golgi membranes.

    PubMed Central

    Tsai, S C; Adamik, R; Haun, R S; Moss, J; Vaughan, M

    1992-01-01

    Six mammalian ADP-ribosylation factors (ARFs) identified by cDNA cloning were expressed as recombinant proteins (rARFs) that stimulated cholera toxin ADP-ribosyltransferase activity. Microsequencing of soluble ARFs I and II (sARFs I and II), purified from bovine brain, established that they are ARFs 1 and 3, respectively. Rabbit antibodies (IgG) against sARF II reacted similarly with ARFs 1, 2, and 3 (class I) on Western blots. ARFs 1 and 3 were distinguished by their electrophoretic mobilities. Antiserum against rARF 5 cross-reacted partially with rARF 4 but not detectably with rARF 6 and minimally with class I ARFs. Guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma S]) increased recovery of ARF activity and immunoreactivity in organelle fractions separated by density gradient centrifugation, after incubation of rat brain homogenate with ATP and a regenerating system. ARF 1 accumulated in microsomes plus Golgi and Golgi fractions, whereas ARF 5 seemed to localize more specifically in Golgi; the smaller increment in ARF 3 was distributed more evenly among fractions. On incubation of Golgi with a crude ARF fraction, GTP[gamma S], and an ATP-regenerating system, association of ARF activity with Golgi increased with increasing ATP concentration paralleled by increases in immunoreactive ARFs 1 and 5 and, to a lesser degree, ARF 3. Golgi incubated with GTP[gamma S] and purified ARF 1 or 3 bound more ARF 1 than ARF 3. Based on immunoreactivity and assay of ARF activity, individual ARFs 1, 3, and 5 appeared to behave independently and selectively in their GTP-dependent association with Golgi in vitro. Images PMID:1409634

  16. Poly(ADP-ribosyl)ation of Methyl CpG Binding Domain Protein 2 Regulates Chromatin Structure*

    PubMed Central

    Becker, Annette; Zhang, Peng; Allmann, Lena; Meilinger, Daniela; Bertulat, Bianca; Eck, Daniel; Hofstaetter, Maria; Bartolomei, Giody; Hottiger, Michael O.; Schreiber, Valérie; Leonhardt, Heinrich; Cardoso, M. Cristina

    2016-01-01

    The epigenetic information encoded in the genomic DNA methylation pattern is translated by methylcytosine binding proteins like MeCP2 into chromatin topology and structure and gene activity states. We have shown previously that the MeCP2 level increases during differentiation and that it causes large-scale chromatin reorganization, which is disturbed by MeCP2 Rett syndrome mutations. Phosphorylation and other posttranslational modifications of MeCP2 have been described recently to modulate its function. Here we show poly(ADP-ribosyl)ation of endogenous MeCP2 in mouse brain tissue. Consequently, we found that MeCP2 induced aggregation of pericentric heterochromatin and that its chromatin accumulation was enhanced in poly(ADP-ribose) polymerase (PARP) 1−/− compared with wild-type cells. We mapped the poly(ADP-ribosyl)ation domains and engineered MeCP2 mutation constructs to further analyze potential effects on DNA binding affinity and large-scale chromatin remodeling. Single or double deletion of the poly(ADP-ribosyl)ated regions and PARP inhibition increased the heterochromatin clustering ability of MeCP2. Increased chromatin clustering may reflect increased binding affinity. In agreement with this hypothesis, we found that PARP-1 deficiency significantly increased the chromatin binding affinity of MeCP2 in vivo. These data provide novel mechanistic insights into the regulation of MeCP2-mediated, higher-order chromatin architecture and suggest therapeutic opportunities to manipulate MeCP2 function. PMID:26772194

  17. The ARTT motif and a unified structural understanding of substraterecognition in ADP ribosylating bacterial toxins and eukaryotic ADPribosyltransferases

    SciTech Connect

    Han, S.; Tainer, J.A.

    2001-08-01

    ADP-ribosylation is a widely occurring and biologically critical covalent chemical modification process in pathogenic mechanisms, intracellular signaling systems, DNA repair, and cell division. The reaction is catalyzed by ADP-ribosyltransferases, which transfer the ADP-ribose moiety of NAD to a target protein with nicotinamide release. A family of bacterial toxins and eukaryotic enzymes has been termed the mono-ADP-ribosyltransferases, in distinction to the poly-ADP-ribosyltransferases, which catalyze the addition of multiple ADP-ribose groups to the carboxyl terminus of eukaryotic nucleoproteins. Despite the limited primary sequence homology among the different ADP-ribosyltransferases, a central cleft bearing NAD-binding pocket formed by the two perpendicular b-sheet core has been remarkably conserved between bacterial toxins and eukaryotic mono- and poly-ADP-ribosyltransferases. The majority of bacterial toxins and eukaryotic mono-ADP-ribosyltransferases are characterized by conserved His and catalytic Glu residues. In contrast, Diphtheria toxin, Pseudomonas exotoxin A, and eukaryotic poly-ADP-ribosyltransferases are characterized by conserved Arg and catalytic Glu residues. The NAD-binding core of a binary toxin and a C3-like toxin family identified an ARTT motif (ADP-ribosylating turn-turn motif) that is implicated in substrate specificity and recognition by structural and mutagenic studies. Here we apply structure-based sequence alignment and comparative structural analyses of all known structures of ADP-ribosyltransfeases to suggest that this ARTT motif is functionally important in many ADP-ribosylating enzymes that bear a NAD binding cleft as characterized by conserved Arg and catalytic Glu residues. Overall, structure-based sequence analysis reveals common core structures and conserved active sites of ADP-ribosyltransferases to support similar NAD binding mechanisms but differing mechanisms of target protein binding via sequence variations within the ARTT

  18. Arsenite induced poly(ADP-ribosyl)ation of tumor suppressor P53 in human skin keratinocytes as a possible mechanism for carcinogenesis associated with arsenic exposure.

    PubMed

    Komissarova, Elena V; Rossman, Toby G

    2010-03-15

    Arsenite is an environmental pollutant. Exposure to inorganic arsenic in drinking water is associated with elevated cancer risk, especially in skin. Arsenite alone does not cause skin cancer in animals, but arsenite can enhance the carcinogenicity of solar UV. Arsenite is not a significant mutagen at non-toxic concentrations, but it enhances the mutagenicity of other carcinogens. The tumor suppressor protein P53 and nuclear enzyme PARP-1 are both key players in DNA damage response. This laboratory demonstrated earlier that in cells treated with arsenite, the P53-dependent increase in p21(WAF1/CIP1) expression, normally a block to cell cycle progression after DNA damage, is deficient. Here we show that although long-term exposure of human keratinocytes (HaCaT) to a nontoxic concentration (0.1 microM) of arsenite decreases the level of global protein poly(ADP-ribosyl)ation, it increases poly(ADP-ribosyl)ation of P53 protein and PARP-1 protein abundance. We also demonstrate that exposure to 0.1 microM arsenite depresses the constitutive expression of p21 mRNA and P21 protein in HaCaT cells. Poly(ADP-ribosyl)ation of P53 is reported to block its activation, DNA binding and its functioning as a transcription factor. Our results suggest that arsenite's interference with activation of P53 via poly(ADP-ribosyl)ation may play a role in the comutagenic and cocarcinogenic effects of arsenite.

  19. Arsenite induced poly(ADP-ribosyl)ation of tumor suppressor P53 in human skin keratinocytes as a possible mechanism for carcinogenesis associated with arsenic exposure

    SciTech Connect

    Komissarova, Elena V.; Rossman, Toby G.

    2010-03-15

    Arsenite is an environmental pollutant. Exposure to inorganic arsenic in drinking water is associated with elevated cancer risk, especially in skin. Arsenite alone does not cause skin cancer in animals, but arsenite can enhance the carcinogenicity of solar UV. Arsenite is not a significant mutagen at non-toxic concentrations, but it enhances the mutagenicity of other carcinogens. The tumor suppressor protein P53 and nuclear enzyme PARP-1 are both key players in DNA damage response. This laboratory demonstrated earlier that in cells treated with arsenite, the P53-dependent increase in p21{sup WAF1/CIP1} expression, normally a block to cell cycle progression after DNA damage, is deficient. Here we show that although long-term exposure of human keratinocytes (HaCaT) to a nontoxic concentration (0.1 muM) of arsenite decreases the level of global protein poly(ADP-ribosyl)ation, it increases poly(ADP-ribosyl)ation of P53 protein and PARP-1 protein abundance. We also demonstrate that exposure to 0.1 muM arsenite depresses the constitutive expression of p21 mRNA and P21 protein in HaCaT cells. Poly(ADP-ribosyl)ation of P53 is reported to block its activation, DNA binding and its functioning as a transcription factor. Our results suggest that arsenite's interference with activation of P53 via poly(ADP-ribosyl)ation may play a role in the comutagenic and cocarcinogenic effects of arsenite.

  20. ADP-ribosylation of dinitrogenase reductase in Azospirillum brasilense is regulated by AmtB-dependent membrane sequestration of DraG.

    PubMed

    Huergo, Luciano F; Souza, Emanuel M; Araujo, Mariana S; Pedrosa, Fábio O; Chubatsu, Leda S; Steffens, Maria B R; Merrick, Mike

    2006-01-01

    Nitrogen fixation in some diazotrophic bacteria is regulated by mono-ADP-ribosylation of dinitrogenase reductase (NifH) that occurs in response to addition of ammonium to the extracellular medium. This process is mediated by dinitrogenase reductase ADP-ribosyltransferase (DraT) and reversed by dinitrogenase reductase glycohydrolase (DraG), but the means by which the activities of these enzymes are regulated are unknown. We have investigated the role of the P(II) proteins (GlnB and GlnZ), the ammonia channel protein AmtB and the cellular localization of DraG in the regulation of the NifH-modification process in Azospirillum brasilense. GlnB, GlnZ and DraG were all membrane-associated after an ammonium shock, and both this membrane sequestration and ADP-ribosylation of NifH were defective in an amtB mutant. We now propose a model in which membrane association of DraG after an ammonium shock creates a physical separation from its cytoplasmic substrate NifH thereby inhibiting ADP-ribosyl-removal. Our observations identify a novel role for an ammonia channel (Amt) protein in the regulation of bacterial nitrogen metabolism by mediating membrane sequestration of a protein other than a P(II) family member. They also suggest a model for control of ADP-ribosylation that is likely to be applicable to all diazotrophs that exhibit such post-translational regulation of nitrogenase.

  1. Structure of an ADP-ribosylation factor, ARF1, from Entamoeba histolytica bound to Mg(2+)-GDP.

    PubMed

    Serbzhinskiy, Dmitry A; Clifton, Matthew C; Sankaran, Banumathi; Staker, Bart L; Edwards, Thomas E; Myler, Peter J

    2015-05-01

    Entamoeba histolytica is the etiological agent of amebiasis, a diarrheal disease which causes amoebic liver abscesses and amoebic colitis. Approximately 50 million people are infected worldwide with E. histolytica. With only 10% of infected people developing symptomatic amebiasis, there are still an estimated 100,000 deaths each year. Because of the emergence of resistant strains of the parasite, it is necessary to find a treatment which would be a proper response to this challenge. ADP-ribosylation factor (ARF) is a member of the ARF family of GTP-binding proteins. These proteins are ubiquitous in eukaryotic cells; they generally associate with cell membranes and regulate vesicular traffic and intracellular signalling. The crystal structure of ARF1 from E. histolytica has been determined bound to magnesium and GDP at 1.8 Å resolution. Comparison with other structures of eukaryotic ARF proteins shows a highly conserved structure and supports the interswitch toggle mechanism of communicating the conformational state to partner proteins.

  2. Characterization of transducin from bovine retinal rod outer segments: mechanism and effects of cholera toxin-catalyzed adp-ribosylation

    SciTech Connect

    Navon, S.E.; Fung, B.K.K.

    1984-05-25

    Transducin, a guanine nucleotide-binding protein consisting of two subunits (T/sub ..cap alpha../ and T/sub ..beta gamma../), mediates the signal coupling between rhodopsin and a membrane-bound cyclic GMP phosphodiesterase in retinal rod outer segments. The T/sub ..cap alpha../ subunit is an activator of the phosphodiesterase, and the function of the T/sub ..beta gamma../ subunit is to physically link T/sub ..cap alpha../ with photolyzed rhodopsin. In this study, the mechanism of cholera toxin-catalyzed ADP-ribosylation of T/sub ..cap alpha../ has been examined in a reconstituted system consisting of purified transducin and stripped rod outer segment membranes. Limited proteolysis of the labeled T/sub ..cap alpha../ with trypsin indicated that the inserted ADP-ribose is located exclusively on a single proteolytic fragment with an apparent molecular weight of 23,000. Maximal incorporation of ADP-ribose was achieved when guanosine 5'-(..beta..,..gamma..-im ido)triphosphate (Gpp(NH)p) and T/sub ..beta gamma../ were present at concentrations equal to that of T/sub ..cap alpha../ and when rhodopsin was continuously irradiated with visible light in the 400-500 nm region. The stimulating effect of illumination was related to the direct interaction of the retinal chromophore with opsin. These findings strongly suggest that a transient protein complex consisting of T/sub ..cap alpha../xGpp(NH)p, T/sub ..beta gamma../, and a photointermediate of rhodopsin is the required substrate for cholera toxin. Single turnover kinetic measurements demonstrated that the ADP-ribosylation of T/sub ..cap alpha../ coincided with the appearance of a population of transducin molecules having a very slow rate of GTP hydrolysis. The hydrolysis rate of the bound GTP for this population was 1.1 x 10/sup -3//s, which was 22-fold slower than the rate for the unmodified transducin. 30 references, 9 figures, 1 table.

  3. Pertussis toxin-catalyzed ADP-ribosylation of a G protein in mouse oocytes, eggs, and preimplantation embryos: Developmental changes and possible functional roles

    SciTech Connect

    Jones, J.; Schultz, R.M. )

    1990-06-01

    G proteins, which in many somatic cells serve as mediators of signal transduction, were identified in preimplantation mouse embryos by their capacity to undergo pertussis toxin-catalyzed ADP-ribosylation. Two pertussis toxin (PT) substrates with Mr = 38,000 and 39,000 (alpha 38 and alpha 39) are present in approximately equal amounts. Relative to the amount in freshly isolated germinal vesicle (GV)-intact oocytes, the amount of PT-catalyzed ADP-ribosylation of alpha 38-39 falls during oocyte maturation, rises between the one- and two-cell stages, falls by the eight-cell and morula stages, and increases again by the blastocyst stage. The decrease in PT-catalyzed ADP-ribosylation of alpha 38-39 that occurs during oocyte maturation, however, does not require germinal vesicle breakdown (GVBD), since inhibiting GVBD with 3-isobutyl-1-methyl xanthine (IBMX) does not prevent the decrease in the extent of PT-catalyzed ADP-ribosylation. A biologically active phorbol diester (12-O-tetradecanoyl phorbol 13-acetate), but not an inactive one (4 alpha-phorbol 12,13-didecanoate, 4 alpha-PDD), totally inhibits the increase in PT-catalyzed ADP-ribosylation of alpha 38-39 that occurs between the one- and two-cell stage; TPA inhibits cleavage, but not transcriptional activation, which occurs in the two-cell embryo. In contrast, cytochalasin D, genistein, or aphidicolin, each of which inhibits cleavage of one-cell embryos, or alpha-amanitin or H8, each of which inhibits transcriptional activation but not cleavage of one-cell embryos, have little or inhibitory effects on the increase in PT-catalyzed ADP-ribosylation of alpha 38-39. Results of immunoblotting experiments using an antibody that is highly specific for alpha il-3 reveal the presence of a cross-reactive species of Mr = 38,000 (alpha 38) in the GV-intact oocyte, metaphase II-arrested egg, and one-, two-cell embryos.

  4. Differential expression during development of ADP-ribosylation factors, 20-kDa guanine nucleotide-binding protein activators of cholera toxin.

    PubMed

    Tsai, S C; Adamik, R; Tsuchiya, M; Chang, P P; Moss, J; Vaughan, M

    1991-05-01

    Cholera toxin exerts its effects on cells in large part through the ADP-ribosylation of guanine nucleotide-binding proteins. Toxin-catalyzed ADP-ribosylation is enhanced by approximately 20-kDa guanine nucleotide-binding proteins termed ADP-ribosylation factors (ARFs), which are allosteric activators of the toxin catalytic unit. Rabbit antiserum against a purified bovine brain ARF (sARF II) reacted on immunoblots with two approximately 20-kDa ARF-like proteins (sARF I and II) in tissue extracts from bovine, rat, frog, and chicken. Levels of ARF were higher in brain than in non-neural tissues. In rat brain, on the second postnatal day, amounts of sARF I and II were similar. By the 10th postnatal day and thereafter, sARF II predominated. Relative levels of ARF determined by immunoreactivity were in agreement with levels assessed in functional assays of cholera toxin-catalyzed ADP-ribosylation. Based on nucleotide and deduced amino acid sequences of human and bovine cDNAs, there appear to be at least six different ARF-like genes. Northern blots of rat brain poly(A)+ RNA were hybridized with cDNA and oligonucleotide probes specific for each of the human and bovine ARF genes. From the second to the 27th postnatal day, ARF 3 mRNA increased, whereas mRNAs for ARFs 2 and 4 decreased; and those for ARFs 1, 5, and 6 were apparently unchanged. Partial amino acid sequence of sARF II is consistent with it being either the ARF 1 or 3 gene product. The developmental changes in rat brain ARF parallel neuronal maturation and synapse formation.

  5. ARTC1-mediated ADP-ribosylation of GRP78/BiP: a new player in endoplasmic-reticulum stress responses.

    PubMed

    Fabrizio, Gaia; Di Paola, Simone; Stilla, Annalisa; Giannotta, Monica; Ruggiero, Carmen; Menzel, Stephan; Koch-Nolte, Friedrich; Sallese, Michele; Di Girolamo, Maria

    2015-03-01

    Protein mono-ADP-ribosylation is a reversible post-translational modification of cellular proteins. This scheme of amino-acid modification is used not only by bacterial toxins to attack host cells, but also by endogenous ADP-ribosyltransferases (ARTs) in mammalian cells. These latter ARTs include members of three different families of proteins: the well characterised arginine-specific ecto-enzymes (ARTCs), two sirtuins, and some members of the poly(ADP-ribose) polymerase (PARP/ARTD) family. In the present study, we demonstrate that human ARTC1 is localised to the endoplasmic reticulum (ER), in contrast to the previously characterised ARTC proteins, which are typical GPI-anchored ecto-enzymes. Moreover, using the "macro domain" cognitive binding module to identify ADP-ribosylated proteins, we show here that the ER luminal chaperone GRP78/BiP (glucose-regulated protein of 78 kDa/immunoglobulin heavy-chain-binding protein) is a cellular target of human ARTC1 and hamster ARTC2. We further developed a procedure to visualise ADP-ribosylated proteins using immunofluorescence. With this approach, in cells overexpressing ARTC1, we detected staining of the ER that co-localises with GRP78/BiP, thus confirming that this modification occurs in living cells. In line with the key role of GRP78/BiP in the ER stress response system, we provide evidence here that ARTC1 is activated during the ER stress response, which results in acute ADP-ribosylation of GRP78/BiP paralleling translational inhibition. Thus, this identification of ARTC1 as a regulator of GRP78/BiP defines a novel, previously unsuspected, player in GRP78-mediated ER stress responses.

  6. The NarE protein of Neisseria gonorrhoeae catalyzes ADP-ribosylation of several ADP-ribose acceptors despite an N-terminal deletion.

    PubMed

    Rodas, Paula I; Álamos-Musre, A Said; Álvarez, Francisca P; Escobar, Alejandro; Tapia, Cecilia V; Osorio, Eduardo; Otero, Carolina; Calderón, Iván L; Fuentes, Juan A; Gil, Fernando; Paredes-Sabja, Daniel; Christodoulides, Myron

    2016-09-01

    The ADP-ribosylating enzymes are encoded in many pathogenic bacteria in order to affect essential functions of the host. In this study, we show that Neisseria gonorrhoeae possess a locus that corresponds to the ADP-ribosyltransferase NarE, a previously characterized enzyme in N. meningitidis The 291 bp coding sequence of gonococcal narE shares 100% identity with part of the coding sequence of the meningococcal narE gene due to a frameshift previously described, thus leading to a 49-amino-acid deletion at the N-terminus of gonococcal NarE protein. However, we found a promoter region and a GTG start codon, which allowed expression of the protein as demonstrated by RT-PCR and western blot analyses. Using a gonococcal NarE-6xHis fusion protein, we demonstrated that the gonococcal enzyme underwent auto-ADP-ribosylation but to a lower extent than meningococcal NarE. We also observed that gonoccocal NarE exhibited ADP-ribosyltransferase activity using agmatine and cell-free host proteins as ADP-ribose acceptors, but its activity was inhibited by human β-defensins. Taken together, our results showed that NarE of Neisseria gonorrhoeae is a functional enzyme that possesses key features of bacterial ADP-ribosylating enzymes.

  7. The NarE protein of Neisseria gonorrhoeae catalyzes ADP-ribosylation of several ADP-ribose acceptors despite an N-terminal deletion.

    PubMed

    Rodas, Paula I; Álamos-Musre, A Said; Álvarez, Francisca P; Escobar, Alejandro; Tapia, Cecilia V; Osorio, Eduardo; Otero, Carolina; Calderón, Iván L; Fuentes, Juan A; Gil, Fernando; Paredes-Sabja, Daniel; Christodoulides, Myron

    2016-09-01

    The ADP-ribosylating enzymes are encoded in many pathogenic bacteria in order to affect essential functions of the host. In this study, we show that Neisseria gonorrhoeae possess a locus that corresponds to the ADP-ribosyltransferase NarE, a previously characterized enzyme in N. meningitidis The 291 bp coding sequence of gonococcal narE shares 100% identity with part of the coding sequence of the meningococcal narE gene due to a frameshift previously described, thus leading to a 49-amino-acid deletion at the N-terminus of gonococcal NarE protein. However, we found a promoter region and a GTG start codon, which allowed expression of the protein as demonstrated by RT-PCR and western blot analyses. Using a gonococcal NarE-6xHis fusion protein, we demonstrated that the gonococcal enzyme underwent auto-ADP-ribosylation but to a lower extent than meningococcal NarE. We also observed that gonoccocal NarE exhibited ADP-ribosyltransferase activity using agmatine and cell-free host proteins as ADP-ribose acceptors, but its activity was inhibited by human β-defensins. Taken together, our results showed that NarE of Neisseria gonorrhoeae is a functional enzyme that possesses key features of bacterial ADP-ribosylating enzymes. PMID:27465490

  8. Molecular cloning, characterization, and expression of human ADP-ribosylation factors: Two guanine nucleotide-dependent activators of cholera toxin

    SciTech Connect

    Bobak, D.A.; Nightingale, M.S.; Murtagh, J.J.; Price, S.R.; Moss, J.; Vaughan, M. )

    1989-08-01

    ADP-ribosylation factors (ARFs) are small guanine nucleotide-binding proteins that enhance the enzymatic activities of cholera toxin. Two ARF cDNAs, ARF1 and ARF3, were cloned from a human cerebellum library. Based on deduced amino acid sequences and patterns of hybridization of cDNA and oligonucleotide probes with mammalian brain poly(A){sup +} RNA, human ARF1 is the homologue of bovine ARF1. Human ARF3, which differs from bovine ARF1 and bovine ARF2, appears to represent a newly identified third type of ARF. Hybridization patterns of human ARF cDNA and clone-specific oligonucleotides with poly(A){sup +} RNA are consistent with the presence of at least two, and perhaps four, separate ARF messages in human brain. In vitro translation of ARF1, ARF2, and ARF3 produced proteins that behaved, by SDS/PAGE, similar to a purified soluble brain ARF. Deduced amino acid sequences of human ARF1 and ARF3 contain regions, similar to those in other G proteins, that are believed to be involved in GTP binding and hydrolysis. ARFS also exhibit a modest degree of homology with a bovine phospholipase C. The observations reported here support the conclusion that the ARFs are members of a multigene family of small guanine nucleotide-binding proteins. Definition of the regulation of ARF mRNAs and of function(s) of recombinant ARF proteins will aid in the elucidation of the physiologic role(s) of ARFs.

  9. Identification of a brefeldin A-insensitive guanine nucleotide-exchange protein for ADP-ribosylation factor in bovine brain.

    PubMed Central

    Tsai, S C; Adamik, R; Moss, J; Vaughan, M

    1994-01-01

    ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that participate in vesicular transport in the Golgi and other intracellular compartments and stimulate cholera toxin ADP-ribosyltransferase activity. ARFs are active in the GTP-bound form; hydrolysis of bound GTP to GDP, possibly with the assistance of a GTP hydrolysis (GTPase)-activating protein results in inactivation. Exchange of GDP for GTP and reactivation were shown by other workers to be enhanced by Golgi membranes in a brefeldin A-sensitive reaction, leading to the proposal that the guanine nucleotide-exchange protein (GEP) was a target of brefeldin A. In the studies reported here, a soluble GEP was partially purified from bovine brain. Exchange of nucleotide on ARFs 1 and 3, based on increased ARF activity in a toxin assay and stimulation of binding of guanosine 5'-[gamma-[35S]thio]triphosphate, was dependent on phospholipids, with phosphatidylserine being more effective than cardiolipin. GEP appeared to increase the rate of nucleotide exchange but did not affect the affinity of ARF for GTP. Whereas the crude GEP had a size of approximately 700 kDa, the partially purified GEP behaved on Ultrogel AcA 54 as a protein of 60 kDa. With purification, the GEP activity became insensitive to brefeldin A, consistent with the conclusion that, in contrast to earlier inferences, the exchange protein is not itself the target of brefeldin A. PMID:8159707

  10. PARP-2 regulates cell cycle-related genes through histone deacetylation and methylation independently of poly(ADP-ribosyl)ation

    SciTech Connect

    Liang, Ya-Chen; Hsu, Chiao-Yu; Yao, Ya-Li; Yang, Wen-Ming

    2013-02-01

    Highlights: ► PARP-2 acts as a transcription co-repressor independently of PARylation activity. ► PARP-2 recruits HDAC5, 7, and G9a and generates repressive chromatin. ► PARP-2 is recruited to the c-MYC promoter by DNA-binding factor YY1. ► PARP-2 represses cell cycle-related genes and alters cell cycle progression. -- Abstract: Poly(ADP-ribose) polymerase-2 (PARP-2) catalyzes poly(ADP-ribosyl)ation (PARylation) and regulates numerous nuclear processes, including transcription. Depletion of PARP-2 alters the activity of transcription factors and global gene expression. However, the molecular action of how PARP-2 controls the transcription of target promoters remains unclear. Here we report that PARP-2 possesses transcriptional repression activity independently of its enzymatic activity. PARP-2 interacts and recruits histone deacetylases HDAC5 and HDAC7, and histone methyltransferase G9a to the promoters of cell cycle-related genes, generating repressive chromatin signatures. Our findings propose a novel mechanism of PARP-2 in transcriptional regulation involving specific protein–protein interactions and highlight the importance of PARP-2 in the regulation of cell cycle progression.

  11. Total synthesis of AMF-26, an antitumor agent for inhibition of the Golgi system, targeting ADP-ribosylation factor 1.

    PubMed

    Shiina, Isamu; Umezaki, Yuma; Ohashi, Yoshimi; Yamazaki, Yuta; Dan, Shingo; Yamori, Takao

    2013-01-10

    An effective method for the total synthesis of 1 (AMF-26), a potentially promising new anticancer drug that disrupts the Golgi system by inhibiting the ADP-ribosylation factor 1 (Arf1) activation, has been developed for the first time. The construction of the chiral linear precursor (a key to the synthesis) was achieved by the asymmetric aldol reaction followed by the computer-assisted predictive stereoselective intramolecular Diels-Alder reaction. The global antitumor activity of the totally synthetic 1 against a variety of human cancer cells was assessed using a panel of 39 human cancer cell lines (JFCR39), and it was shown that the synthetic 1 strongly inhibited the growth of several cancer cell lines at concentrations of less than 0.04 μM. Biological assays of novel derivatives, 26 and 31, which have different side-chains at the C-4 positions in the Δ¹,²-octalin backbone, disclosed the importance of the suitable structure of the side-chain containing conjugated multidouble bonds.

  12. Structure, organization and evolution of ADP-ribosylation factors in rice and foxtail millet, and their expression in rice

    PubMed Central

    Muthamilarasan, Mehanathan; Mangu, Venkata R.; Zandkarimi, Hana; Prasad, Manoj; Baisakh, Niranjan

    2016-01-01

    ADP-ribosylation factors (ARFs) have been reported to function in diverse physiological and molecular activities. Recent evidences also demonstrate the involvement of ARFs in conferring tolerance to biotic and abiotic stresses in plant species. In the present study, 23 and 25 ARF proteins were identified in C3 model- rice and C4 model- foxtail millet, respectively. These proteins are classified into four classes (I–IV) based on phylogenetic analysis, with ARFs in classes I–III and ARF-like proteins (ARLs) in class IV. Sequence alignment and domain analysis revealed the presence of conserved and additional motifs, which may contribute to neo- and sub-functionalization of these proteins. Promoter analysis showed the presence of several cis-regulatory elements related to stress and hormone response, indicating their role in stress regulatory network. Expression analysis of rice ARFs and ARLs in different tissues, stresses and abscisic acid treatment highlighted temporal and spatial diversification of gene expression. Five rice cultivars screened for allelic variations in OsARF genes showed the presence of allelic polymorphisms in few gene loci. Altogether, the study provides insights on characteristics of ARF/ARL genes in rice and foxtail millet, which could be deployed for further functional analysis to extrapolate their precise roles in abiotic stress responses. PMID:27097755

  13. ADP-ribosylation Factor-related Protein 1 Interacts with NS5A and Regulates Hepatitis C Virus Propagation.

    PubMed

    Lim, Yun-Sook; Ngo, Huong T T; Lee, Jihye; Son, Kidong; Park, Eun-Mee; Hwang, Soon B

    2016-01-01

    The life cycle of hepatitis C virus (HCV) is tightly coupled to the lipid metabolism of host cells. In order to identify host factors involved in HCV propagation, we have previously screened a small interfering RNA (siRNA) library targeting host genes that control lipid metabolism and lipid droplet (LD) formation using cell culture-grown HCV (HCVcc)-infected cells. In this study, we selected and characterized the gene encoding ADP-ribosylation factor-related protein 1 (ARFRP1). ARFRP1 is essential for LD growth and is involved in the regulation of lipolysis. siRNA-mediated knockdown of ARFRP1 significantly inhibited HCV replication in both subgenomic replicon cells and HCVcc-infected cells. ARFRP1 interacted with NS5A and NS5A partially colocalized with LD. Silencing of ARFRP1 abrogated HCV-induced LD growth and viral protein expressions. Moreover, ARFRP1 recruited synaptosomal-associated protein 23 (SNAP23) to sites in close proximity to LDs in HCV-infected cells. Silencing of ARFRP1 ablated relocalization of SNAP23 to LD. These data indicate that HCV regulates ARFRP1 for LD growth to facilitate viral propagation and thus ARFRP1 may be a potential target for antiviral therapy. PMID:27550144

  14. ADP-ribosylation Factor-related Protein 1 Interacts with NS5A and Regulates Hepatitis C Virus Propagation

    PubMed Central

    Lim, Yun-Sook; Ngo, Huong T. T.; Lee, Jihye; Son, Kidong; Park, Eun-Mee; Hwang, Soon B.

    2016-01-01

    The life cycle of hepatitis C virus (HCV) is tightly coupled to the lipid metabolism of host cells. In order to identify host factors involved in HCV propagation, we have previously screened a small interfering RNA (siRNA) library targeting host genes that control lipid metabolism and lipid droplet (LD) formation using cell culture-grown HCV (HCVcc)-infected cells. In this study, we selected and characterized the gene encoding ADP-ribosylation factor-related protein 1 (ARFRP1). ARFRP1 is essential for LD growth and is involved in the regulation of lipolysis. siRNA-mediated knockdown of ARFRP1 significantly inhibited HCV replication in both subgenomic replicon cells and HCVcc-infected cells. ARFRP1 interacted with NS5A and NS5A partially colocalized with LD. Silencing of ARFRP1 abrogated HCV-induced LD growth and viral protein expressions. Moreover, ARFRP1 recruited synaptosomal-associated protein 23 (SNAP23) to sites in close proximity to LDs in HCV-infected cells. Silencing of ARFRP1 ablated relocalization of SNAP23 to LD. These data indicate that HCV regulates ARFRP1 for LD growth to facilitate viral propagation and thus ARFRP1 may be a potential target for antiviral therapy. PMID:27550144

  15. Role of CTCF poly(ADP-Ribosyl)ation in the regulation of apoptosis in breast cancer cells

    PubMed Central

    Venkatraman, Bhooma; Klenova, Elena

    2015-01-01

    Introduction: CTCF is a candidate tumor suppressor gene encoding a multifunctional transcription factor. CTCF function is controlled by posttranslational modification and interaction with other proteins. Research findings suggested that CTCF function can be regulated by poly(ADP-ribosyl)ation (PARlation) and has specific anti-apoptotic function in breast cancer cells. The aim of this study is to assess the effect of CTCF-wild type (WT) and CTCF complete mutant, which is deficient of PARlation in regulating apoptosis in breast cancer cells. Materials and Methods: The effect of CTCF-WT and CTCF complete mutant was expressed in breast cancer cell-lines by DNA-mediated transfection technique monitored by enhanced green fluorescent protein fluorescence. Evaluation of apoptotic cell death was carried out with immunohistochemical staining using 4’-6’-diamino-2 phenylindole and scoring by fluorescent microscopy. Results: CTCF-WT supports survival of breast cancer cells and was observed that CTCF complete mutant interferes with the functions of the CTCF-WT and there was a considerable apoptotic cell death in the breast cancer cell lines such as MDA-MB-435, CAMA-1 and MCF-7. Conclusion: The study enlighten CTCF as a Biological Marker for breast cancer and the role of CTCF PARlation may be involved in breast carcinogenesis. PMID:25810575

  16. Poly-ADP-ribosylation-mediated degradation of ARTD1 by the NLRP3 inflammasome is a prerequisite for osteoclast maturation

    PubMed Central

    Wang, C; Qu, C; Alippe, Y; Bonar, S L; Civitelli, R; Abu-Amer, Y; Hottiger, M O; Mbalaviele, G

    2016-01-01

    Evidence implicates ARTD1 in cell differentiation, but its role in skeletal metabolism remains unknown. Osteoclasts (OC), the bone-resorbing cells, differentiate from macrophages under the influence of macrophage colony-stimulating factor (M-CSF) and receptor-activator of NF-κB ligand (RANKL). We found that M-CSF induced ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1) auto-ADP-ribosylation in macrophages, a modification that marked ARTD1 for cleavage, and subsequently, for degradation upon RANKL exposure. We established that ARTD1 proteolysis was NLRP3 inflammasome-dependent, and occurred via the proteasome pathway. Since ARTD1 is cleaved at aspartate214, we studied the impact of ARTD1 rendered uncleavable by D214N substitution (ARTD1D214N) on skeletal homeostasis. ARTD1D214N, unlike wild-type ARTD1, was resistant to cleavage and degradation during osteoclastogenesis. As a result, ARTD1D214N altered histone modification and promoted the abundance of the repressors of osteoclastogenesis by interfering with the expression of B lymphocyte-induced maturation protein 1 (Blimp1), the master regulator of anti-osteoclastogenic transcription factors. Importantly, ARTD1D214N-expressing mice exhibited higher bone mass compared with controls, owing to decreased osteoclastogenesis while bone formation was unaffected. Thus, unless it is degraded, ARTD1 represses OC development through transcriptional regulation. PMID:27010854

  17. The TITAN5 gene of Arabidopsis encodes a protein related to the ADP ribosylation factor family of GTP binding proteins.

    PubMed

    McElver, J; Patton, D; Rumbaugh, M; Liu, C; Yang, L J; Meinke, D

    2000-08-01

    The titan (ttn) mutants of Arabidopsis exhibit dramatic alterations in mitosis and cell cycle control during seed development. Endosperm development in these mutants is characterized by the formation of giant polyploid nuclei with enlarged nucleoli. Embryo development is accompanied by significant cell enlargement in some mutants (ttn1 and ttn5) but not others (ttn2 and ttn3). We describe here the molecular cloning of TTN5 using a T-DNA-tagged allele. A second allele with a similar phenotype contains a nonsense mutation in the same coding region. The predicted protein is related to ADP ribosylation factors (ARFs), members of the RAS family of small GTP binding proteins that regulate various cellular functions in eukaryotes. TTN5 is most closely related in sequence to the ARL2 class of ARF-like proteins isolated from humans, rats, and mice. Although the cellular functions of ARL proteins remain unclear, the ttn5 phenotype is consistent with the known roles of ARFs in the regulation of intracellular vesicle transport.

  18. A presynaptic role for the ADP ribosylation factor (ARF)-specific GDP/GTP exchange factor msec7-1.

    PubMed

    Ashery, U; Koch, H; Scheuss, V; Brose, N; Rettig, J

    1999-02-01

    ADP ribosylation factors (ARFs) represent a family of small monomeric G proteins that switch from an inactive, GDP-bound state to an active, GTP-bound state. One member of this family, ARF6, translocates on activation from intracellular compartments to the plasma membrane and has been implicated in regulated exocytosis in neuroendocrine cells. Because GDP release in vivo is rather slow, ARF activation is facilitated by specific guanine nucleotide exchange factors like cytohesin-1 or ARNO. Here we show that msec7-1, a rat homologue of cytohesin-1, translocates ARF6 to the plasma membrane in living cells. Overexpression of msec7-1 leads to an increase in basal synaptic transmission at the Xenopus neuromuscular junction. msec7-1-containing synapses have a 5-fold higher frequency of spontaneous synaptic currents than control synapses. On stimulation, the amplitudes of the resulting evoked postsynaptic currents of msec7-1-overexpressing neurons are increased as well. However, further stimulation leads to a decline in amplitudes approaching the values of control synapses. This transient effect on amplitude is strongly reduced on overexpression of msec7-1E157K, a mutant incapable of translocating ARFs. Our results provide evidence that small G proteins of the ARF family and activating factors like msec7-1 play an important role in synaptic transmission, most likely by making more vesicles available for fusion at the plasma membrane.

  19. Molecular cloning, characterization, and expression of human ADP-ribosylation factors: two guanine nucleotide-dependent activators of cholera toxin.

    PubMed Central

    Bobak, D A; Nightingale, M S; Murtagh, J J; Price, S R; Moss, J; Vaughan, M

    1989-01-01

    ADP-ribosylation factors (ARFs) are small guanine nucleotide-binding proteins that enhance the enzymatic activities of cholera toxin. Two ARF cDNAs, ARF1 and ARF3, were cloned from a human cerebellum library. Based on deduced amino acid sequences and patterns of hybridization of cDNA and oligonucleotide probes with mammalian brain poly(A)+ RNA, human ARF1 is the homologue of bovine ARF1. Human ARF3, which differs from bovine ARF1 and bovine ARF2, appears to represent a newly identified third type of ARF. Hybridization patterns of human ARF cDNA and clone-specific oligonucleotides with poly(A)+ RNA are consistent with the presence of at least two, and perhaps four, separate ARF messages in human brain. In vitro translation of ARF1, ARF2, and ARF3 produced proteins that behaved, by SDS/PAGE, similar to a purified soluble brain ARF. Deduced amino acid sequences of human ARF1 and ARF3 contain regions, similar to those in other G proteins, that are believed to be involved in GTP binding and hydrolysis. ARFs also exhibit a modest degree of homology with a bovine phospholipase C. The observations reported here support the conclusion that the ARFs are members of a multigene family of small guanine nucleotide-binding proteins. Definition of the regulation of ARF mRNAs and of function(s) of recombinant ARF proteins will aid in the elucidation of the physiologic role(s) of ARFs. Images PMID:2474826

  20. ADP Ribosylation Factor 6 (ARF6) Promotes Acrosomal Exocytosis by Modulating Lipid Turnover and Rab3A Activation*

    PubMed Central

    Pelletán, Leonardo E.; Suhaiman, Laila; Vaquer, Cintia C.; Bustos, Matías A.; De Blas, Gerardo A.; Vitale, Nicolas; Mayorga, Luis S.; Belmonte, Silvia A.

    2015-01-01

    Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. ARF6 (ADP-ribosylation factor 6) is a small GTPase implicated in exocytosis, but its downstream effectors remain elusive in this process. We combined biochemical, functional, and microscopy-based methods to show that ARF6 is present in human sperm, localizes to the acrosomal region, and is required for calcium and diacylglycerol-induced exocytosis. Results from pulldown assays show that ARF6 exchanges GDP for GTP in sperm challenged with different exocytic stimuli. Myristoylated and guanosine 5′-3-O-(thio)triphosphate (GTPγS)-loaded ARF6 (active form) added to permeabilized sperm induces acrosome exocytosis even in the absence of extracellular calcium. We explore the ARF6 signaling cascade that promotes secretion. We demonstrate that ARF6 stimulates a sperm phospholipase D activity to produce phosphatidic acid and boosts the synthesis of phosphatidylinositol 4,5-bisphosphate. We present direct evidence showing that active ARF6 increases phospholipase C activity, causing phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate-dependent intra-acrosomal calcium release. We show that active ARF6 increases the exchange of GDP for GTP on Rab3A, a prerequisite for secretion. We propose that exocytic stimuli activate ARF6, which is required for acrosomal calcium efflux and the assembly of the membrane fusion machinery. This report highlights the physiological importance of ARF6 as a key factor for human sperm exocytosis and fertilization. PMID:25713146

  1. Cloning of an ADP-ribosylation factor gene from banana (Musa acuminata) and its expression patterns in postharvest ripening fruit.

    PubMed

    Wang, Yuan; Wu, Jing; Xu, Bi-Yu; Liu, Ju-Hua; Zhang, Jian-Bin; Jia, Cai-Hong; Jin, Zhi-Qiang

    2010-08-15

    A full-length cDNA encoding an ADP-ribosylation factor (ARF) from banana (Musa acuminata) fruit was cloned and named MaArf. It contains an open reading frame encoding a 181-amino-acid polypeptide. Sequence analysis showed that MaArf shared high similarity with ARF of other plant species. The genomic sequence of MaArf was also obtained using polymerase chain reaction (PCR). Sequence analysis showed that MaArf was a split gene containing five exons and four introns in genomic DNA. Reverse-transcriptase PCR was used to analyze the spatial expression of MaArf. The results showed that MaArf was expressed in all the organs examined: root, rhizome, leaf, flower and fruit. Real-time quantitative PCR was used to explore expression patterns of MaArf in postharvest banana. There was differential expression of MaArf associated with ethylene biosynthesis. In naturally ripened banana, expression of MaArf was in accordance with ethylene biosynthesis. However, in 1-methylcyclopropene-treated banana, the expression of MaArf was inhibited and changed little. When treated with ethylene, MaArf expression in banana fruit significantly increased in accordance with ethylene biosynthesis; the peak of MaArf was 3 d after harvest, 11 d earlier than for naturally ripened banana fruits. These results suggest that MaArf is induced by ethylene in regulating postharvest banana ripening. Finally, subcellular localization assays showed the MaArf protein in the cytoplasm. PMID:20435371

  2. Characterization of two novel ADP ribosylation factors from giant freshwater prawn Macrobrachium rosenbergii and their responses to WSSV challenge.

    PubMed

    Ding, Zheng-Feng; Ren, Jie; Tan, Jing-Min; Wang, Zheng; Yin, Shao-Wu; Huang, Ying; Huang, Xin; Wang, Wen; Lan, Jiang-Feng; Ren, Qian

    2015-01-01

    ADP-ribosylation factors (Arfs) are small GTP-binding proteins that have an essential function in intracellular trafficking and organelle structure. To date, little information is available on the Arfs in the economically important giant freshwater prawn Macrobrachium rosenbergii and their relationship to viral infection. Here we identified two Arf genes from M. rosenbergii (MrArf1 and MrArf2) for the first time. Phylogenetic analysis showed that MrArf1, together with MjArf1 from shrimp Marsupenaeus japonicus belonged to Class I Arfs. By contrast, MrArf2 didn't not match any of the Arfs classes of I/II/III, although it could be clustered with an Arf protein from M. japonicas called MjArfn, which may represent an analog of the Arf. MrArf1 was ubiquitously expressed in all the examined tissues, with the highest transcription level in the hepatopancreas, whereas MrArf2 was only highly expressed in the hepatopancreas and exhibited very low levels in the heart, stomach, gills and intestine. The expression level of MrArf1 in the gills was down-regulated post 24 h WSSV challenge, and reached the maximal level at 48 h. MrArf1 in the hepatopancreas went up from 24 to 48 h WSSV challenge. MrArf2 transcript in the gill also went down at 24 h and then was upregulated at 48 h WSSV challenge. MrArf2 increased significantly in the hepatopancreas 24 h after infection and then went down at 48 h WSSV challenge. RNAi results showed that knockdown of MrArf1 or MrArf2 could inhibit the expression of the envelope protein gene vp28 of the WSSV. So, it could be speculated that MrArf1 and MrArf2 might play important roles in the innate immune system against WSSV infection.

  3. GTP but not GDP analogues promote association of ADP-ribosylation factors, 20-kDa protein activators of cholera toxin, with phospholipids and PC-12 cell membranes.

    PubMed

    Walker, M W; Bobak, D A; Tsai, S C; Moss, J; Vaughan, M

    1992-02-15

    ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide-binding proteins initially identified by their ability to enhance cholera toxin ADP-ribosyltransferase activity in the presence of GTP. ARFs have been purified from both membrane and cytosolic fractions. ARF purified from bovine brain cytosol requires phospholipid plus detergent for high affinity guanine nucleotide binding and for optimal enhancement of cholera toxin ADP-ribosyltransferase activity. The phospholipid requirements, combined with a putative role for ARF in vesicular transport, suggested that the soluble protein might interact reversibly with membranes. A polyclonal antibody against purified bovine ARF (sARF II) was used to detect ARF by immunoblot in membrane and soluble fractions from rat pheochromocytoma (PC-12) cell homogenates. ARF was predominantly cytosolic but increased in membranes during incubation of homogenates with nonhydrolyzable GTP analogues guanosine 5'-O-(3-thiotriphosphate), guanylyl-(beta gamma-imido)-diphosphate, and guanylyl-(beta gamma-methylene)-diphosphate, and to a lesser extent, adenosine 5'-O-(3-thiotriphosphate). GTP, GDP, GMP, and ATP were inactive. Cytosolic ARF similarly associated with added phosphatidylserine, phosphatidylinositol, or cardiolipin in GTP gamma S-dependent fashion. ARF binding to phosphatidylserine was reversible and coincident with stimulation of cholera toxin-catalyzed ADP-ribosylation. These observations may reflect a mechanism by which ARF could cycle between soluble and membrane compartments in vivo.

  4. Exocytosis of CTLA-4 is dependent on phospholipase D and ADP ribosylation factor-1 and stimulated during activation of regulatory T cells.

    PubMed

    Mead, Karen I; Zheng, Yong; Manzotti, Claire N; Perry, Laura C A; Liu, Michael K P; Burke, Fiona; Powner, Dale J; Wakelam, Michael J O; Sansom, David M

    2005-04-15

    CTLA-4 is an essential protein in the regulation of T cell responses that interacts with two ligands found on the surface of APCs (CD80 and CD86). CTLA-4 is itself poorly expressed on the T cell surface and is predominantly localized to intracellular compartments. We have studied the mechanisms involved in the delivery of CTLA-4 to the cell surface using a model Chinese hamster ovary cell system and compared this with activated and regulatory human T cells. We have shown that expression of CTLA-4 at the plasma membrane (PM) is controlled by exocytosis of CTLA-4-containing vesicles and followed by rapid endocytosis. Using selective inhibitors and dominant negative mutants, we have shown that exocytosis of CTLA-4 is dependent on the activity of the GTPase ADP ribosylation factor-1 and on phospholipase D activity. CTLA-4 was identified in a perinuclear compartment overlapping with the cis-Golgi marker GM-130 but did not colocalize strongly with lysosomal markers such as CD63 and lysosome-associated membrane protein. In regulatory T cells, activation of phospholipase D was sufficient to trigger release of CTLA-4 to the PM but did not inhibit endocytosis. Taken together, these data suggest that CTLA-4 may be stored in a specialized compartment in regulatory T cells that can be triggered rapidly for deployment to the PM in a phospholipase D- and ADP ribosylation factor-1-dependent manner.

  5. Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the. alpha. subunit of the stimulatory guanine nucleotide-binding regulatory protein

    SciTech Connect

    Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G. )

    1988-08-01

    ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the {alpha} subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost, a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5{prime}-({alpha}-{sup 32}P)triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an {alpha} subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera.

  6. Purification and properties of poly(ADP-ribose)polymerase from Crithidia fasciculata. Automodification and poly(ADP-ribosyl)ation of DNA topoisomerase I.

    PubMed

    Podestá, Dolores; García-Herreros, María I; Cannata, Joaquín J B; Stoppani, Andrés O M; Fernández Villamil, Silvia H

    2004-06-01

    Poly(ADP-ribose)polymerase has been purified more than 160000-fold from Crithidia fasciculata. This is the first PARP isolated to apparent homogeneity from trypanosomatids. The purified enzyme absolutely required DNA for catalytic activity and histones enhanced it 2.5-fold, when the DNA:histone ratio was 1:1.3. The enzyme required no magnesium or any other metal ion cofactor. The apparent molecular mass of 111 kDa, determined by gel filtration would correspond to a dimer of two identical 55-kDa subunits. Activity was inhibited by nicotinamide, 3-aminobenzamide, theophylline, thymidine, xanthine and hypoxanthine but not by adenosine. The enzyme was localized to the cell nucleus. Our findings suggest that covalent poly(ADP-ribosyl)ation of PARP itself or DNA topoisomerase I resulted in the inhibition of their activities and provide an initial biochemical characterization of this covalent post-translational modification in trypanosomatids.

  7. Blockade of PARP activity attenuates poly(ADP-ribosyl)ation but offers only partial neuroprotection against NMDA-induced cell death in the rat retina.

    PubMed

    Goebel, Dennis J; Winkler, Barry S

    2006-09-01

    Recent reports have linked neuronal cell death by necrosis to poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation. It is believed that under stress, the activity of this enzyme is up-regulated, resulting in extensive poly(ADP-ribosyl)ation of nuclear proteins, using NAD(+) as its substrate, which, in turn, leads to the depletion of NAD(+). In efforts to restore the level of NAD(+), depletion of ATP occurs, resulting in the shutdown of ATP-dependent ionic pumps. This results in cell swelling and eventual loss of membrane selectivity, hallmarks of necrosis. Reports from in vitro and in vivo studies in the brain have shown that NMDA receptor activation stimulates PARP activity and that blockade of the enzyme provides substantial neuroprotection. The present study was undertaken to determine whether PARP activity is regulated by NMDA in the rat retina, and whether blockade of PARP activity provides protection against toxic effects of NMDA. Rat retinas exposed to intravitreal injections containing NMDA, with or without the PARP inhibitor N-(6-oxo-5, 6-dihydrophenanthridin-2-yl)-(N,-dimethylamino) acetamide hydrochloride (PJ-34), were assessed for changes in PARP-1 activity as evidenced by poly(ADP-ribosyl)ation (PAR), loss of membrane integrity, morphological indicators of apoptosis and necrosis, and ganglion cell loss. Results showed that: NMDA increased PAR formation in a concentration-dependent manner and caused a decline in retinal ATP levels; PJ-34 blockade attenuated the NMDA-induced formation of PAR and decline in ATP; NMDA induced the loss of membrane selectivity to ethidium bromide (EtBr) in inner retinal neurons, but loss of membrane selectivity was not prevented by blocking PARP activity; cells stained with EtBr, or reacted for TUNEL-labeling, displayed features characteristic of both apoptosis and necrosis. In the presence of PJ-34, greater numbers of cells exhibited apoptotic features; PJ-34 provided partial neuroprotection against NMDA-induced ganglion

  8. Nucleotide sequence and chromosomal localization of the gene for pierisin-1, a DNA ADP-ribosylating protein, in the cabbage butterfly Pieris rapae.

    PubMed

    Yamamoto, Masafumi; Takahashi-Nakaguchi, Azusa; Matsushima-Hibiya, Yuko; Nakano, Tsuyoshi; Totsuka, Yukari; Imanishi, Shigeo; Mitsuhashi, Jun; Watanabe, Masahiko; Nakagama, Hitoshi; Sugimura, Takashi; Wakabayashi, Keiji

    2011-10-01

    Cabbage butterfly, Pieris rapae, contains a unique DNA ADP-ribosylating protein, pierisin-1, which transfers ADP-ribose moiety of NAD to guanine bases of DNA. Pierisin-like proteins are only distributed in subtribes Pierina, Aporiina and Appiadina of the family Pieridae. In this study, we obtained genomic clones carrying the pierisin-1 gene from adult samples of P. rapae by plaque hybridization. The pierisin-1 gene was found to consist of two exons, 0.1-kb exon 1 and 3.9-kb exon 2, and a 2.3-kb intron. In addition, we could demonstrate that the putative promoter in the about 3-kb upstream region from the transcription start site of the gene include a transcriptional activating motif involved in immune pathways and hormonal regulation. We also examined chromosomal localization of the pierisin-1 gene. Fluorescence in situ hybridization (FISH) analysis using Cy3-labeled pierisin-1 genomic clone demonstrated the localization of the gene near the kinetochore in chromosome 9. Thus, we confirmed that the pierisin-1 gene is located in the genome of P. rapae.

  9. GB virus type C E2 protein inhibits human immunodeficiency virus type 1 Gag assembly by downregulating human ADP-ribosylation factor 1

    PubMed Central

    Wang, Chenliang; Timmons, Christine L.; Shao, Qiujia; Kinlock, Ballington L.; Turner, Tiffany M.; Iwamoto, Aikichi; Zhang, Hui; Liu, Huanliang; Liu, Bindong

    2015-01-01

    GB virus type C (GBV-C) glycoprotein E2 protein disrupts HIV-1 assembly and release by inhibiting Gag plasma membrane targeting, however the mechanism by which the GBV-C E2 inhibits Gag trafficking remains unclear. In the present study, we identified ADP-ribosylation factor 1 (ARF1) contributed to the inhibitory effect of GBV-C E2 on HIV-1 Gag membrane targeting. Expression of GBV-C E2 decreased ARF1 expression in a proteasomal degradation-dependent manner. The restoration of ARF1 expression rescued the HIV-1 Gag processing and membrane targeting defect imposed by GBV-C E2. In addition, GBV-C E2 expression also altered Golgi morphology and suppressed protein traffic through the secretory pathway, which are all consistent with a phenotype of disrupting the function of ARF1 protein. Thus, our results indicate that GBV-C E2 inhibits HIV-1 assembly and release by decreasing ARF1, and may provide insights regarding GBV-C E2's potential for a new therapeutic approach for treating HIV-1. PMID:26675377

  10. Solution structure of the cytohesin-1 (B2–1) Sec7 domain and its interaction with the GTPase ADP ribosylation factor 1

    PubMed Central

    Betz, Stephen F.; Schnuchel, Arndt; Wang, Hong; Olejniczak, Edward T.; Meadows, Robert P.; Lipsky, Brian P.; Harris, Edith A. S.; Staunton, Donald E.; Fesik, Stephen W.

    1998-01-01

    Cytohesin-1 (B2–1) is a guanine nucleotide exchange factor for human ADP ribosylation factor (Arf) GTPases, which are important for vesicular protein trafficking and coatamer assembly in the cell. Cytohesin-1 also has been reported to promote cellular adhesion via binding to the β2 integrin cytoplasmic domain. The solution structure of the Sec7 domain of cytohesin-1, which is responsible for both the protein’s guanine nucleotide exchange factor function and β2 integrin binding, was determined by NMR spectroscopy. The structure consists of 10 α-helices that form a unique tertiary fold. The binding between the Sec7 domain and a soluble, truncated version of human Arf-1 was investigated by examining 1H-15N and 1H-13C chemical shift changes between the native protein and the Sec7/Arf-1 complex. We show that the binding to Arf-1 occurs through a large surface on the C-terminal subdomain that is composed of both hydrophobic and polar residues. Structure-based mutational analysis of the cytohesin-1 Sec7 domain has been used to identify residues important for binding to Arf and for mediating nucleotide exchange. Investigations into the interaction between the Sec7 domain and the β2 integrin cytoplasmic domain suggest that the two proteins do not interact in the solution phase. PMID:9653114

  11. The Structure of RalF, an ADP-Ribosylation Factor Guanine Nucleotide Exchange Factor from Legionella pneumophila, Reveals the Presence of a Cap over the Active Site

    SciTech Connect

    Amor,J.; Swails, J.; Zhu, X.; Roy, C.; Nagai, H.; Ingmundson, A.; Cheng, X.; Kahn, R.

    2005-01-01

    The Legionella pneumophila protein RalF is secreted into host cytosol via the Dot/Icm type IV transporter where it acts to recruit ADP-ribosylation factor (Arf) to pathogen-containing phagosomes in the establishment of a replicative organelle. The presence in RalF of the Sec7 domain, present in all Arf guanine nucleotide exchange factors, has suggested that recruitment of Arf is an early step in pathogenesis. We have determined the crystal structure of RalF and of the isolated Sec7 domain and found that RalF is made up of two domains. The Sec7 domain is homologous to mammalian Sec7 domains. The C-terminal domain forms a cap over the active site in the Sec7 domain and contains a conserved folding motif, previously observed in adaptor subunits of vesicle coat complexes. The importance of the capping domain and of the glutamate in the 'glutamic finger,' conserved in all Sec7 domains, to RalF functions was examined using three different assays. These data highlight the functional importance of domains other than Sec7 in Arf guanine nucleotide exchange factors to biological activities and suggest novel mechanisms of regulation of those activities.

  12. ADP-ribosylation factor arf6p may function as a molecular switch of new end take off in fission yeast

    SciTech Connect

    Fujita, Atsushi

    2008-02-01

    Small GTPases act as molecular switches in a wide variety of cellular processes. In fission yeast Schizosaccharomyces pombe, the directions of cell growth change from a monopolar manner to a bipolar manner, which is known as 'New End Take Off' (NETO). Here I report the identification of a gene, arf6{sup +}, encoding an ADP-ribosylation factor small GTPase, that may be essential for NETO. arf6{delta} cells completely fail to undergo NETO. arf6p localizes at both cell ends and presumptive septa in a cell-cycle dependent manner. And its polarized localization is not dependent on microtubules, actin cytoskeletons and some NETO factors (bud6p, for3p, tea1p, tea3p, and tea4p). Notably, overexpression of a fast GDP/GTP-cycling mutant of arf6p can advance the timing of NETO. These findings suggest that arf6p functions as a molecular switch for the activation of NETO in fission yeast.

  13. ADP Ribosylation Factor 6 Regulates Neuronal Migration in the Developing Cerebral Cortex through FIP3/Arfophilin-1-dependent Endosomal Trafficking of N-cadherin.

    PubMed

    Hara, Yoshinobu; Fukaya, Masahiro; Hayashi, Kanehiro; Kawauchi, Takeshi; Nakajima, Kazunori; Sakagami, Hiroyuki

    2016-01-01

    During neural development, endosomal trafficking controls cell shape and motility through the polarized transport of membrane proteins related to cell-cell and cell-extracellular matrix interactions. ADP ribosylation factor 6 (Arf6) is a critical small GTPase that regulates membrane trafficking between the plasma membrane and endosomes. We herein demonstrated that the knockdown of endogenous Arf6 in mouse cerebral cortices led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin and syntaxin12 in migrating neurons. Rescue experiments with separation-of-function Arf6 mutants identified Rab11 family-interacting protein 3 (FIP3)/Arfophilin-1, a dual effector for Arf6 and Rab11, as a downstream effector of Arf6 in migrating neurons. The knockdown of FIP3 led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin in migrating neurons, similar to that of Arf6, which could be rescued by the coexpression of wild-type FIP3 but not FIP3 mutants lacking the binding site for Arf6 or Rab11. These results suggest that Arf6 regulates cortical neuronal migration in the intermediate zone through the FIP3-dependent endosomal trafficking. PMID:27622210

  14. Functional Characterization of an Extended Binding Component of the Actin-ADP-Ribosylating C2 Toxin Detected in Clostridium botulinum Strain (C) 2300 ▿

    PubMed Central

    Sterthoff, Charlott; Lang, Alexander E.; Schwan, Carsten; Tauch, Andreas; Aktories, Klaus

    2010-01-01

    Clostridium botulinum C2 toxin consists of the binding component C2II and the enzyme component C2I, which ADP-ribosylates G-actin of eukaryotic cells. Trypsin-activated C2II (C2IIa) forms heptamers that mediate cell binding and translocation of C2I from acidic endosomes into the cytosol of target cells. By genome sequencing of C. botulinum strain (C) 2300, we found that C2II from this strain carries a C-terminal extension of 129 amino acids, unlike its homologous counterparts from strains (C) 203U28, (C) 468, and (D) 1873. This extension shows a high similarity to the C-terminal receptor-binding domain of C2II and is presumably the result of a duplication of this domain. The C2II extension facilitates the binding to cell surface receptors, which leads to an increased intoxication efficiency compared to that of C2II proteins from other C. botulinum strains. PMID:20145093

  15. ADP Ribosylation Factor 6 Regulates Neuronal Migration in the Developing Cerebral Cortex through FIP3/Arfophilin-1-dependent Endosomal Trafficking of N-cadherin

    PubMed Central

    Hara, Yoshinobu; Fukaya, Masahiro

    2016-01-01

    Abstract During neural development, endosomal trafficking controls cell shape and motility through the polarized transport of membrane proteins related to cell–cell and cell–extracellular matrix interactions. ADP ribosylation factor 6 (Arf6) is a critical small GTPase that regulates membrane trafficking between the plasma membrane and endosomes. We herein demonstrated that the knockdown of endogenous Arf6 in mouse cerebral cortices led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin and syntaxin12 in migrating neurons. Rescue experiments with separation-of-function Arf6 mutants identified Rab11 family-interacting protein 3 (FIP3)/Arfophilin-1, a dual effector for Arf6 and Rab11, as a downstream effector of Arf6 in migrating neurons. The knockdown of FIP3 led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin in migrating neurons, similar to that of Arf6, which could be rescued by the coexpression of wild-type FIP3 but not FIP3 mutants lacking the binding site for Arf6 or Rab11. These results suggest that Arf6 regulates cortical neuronal migration in the intermediate zone through the FIP3-dependent endosomal trafficking.

  16. ADP Ribosylation Factor 6 Regulates Neuronal Migration in the Developing Cerebral Cortex through FIP3/Arfophilin-1-dependent Endosomal Trafficking of N-cadherin

    PubMed Central

    Hara, Yoshinobu; Fukaya, Masahiro

    2016-01-01

    Abstract During neural development, endosomal trafficking controls cell shape and motility through the polarized transport of membrane proteins related to cell–cell and cell–extracellular matrix interactions. ADP ribosylation factor 6 (Arf6) is a critical small GTPase that regulates membrane trafficking between the plasma membrane and endosomes. We herein demonstrated that the knockdown of endogenous Arf6 in mouse cerebral cortices led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin and syntaxin12 in migrating neurons. Rescue experiments with separation-of-function Arf6 mutants identified Rab11 family-interacting protein 3 (FIP3)/Arfophilin-1, a dual effector for Arf6 and Rab11, as a downstream effector of Arf6 in migrating neurons. The knockdown of FIP3 led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin in migrating neurons, similar to that of Arf6, which could be rescued by the coexpression of wild-type FIP3 but not FIP3 mutants lacking the binding site for Arf6 or Rab11. These results suggest that Arf6 regulates cortical neuronal migration in the intermediate zone through the FIP3-dependent endosomal trafficking. PMID:27622210

  17. ADP-Ribosylation Factor 6 Regulates Mammalian Myoblast Fusion through Phospholipase D1 and Phosphatidylinositol 4,5-Bisphosphate Signaling Pathways

    PubMed Central

    Bach, Anne-Sophie; Enjalbert, Sandrine; Comunale, Franck; Bodin, Stéphane; Vitale, Nicolas; Charrasse, Sophie

    2010-01-01

    Myoblast fusion is an essential step during myoblast differentiation that remains poorly understood. M-cadherin–dependent pathways that signal through Rac1 GTPase activation via the Rho-guanine nucleotide exchange factor (GEF) Trio are important for myoblast fusion. The ADP-ribosylation factor (ARF)6 GTPase has been shown to bind to Trio and to regulate Rac1 activity. Moreover, Loner/GEP100/BRAG2, a GEF of ARF6, has been involved in mammalian and Drosophila myoblast fusion, but the specific role of ARF6 has been not fully analyzed. Here, we show that ARF6 activity is increased at the time of myoblast fusion and is required for its implementation in mouse C2C12 myoblasts. Specifically, at the onset of myoblast fusion, ARF6 is associated with the multiproteic complex that contains M-cadherin, Trio, and Rac1 and accumulates at sites of myoblast fusion. ARF6 silencing inhibits the association of Trio and Rac1 with M-cadherin. Moreover, we demonstrate that ARF6 regulates myoblast fusion through phospholipase D (PLD) activation and phosphatidylinositol 4,5-bis-phosphate production. Together, these data indicate that ARF6 is a critical regulator of C2C12 myoblast fusion and participates in the regulation of PLD activities that trigger both phospholipids production and actin cytoskeleton reorganization at fusion sites. PMID:20505075

  18. ADP-ribosylation factor 1 controls the activation of the phosphatidylinositol 3-kinase pathway to regulate epidermal growth factor-dependent growth and migration of breast cancer cells.

    PubMed

    Boulay, Pierre-Luc; Cotton, Mathieu; Melançon, Paul; Claing, Audrey

    2008-12-26

    Activation of intracellular signaling pathways by growth factors is one of the major causes of cancer development and progression. Recent studies have demonstrated that monomeric G proteins of the Ras family are key regulators of cell proliferation, migration, and invasion. Using an invasive breast cancer cell lines, we demonstrate that the ADP-ribosylation factor 1 (ARF1), a small GTPase classically associated with the Golgi, is an important regulator of the biological effects induced by epidermal growth factor. Here, we show that this ARF isoform is activated following epidermal growth factor stimulation and that, in MDA-MB-231 cells, ARF1 is found in dynamic plasma membrane ruffles. Inhibition of endogenous ARF1 expression results in the inhibition of breast cancer cell migration and proliferation. The underlying mechanism involves the activation of the phosphatidylinositol 3-kinase pathway. Our data demonstrate that depletion of ARF1 markedly impairs the recruitment of the phosphatidylinositol 3-kinase catalytic subunit (p110alpha) to the plasma membrane, and the association of the regulatory subunit (p85alpha) to the activated receptor. These results uncover a novel molecular mechanism by which ARF1 regulates breast cancer cell growth and invasion during cancer progression.

  19. A Luman/CREB3–ADP-ribosylation factor 4 (ARF4) signaling pathway mediates the response to Golgi stress and susceptibility to pathogens

    PubMed Central

    Reiling, Jan H.; Olive, Andrew J.; Sanyal, Sumana; Carette, Jan E.; Brummelkamp, Thijn R.; Ploegh, Hidde L.; Starnbach, Michael N.; Sabatini, David M.

    2014-01-01

    SUMMARY Treatment of cells with Brefeldin A (BFA) blocks secretory vesicle transport and causes a collapse of the Golgi apparatus. To gain more insight into the cellular mechanisms mediating BFA toxicity, we conducted a genome-wide haploid genetic screen that led to the identification of the small G protein ADP-ribosylation factor 4 (ARF4). ARF4 depletion preserves viability, Golgi integrity and cargo trafficking in the presence of BFA, and these effects depend on the guanine nucleotide exchange factor GBF1 and other ARF isoforms including ARF1 and ARF5. ARF4 knockdown cells show increased resistance to several human pathogens including Chlamydia trachomatis and Shigella flexneri. Furthermore, ARF4 expression is induced when cells are exposed to several Golgi-disturbing agents and requires the CREB3/Luman transcription factor whose downregulation mimics ARF4 loss. Thus, we have uncovered a CREB3–ARF4 signaling cascade that may be part of a Golgi stress response set in motion by stimuli compromising Golgi capacity. PMID:24185178

  20. Selective amplification of an mRNA and related pseudogene for a human ADP-ribosylation factor, a guanine nucleotide-dependent protein activator of cholera toxin

    SciTech Connect

    Monaco, L.; Murtagh, J.J.; Newman, K.B.; Tsai, Su-Chen; Moss, J.; Vaughan, M. )

    1990-03-01

    ADP-ribosylation factors (ARFs) are {approx}20-kDa proteins that act as GTP-dependent allosteric activators of cholera toxin. With deoxyinosine-containing degenerate oligonucleotide primers corresponding to conserved GTP-binding domains in ARFs, the polymerase chain reaction (PCR) was used to amplify simultaneously from human DNA portions of three ARF genes that include codons for 102 amino acids, with intervening sequences. Amplification products that differed in size because of differences in intron sizes were separated by agarose gel electrophoresis. One amplified DNA contained no introns and had a sequence different from those of known AFRs. Based on this sequence, selective oligonucleotide probes were prepared and used to isolate clone {Psi}ARF 4, a putative ARF pseudogene, from a human genomic library in {lambda} phage EMBL3. Reverse transcription-PCR was then used to clone from human poly(A){sup +} RNA the cDNA corresponding to the expressed homolog of {Psi}ARF 4, referred to as human ARF 4. It appears that {Psi}ARF 4 arose during human evolution by integration of processed ARF 4 mRNA into the genome. Human ARF 4 differs from previously identified mammalian ARFs 1, 2, and 3. Hybridization of ARF 4-specific oligonucleotide probes with human, bovine, and rat RNA revealed a single 1.8-kilobase mRNA, which was clearly distinguished from the 1.9-kilobase mRNA for ARF 1 in these tissues. The PCR provides a powerful tool for investigating diversity in this and other multigene families, especially with primers targeted at domains believed to have functional significance.

  1. AMF-26, a novel inhibitor of the Golgi system, targeting ADP-ribosylation factor 1 (Arf1) with potential for cancer therapy.

    PubMed

    Ohashi, Yoshimi; Iijima, Hiroshi; Yamaotsu, Noriyuki; Yamazaki, Kanami; Sato, Shigeo; Okamura, Mutsumi; Sugimoto, Kenji; Dan, Shingo; Hirono, Shuichi; Yamori, Takao

    2012-02-01

    ADP-ribosylation factor 1 (Arf1) plays a major role in mediating vesicular transport. Brefeldin A (BFA), a known inhibitor of the Arf1-guanine nucleotide exchange factor (GEF) interaction, is highly cytotoxic. Therefore, interaction of Arf1 with ArfGEF is an attractive target for cancer treatment. However, BFA and its derivatives have not progressed beyond the pre-clinical stage of drug development because of their poor bioavailability. Here, we aimed to identify novel inhibitors of the Arf1-ArfGEF interaction that display potent antitumor activity in vivo but with a chemical structure distinct from that of BFA. We exploited a panel of 39 cell lines (termed JFCR39) coupled with a drug sensitivity data base and COMPARE algorithm, resulting in the identification of a possible novel Arf1-ArfGEF inhibitor AMF-26, which differed structurally from BFA. By using a pulldown assay with GGA3-conjugated beads, we demonstrated that AMF-26 inhibited Arf1 activation. Subsequently, AMF-26 induced Golgi disruption, apoptosis, and cell growth inhibition. Computer modeling/molecular dynamics (MD) simulation suggested that AMF-26 bound to the contact surface of the Arf1-Sec7 domain where BFA bound. AMF-26 affected membrane traffic, including the cis-Golgi and trans-Golgi networks, and the endosomal systems. Furthermore, using AMF-26 and its derivatives, we demonstrated that there was a significant correlation between cell growth inhibition and Golgi disruption. In addition, orally administrated AMF-26 (83 mg/kg of body weight; 5 days) induced complete regression of human breast cancer BSY-1 xenografts in vivo, suggesting that AMF-26 is a novel anticancer drug candidate that inhibits the Golgi system, targeting Arf1 activation.

  2. Functional genomic analysis of the ADP-ribosylation factor family of GTPases: phylogeny among diverse eukaryotes and function in C. elegans.

    PubMed

    Li, Yawei; Kelly, William G; Logsdon, John M; Schurko, Andrew M; Harfe, Brian D; Hill-Harfe, Katherine L; Kahn, Richard A

    2004-12-01

    ADP-ribosylation factor (Arf) and Arf-like (Arl) proteins are a family of highly conserved 21 kDa GTPases that emerged early in the evolution of eukaryotes. These proteins serve regulatory roles in vesicular traffic, lipid metabolism, microtubule dynamics, development, and likely other cellular processes. We found evidence for the presence of 6 Arf family members in the protist Giardia lamblia and 22 members in mammals. A phylogenetic analysis was performed to delineate the evolutionary relationships among Arf family members and to attempt to organize them by both their evolutionary origins and functions in cells and/or organisms. The approximately 100 protein sequences analyzed from animals, fungi, plants, and protists clustered into 11 groups, including Arfs, nine Arls, and Sar proteins. To begin functional analyses of the family in a metazoan model organism, we examined roles for all three C. elegans Arfs (Arf-1, Arf-3, and Arf-6) and three Arls (Arl-1, Arl-2, and Arl-3) by use of RNA-mediated interference (RNAi). Injection of double-stranded RNA (dsRNA) encoding Arf-1 or Arf-3 into N2 hermaphrodites produced embryonic lethality in their offspring and, later, sterility in the injected animals themselves. Injection of Arl-2 dsRNA resulted in a disorganized germline and sterility in early offspring, with later offspring exhibiting an early embryonic arrest. Thus, of the six Arf family members examined in C. elegans, at least three are required for embryogenesis. These data represent the first analysis of the role(s) of multiple members of this family in the development of a multicellular organism.

  3. Characterization of ADP ribosylation factor 1 gene from Exopalaemon carinicauda and its immune response to pathogens challenge and ammonia-N stress.

    PubMed

    Duan, Yafei; Li, Jian; Zhang, Zhe; Li, Jitao; Liu, Ping

    2016-08-01

    ADP ribosylation factors (Arf), as highly conserved small guanosine triphosphate (GTP)-binding proteins, participates in intracellular trafficking and organelle structure. In this study, a full-length cDNA of Arf1 (designated EcArf1) was cloned from Exopalaemon carinicauda by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EcArf1 was 1428 bp, which contains an open reading frame (ORF) of 549 bp, encoding a 182 amino-acid polypeptide with the predicted molecular weight of 20.69 kDa and estimated isoelectric point was 7.24. Sequence analysis revealed that the conserved Arf protein family signatures were identified in EcArf1. The deduced amino acid sequence of EcArf1 shared high identity (95%-98%) with that of other species and clustered together with Arf1 of other shrimp in the NJ phylogenetic tree, indicating that EcArf1 should be a member of the Arf1 family. Quantitative real-time RT-qPCR analysis indicated that EcArf1 was expressed in hemocytes, hepatopancreas, gills, muscle, ovary, intestine, stomach and heart, and the most abundant level was in hemocytes and gills, which were also the two main target tissues of pathogen infection and environmental stress. After Vibrio parahaemolyticus challenge, EcArf1 transcripts level significantly increased in hemocytes and hepatopancreas at 3 h and 6 h, respectively. The expression of EcArf1 in hemocytes and hepatopancreas significantly up-regulated at 12 h and 6 h respectively, and down-regulated at 72 h and 48 h, respectively. EcArf1 expression in hepatopancreas and gills both significantly increased at 6 h and decreased at 24 h under ammonia-N stress. The results suggested that EcArf1 might be involved in immune responses to pathogens (V. parahaemolyticus and WSSV) challenge and ammonia-N stress in E. carinicauda. PMID:27231192

  4. Characterization of ADP ribosylation factor 1 gene from Exopalaemon carinicauda and its immune response to pathogens challenge and ammonia-N stress.

    PubMed

    Duan, Yafei; Li, Jian; Zhang, Zhe; Li, Jitao; Liu, Ping

    2016-08-01

    ADP ribosylation factors (Arf), as highly conserved small guanosine triphosphate (GTP)-binding proteins, participates in intracellular trafficking and organelle structure. In this study, a full-length cDNA of Arf1 (designated EcArf1) was cloned from Exopalaemon carinicauda by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EcArf1 was 1428 bp, which contains an open reading frame (ORF) of 549 bp, encoding a 182 amino-acid polypeptide with the predicted molecular weight of 20.69 kDa and estimated isoelectric point was 7.24. Sequence analysis revealed that the conserved Arf protein family signatures were identified in EcArf1. The deduced amino acid sequence of EcArf1 shared high identity (95%-98%) with that of other species and clustered together with Arf1 of other shrimp in the NJ phylogenetic tree, indicating that EcArf1 should be a member of the Arf1 family. Quantitative real-time RT-qPCR analysis indicated that EcArf1 was expressed in hemocytes, hepatopancreas, gills, muscle, ovary, intestine, stomach and heart, and the most abundant level was in hemocytes and gills, which were also the two main target tissues of pathogen infection and environmental stress. After Vibrio parahaemolyticus challenge, EcArf1 transcripts level significantly increased in hemocytes and hepatopancreas at 3 h and 6 h, respectively. The expression of EcArf1 in hemocytes and hepatopancreas significantly up-regulated at 12 h and 6 h respectively, and down-regulated at 72 h and 48 h, respectively. EcArf1 expression in hepatopancreas and gills both significantly increased at 6 h and decreased at 24 h under ammonia-N stress. The results suggested that EcArf1 might be involved in immune responses to pathogens (V. parahaemolyticus and WSSV) challenge and ammonia-N stress in E. carinicauda.

  5. Adenosine Diphosphate (ADP)-Ribosylation of the Guanosine Triphosphatase (GTPase) Rho in Resting Peripheral Blood Human T Lymphocytes Results in Pseudopodial Extension and the Inhibition of  T Cell Activation

    PubMed Central

    Woodside, Darren G.; Wooten, David K.; McIntyre, Bradley W.

    1998-01-01

    Scrape loading Clostridium botulinum C3 exoenzyme into primary peripheral blood human T lymphocytes (PB T cells) efficiently adenosine diphosphate (ADP)-ribosylates and thus inactivates the guanosine triphosphatase (GTPase) Rho. Basal adhesion of PB T cells to the β1 integrin substrate fibronectin (Fn) was not inhibited by inactivation of Rho, nor was upregulation of adhesion using phorbol myristate acetate (PMA; 10 ng/ml) or Mn++ (1 mM) affected. Whereas untreated PB T cells adherent to Fn remain spherical, C3-treated PB T cells extend F-actin–containing pseudopodia. Inactivation of Rho delayed the kinetics of PMA-dependent PB T cell homotypic aggregation, a process involving integrin αLβ2. Although C3 treatment of PB T cells did not prevent adhesion to the β1 integrin substrate Fn, it did inhibit β1 integrin/CD3-mediated costimulation of proliferation. Analysis of intracellular cytokine production at the single cell level demonstrated that ADP-ribosylation of Rho inhibited β1 integrin/ CD3 and CD28/CD3 costimulation of IL-2 production within 6 h of activation. Strikingly, IL-2 production induced by PMA and ionomycin was unaffected by C3 treatment. Thus, the GTPase Rho is a novel regulator of T lymphocyte cytoarchitecture, and functional Rho is required for very early events regulating costimulation of IL-2 production in PB T cells. PMID:9763600

  6. Hemin-dependent induction and internalization of CD38 in K562 cells.

    PubMed

    Yalcintepe, Leman; Ercelen, Sebnem; Adin-Cinar, Suzan; Badur, Selim; Tiryaki, Demir; Bermek, Engin

    2003-10-01

    The cell surface antigen, CD38, is a bifunctional ecto-enzyme, which is predominantly expressed on hematopoietic cells during differentiation. In the present study, it is shown that hemin treatment of K562 cells gives rise to induction of enzymatic activities inherent to CD38. GDP-ribosyl cyclase activity, an indicator of CD38, increased initially in response to hemin in a time-dependent manner, reached a maximum level on the 5th day and, thereafter, declined sharply to the initial level. The increase in NAD(+) glycohydrolase and ADP-ribose uptake activities followed a similar time course. However, the decline in the latter activities after the 5th day of induction appeared to be rather slow in contrast to GDP-ribosyl cyclase activity. The time course of these changes was well correlated with the FACScan findings obtained by use of anti-CD38 monoclonal antibody. SDS-PAGE and Western blot analyses by use of the monoclonal antibody OKT10 revealed a transient hemin-dependent appearance of a 43 kDa membrane protein with maximum signal intensity on the first 4 days of incubation. There was subsequently a gradual decrease on the 5th day, concomitant with a reciprocal increase in activity of the internalized protein fraction. The results together indicated that hemin-induced expression of CD38 was followed by its down-regulation.

  7. Human serotonin1B receptor expression in Sf9 cells: phosphorylation, palmitoylation, and adenylyl cyclase inhibition.

    PubMed

    Ng, G Y; George, S R; Zastawny, R L; Caron, M; Bouvier, M; Dennis, M; O'Dowd, B F

    1993-11-01

    Analysis of the primary protein structure of the human serotonin1B (5-HT1B) receptor reveals consensus sites for phosphorylation and a putative site for palmitoylation. To investigate these posttranslational modifications, we have expressed a c-myc epitope-tagged 5-HT1B (m5-HT1B) receptor in Sf9 cells. This strategy enabled receptors to be detected by immunoblot analysis and purified by immunoprecipitation using a monoclonal antibody, 9E10, specific for the c-myc epitope. Agonist radioligand [3H]5-HT binding studies showed that the expressed 5-HT1B and m5-HT1B receptors displayed the characteristic pharmacological profile of the neuronal 5-HT1B receptor. The expressed receptors displayed both high- and low-affinity states for [3H]5-HT, suggesting that the receptors were coupled to endogenous G-proteins. Indeed, agonist binding to the high-affinity receptor state was regulated in the presence of GTP gamma S, Gpp(NH)p, and pertussis toxin. [32P]ADP-ribosylation experiments identified a major approximately 41-kDa ADP-ribosylated protein present in Sf9 membranes that comigrated with partially purified bovine brain Gi alpha/G(o) alpha subunits. Measurements of adenylyl cyclase activity in membranes from cells expressing m5-HT1B receptors showed that serotonergic agonists mediated the inhibition of adenylyl cyclase activity with a rank order of potency comparable to their affinity constants. Immunoblot analysis of membranes prepared from cells expressing m5-HT1B receptors and photoaffinity labeling of the immunoprecipitated material revealed photolabeled species at approximately 95 and at approximately 42 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. Impaired learning and memory in CD38 null mutant mice.

    PubMed

    Kim, Somi; Kim, TaeHyun; Lee, Hye-Ryeon; Jang, Eun-Hye; Ryu, Hyun-Hee; Kang, Minkyung; Rah, So-Young; Yoo, Juyoun; Lee, Bolam; Kim, Jae-Ick; Lim, Chae Seok; Kim, Sang Jeong; Kim, Uh-Hyun; Lee, Yong-Seok; Kaang, Bong-Kiun

    2016-02-09

    CD38 is an enzyme that catalyzes the formation of cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate, both of which are involved in the mobilization of Ca(2+) from intracellular stores. Recently, CD38 has been shown to regulate oxytocin release from hypothalamic neurons. Importantly, CD38 mutations are associated with autism spectrum disorders (ASD) and CD38 knockout (CD38(-/-)) mice display ASD-like behavioral phenotypes including deficient parental behavior and poor social recognition memory. Although ASD and learning deficits commonly co-occur, the role of CD38 in learning and memory has not been investigated. We report that CD38(-/-) mice show deficits in various learning and memory tasks such as the Morris water maze, contextual fear conditioning, and the object recognition test. However, either long-term potentiation or long-term depression is not impaired in the hippocampus of CD38(-/-) mice. Our results provide convincing evidence that CD38(-/-) mice show deficits in various learning and memory tasks including spatial and non-spatial memory tasks. Our data demonstrate that CD38 is critical for regulating hippocampus-dependent learning and memory without modulating synaptic plasticity.

  9. Immunochemical analysis of poly(ADP-ribosyl)ation in HaCaT keratinocytes induced by the mono-alkylating agent 2-chloroethyl ethyl sulfide (CEES): Impact of experimental conditions.

    PubMed

    Debiak, Malgorzata; Lex, Kirsten; Ponath, Viviane; Burckhardt-Boer, Waltraud; Thiermann, Horst; Steinritz, Dirk; Schmidt, Annette; Mangerich, Aswin; Bürkle, Alexander

    2016-02-26

    Sulfur mustard (SM) is a bifunctional alkylating agent with a long history of use as a chemical weapon. Although its last military use is dated for the eighties of the last century, a potential use in terroristic attacks against civilians remains a significant threat. Thus, improving medical therapy of mustard exposed individuals is still of particular interest. PARP inhibitors were recently brought into the focus as a potential countermeasure for mustard-induced pathologies, supported by the availability of efficient compounds successfully tested in cancer therapy. PARP activation after SM treatment was reported in several cell types and tissues under various conditions; however, a detailed characterization of this phenomenon is still missing. This study provides the basis for such studies by developing and optimizing experimental conditions to investigate poly(ADP-ribosyl)ation (PARylation) in HaCaT keratinocytes upon treatment with the monofunctional alkylating agent 2-chloroethyl ethyl sulfide ("half mustard", CEES). By using an immunofluorescence-based approach, we show that optimization of experimental conditions with regards to the type of solvent, dilution factors and treatment procedure is essential to obtain a homogenous PAR staining in HaCaT cell cultures. Furthermore, we demonstrate that different CEES treatment protocols significantly influence the cytotoxicity profiles of treated cells. Using an optimized treatment protocol, our data reveals that CEES induces a dose- and time-dependent dynamic PARylation response in HaCaT cells that could be completely blocked by treating cells with the clinically relevant pharmacological PARP inhibitor ABT888 (also known as veliparib). Finally, siRNA experiments show that CEES-induced PAR formation is predominantly due to the activation of PARP1. In conclusion, this study provides a detailed analysis of the CEES-induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to study the

  10. Ligation of CD38 suppresses human B lymphopoiesis

    PubMed Central

    1995-01-01

    CD38 is a transmembrane glycoprotein expressed in many cell types, including lymphoid progenitors and activated lymphocytes. High levels of CD38 expression on immature lymphoid cells suggest its role in the regulation of cell growth and differentiation, but there is no evidence demonstrating a functional activity of CD38 on these cells. We used stroma-supported cultures of B cell progenitors and anti-CD38 monoclonal antibodies (T16 and IB4) to study CD38 function. In cultures of normal bone marrow CD19+ cells (n = 5), addition of anti-CD38 markedly reduced the number of cells recovered after 7 d. Cell loss was greatest among CD19+ sIg- B cell progenitors (mean cell recovery +/- SD = 7.2 +/- 11.7% of recovery in control cultures) and extended to CD19+CD34+ B cells (the most immature subset; 7.6 +/- 2.2%). In contrast, CD38 ligation did not substantially affect cell numbers in cultures of normal peripheral blood or tonsillar B cells. In stroma- supported cultures of 22 B-lineage acute lymphoblastic leukemia cases, anti-CD38 suppressed recovery of CD19+ sIg- leukemic cells. CD38 ligation also suppressed the growth of immature lymphoid cell lines cultured on stroma and, in some cases, in the presence of stroma- derived cytokines (interleukin [IL] 7, IL-3, and/or stem cell factor), but did not inhibit growth in stroma- or cytokine-free cultures. DNA content and DNA fragmentation studies showed that CD38 ligation of stroma-supported cells resulted in both inhibition of DNA synthesis and induction of apoptosis. It is known that CD38 catalyzes nicotinamide adenine dinucleotide (NAD+) hydrolysis into cyclic ADP-ribose (cADPR) and ADPR. However, no changes in NAD+ hydrolysis or cADPR and ADPR production after CD38 ligation were found by high-performance liquid chromatography; addition of NAD+, ADPR, or cADPR to cultures of lymphoid progenitors did not offset the inhibitory effects of anti- CD38. Thus, anti-CD38 does not suppress B lymphopoiesis by altering the enzymatic

  11. Immuno-targeting the multifunctional CD38 using nanobody

    PubMed Central

    Li, Ting; Qi, Shali; Unger, Mandy; Hou, Yun Nan; Deng, Qi Wen; Liu, Jun; Lam, Connie M. C.; Wang, Xian Wang; Xin, Du; Zhang, Peng; Koch-Nolte, Friedrich; Hao, Quan; Zhang, Hongmin; Lee, Hon Cheung; Zhao, Yong Juan

    2016-01-01

    CD38, as a cell surface antigen is highly expressed in several hematologic malignancies including multiple myeloma (MM) and has been proven to be a good target for immunotherapy of the disease. CD38 is also a signaling enzyme responsible for the metabolism of two novel calcium messenger molecules. To be able to target this multifunctional protein, we generated a series of nanobodies against CD38 with high affinities. Crystal structures of the complexes of CD38 with the nanobodies were solved, identifying three separate epitopes on the carboxyl domain. Chromobodies, engineered by tagging the nanobody with fluorescence proteins, provide fast, simple and versatile tools for quantifying CD38 expression. Results confirmed that CD38 was highly expressed in malignant MM cells compared with normal white blood cells. The immunotoxin constructed by splicing the nanobody with a bacterial toxin, PE38 shows highly selective cytotoxicity against patient-derived MM cells as well as the cell lines, with half maximal effective concentration reaching as low as 10−11 molar. The effectiveness of the immunotoxin can be further increased by stimulating CD38 expression using retinoid acid. These results set the stage for the development of clinical therapeutics as well as diagnostic screening for myeloma. PMID:27251573

  12. Immuno-targeting the multifunctional CD38 using nanobody.

    PubMed

    Li, Ting; Qi, Shali; Unger, Mandy; Hou, Yun Nan; Deng, Qi Wen; Liu, Jun; Lam, Connie M C; Wang, Xian Wang; Xin, Du; Zhang, Peng; Koch-Nolte, Friedrich; Hao, Quan; Zhang, Hongmin; Lee, Hon Cheung; Zhao, Yong Juan

    2016-01-01

    CD38, as a cell surface antigen is highly expressed in several hematologic malignancies including multiple myeloma (MM) and has been proven to be a good target for immunotherapy of the disease. CD38 is also a signaling enzyme responsible for the metabolism of two novel calcium messenger molecules. To be able to target this multifunctional protein, we generated a series of nanobodies against CD38 with high affinities. Crystal structures of the complexes of CD38 with the nanobodies were solved, identifying three separate epitopes on the carboxyl domain. Chromobodies, engineered by tagging the nanobody with fluorescence proteins, provide fast, simple and versatile tools for quantifying CD38 expression. Results confirmed that CD38 was highly expressed in malignant MM cells compared with normal white blood cells. The immunotoxin constructed by splicing the nanobody with a bacterial toxin, PE38 shows highly selective cytotoxicity against patient-derived MM cells as well as the cell lines, with half maximal effective concentration reaching as low as 10(-11) molar. The effectiveness of the immunotoxin can be further increased by stimulating CD38 expression using retinoid acid. These results set the stage for the development of clinical therapeutics as well as diagnostic screening for myeloma. PMID:27251573

  13. Seminal CD38 is a pivotal regulator for fetomaternal tolerance.

    PubMed

    Kim, Byung-Ju; Choi, Yun-Min; Rah, So-Young; Park, Dae-Ryoung; Park, Seon-Ah; Chung, Yun-Jo; Park, Seung-Moon; Park, Jong Kwan; Jang, Kyu Yun; Kim, Uh-Hyun

    2015-02-01

    A successful pregnancy depends on a complex process that establishes fetomaternal tolerance. Seminal plasma is known to induce maternal immune tolerance to paternal alloantigens, but the seminal factors that regulate maternal immunity have yet to be characterized. Here, we show that a soluble form of CD38 (sCD38) released from seminal vesicles to the seminal plasma plays a crucial role in inducing tolerogenic dendritic cells and CD4(+) forkhead box P3(+) (Foxp3(+)) regulatory T cells (Tregs), thereby enhancing maternal immune tolerance and protecting the semiallogeneic fetus from resorption. The abortion rate in BALB/c females mated with C57BL/6 Cd38(-/-) males was high compared with that in females mated with Cd38(+/+) males, and this was associated with a reduced proportion of Tregs within the CD4(+) T-cell pool. Direct intravaginal injection of sCD38 to CBA/J pregnant mice at preimplantation increased Tregs and pregnancy rates in mice under abortive sonic stress from 48 h after mating until euthanasia. Thus, sCD38 released from seminal vesicles to the seminal plasma acts as an immunoregulatory factor to protect semiallogeneic fetuses from maternal immune responses.

  14. Computational characterization for catalytic activities of human CD38's wild type, E226 and E146 mutants.

    PubMed

    Nguyen, My H; Dang, Van U; Luu, Boi V

    2010-06-01

    A series of the complexes of human CD38's wild type, E226 and E146 mutants as well have been simulated. The biosoftwares well simulate the penetration of nicotinamide-adenine-dinucleotide (NAD) into the active site. The nicotinamide end of NAD penetrates deep into the active site consistent with cleavage of the nicotinamide-glycosidic bond which is the first step of catalysis creating a Michaelis complex regarded as the intermediate product of NAD cyclase and hydrolysis reaction. The breaking down hydrogen bond between 2'-3' OH ribosyl and the residues replaced Glu(226) makes NAD to be less constrained in active site and nicotinamide (NA) becomes more difficult to be cleaved and eliminates the mutant catalytic activities. The large majority of the substrate NAD is hydrolyzed to ADPR while the conversion of NAD to cADPR is not the dominant reaction catalyzed by wild-type human CD38. The more strongly kept ribosyl group by hydrogen bonds the more NADase and the less cyclase activity. Breaking hydrogen bonds of ribosyl 2'- and 3'-OH by mutation will loosen it to promote the cyclase. The cyclic adenosine diphosphate-ribose (cADPR) could also penetrate deeply into active site to make some hydrogen bonds with Glu(146) and Glu(226); however, its docking poses are affected by a residue located at the entrance of the catalytic pocket (Lys(129)). These results are in good agreement with the previous crystallographic analysis and the experiments quantified the catalytic activities of human CD38 and its mutants.

  15. Structure and functional regulation of the CD38 promoter

    SciTech Connect

    Sun Li; Iqbal, Jameel; Zaidi, Samir; Zhu Linglng; Zhang Xuefeng; Peng Yuanzheng; Moonga, Baljit S.; Zaidi, Mone . E-mail: mone.zaidi@mssm.edu

    2006-03-17

    CD38 has multiple roles in biology, including T lymphocyte signaling, neutrophil migration, neurotransmission, cell proliferation, apoptosis, and bone remodeling. To study the transcriptional control of the CD38 gene, we cloned a putative 1.8 kb promoter fragment from a rabbit genomic DNA library. Primer extension analysis indicated two transcription start sites consistent with the absence of a TATA box. Sequence analysis revealed several AP-1, AP-4, myo-D, GATA, and SP-1 sequences. MC3T3.E1 (osteoblast) or RAW-C3 (osteoclast precursor macrophage) cells were then transfected with the CD38 promoter or its deletion fragments ligated to the luciferase reporter gene. Except for the shortest 41 bp fragment, all fragments showed significant luciferase activity. There was a marked stimulation of basal activity in the 93 bp fragment that contained a GC box and SP-1 site. Furthermore, there were significant differences in the activity of the fragments in MC3T3.E1 and RAW-C3 cells. Intracellular Ca{sup 2+} elevations by ionomycin (10 {mu}M) in MC3T3.E1 cells inhibited promoter activity, except in the short 41 bp. In contrast, cAMP elevation by exposure to forskolin (100 {mu}M) inhibited activation of all fragments, except the 0.6 and 1.2 kb fragments. Finally, TNF-{alpha} stimulated promoter activity in RAW-C3 cells transfected with the 93 bp and 1.0 kb fragments, consistent with the stimulation of CD38 mRNA by TNF-{alpha}. Physiologically, therefore, modulation of the expression of the NAD{sup +}-sensing enzyme, CD38, by Ca{sup 2+}, cAMP, and cytokines, such as TNF-{alpha} may contribute to coupling the intense metabolic activity of osteoclasts and osteoblasts to their respective bone-resorbing and bone-forming functions.

  16. Ontogeny of fetal adenylate cyclase; mechanisms for regulation of beta-adrenergic receptors.

    PubMed

    Maier, J A; Roberts, J M; Jacobs, M M

    1989-11-01

    Transmembrane second messenger signalling systems regulate differentiation, growth and homeostatic responses during fetal development. The beta-adrenergic adenylate cyclase system is the best studied of these and has been used as a model to investigate the control of developmental processes. In tissues such as lung, heart and parotid, beta-adrenergic responsiveness of adenylate cyclase increases during development. In the developing fetal lung beta-receptor concentration increases during gestation or after glucocorticoid treatment, but cannot fully explain enhanced adrenergic responsiveness. To probe developmental and hormonal effects on beta-receptor function, we asked if advancing gestation or glucocorticoid treatment alters beta-receptor-Gs interactions in fetal rabbit lung membrane particulates. Before 25 days gestation, 1-isoproterenol competes for 3H-dihydroalprenolol (DHA), a radiolabelled beta-antagonist, with a single low affinity, later in gestation, high and low affinities of isoproterenol for the beta-receptor are present which can be shifted to the lower affinity by addition of guanyl nucleotide. High affinity binding is precociously induced in 25 days--fetal lung particulates as early as 3 h after maternal betamethasone treatment, but beta-adrenoreceptor concentration in treated fetuses was increased over controls only after 24 h of treatment. Cholera toxin catalyzed ADP ribosylation of membrane particulates showed cholera toxin substrate (Gs) was not altered by glucocorticoid treatment. Stimulation of adenylate cyclase activity with isoproterenol (100mM) and GTP (100mM) resulted in no incremental increase over that produced by GTP (100mM) alone in glucocorticoid treated or control particulates, either early or late in gestation. These data demonstrate that beta-receptor-Gs interactions are not sufficient to produce full agonist responses. Although both beta-adrenergic receptors and Gs are present in fetal rabbit lung early in gestation, interaction

  17. Yeast Golgi-localized, γ-Ear–containing, ADP-Ribosylation Factor-binding Proteins Are but Adaptor Protein-1 Is Not Required for Cell-free Transport of Membrane Proteins from the Trans-Golgi Network to the Prevacuolar Compartment

    PubMed Central

    Abazeed, Mohamed E.

    2008-01-01

    Golgi-localized, γ-Ear–containing, ADP-ribosylation factor-binding proteins (GGAs) and adaptor protein-1 (AP-1) mediate clathrin-dependent trafficking of transmembrane proteins between the trans-Golgi network (TGN) and endosomes. In yeast, the vacuolar sorting receptor Vps10p follows a direct pathway from the TGN to the late endosome/prevacuolar compartment (PVC), whereas, the processing protease Kex2p partitions between the direct pathway and an indirect pathway through the early endosome. To examine the roles of the Ggas and AP-1 in TGN–PVC transport, we used a cell-free assay that measures delivery to the PVC of either Kex2p or a chimeric protein (K-V), in which the Vps10p cytosolic tail replaces the Kex2p tail. Either antibody inhibition or dominant-negative Gga2p completely blocked K-V transport but only partially blocked Kex2p transport. Deletion of APL2, encoding the β subunit of AP-1, did not affect K-V transport but partially blocked Kex2p transport. Residual Kex2p transport seen with apl2Δ membranes was insensitive to dominant-negative Gga2p, suggesting that the apl2Δ mutation causes Kex2p to localize to a compartment that precludes Gga-dependent trafficking. These results suggest that yeast Ggas facilitate the specific and direct delivery of Vps10p and Kex2p from the TGN to the PVC and that AP-1 modulates Kex2p trafficking through a distinct pathway, presumably involving the early endosome. PMID:18784256

  18. Ectocellular in vitro and in vivo metabolism of cADP-ribose in cerebellum.

    PubMed Central

    De Flora, A; Guida, L; Franco, L; Zocchi, E; Pestarino, M; Usai, C; Marchetti, C; Fedele, E; Fontana, G; Raiteri, M

    1996-01-01

    CD38, a type II transmembrane glycoprotein predominantly expressed in blood cells, is a bifunctional ectoenzyme directly involved in the metabolism of cADP-ribose (cADPR). This is a potent Ca2+ mobilizer in several types of cells. The relationship between the ectocellular site of cADPR production and its intracellular calcium-related functions is poorly understood. Cultured rat cerebellar granule cells showed both enzymic activities of CD38, ADP-ribosyl cyclase and cADPR hydrolase, at a ratio of 16 to 1 respectively, and were immunostained by the anti-(human CD38) monoclonal antibody IB4. In these cells externally added cADPR and beta-NAD+ (the precursor of cADPR), but not alpha-NAD+ or ADP-ribose, enhanced the peak of the depolarization-induced rise in intracellular Ca2+ concentration. This effect was inhibited by 1 microM ryanodine, suggesting a potentiation of calcium-induced calcium release by cADPR. CD38 ectoenzyme activities, ADP-ribosyl cyclase and cADPR hydrolase, were also demonstrated in vivo by microdialysis of adult rat cerebellum, where IB4 bound to granule neurons selectively. Trace amounts (11.5 +/- 3.8 nM) of NAD+ were detected by microdialysis sampling and sensitive assays in the basal interstitial fluid of the cerebellum. These results provide a link between ectocellular cADPR turnover and intracellular calcium mobilization in cerebellum. PMID:8973582

  19. Determinants of the membrane orientation of a calcium signaling enzyme CD38.

    PubMed

    Zhao, Yong Juan; Zhu, Wen Jie; Wang, Xian Wang; Zhang, Li-He; Lee, Hon Cheung

    2015-09-01

    CD38 catalyzes the synthesis of two structurally distinct messengers for Ca²⁺-mobilization, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), from cytosolic substrates, NAD and NADP, respectively. CD38 is generally thought of as a type II membrane protein with its catalytic site facing outside. We recently showed that CD38 exists, instead, in two opposite membrane orientations. The determinant for the membrane topology is unknown. Here, specific antibodies against type III CD38 were designed and produced. We show that mutating the positively charged residues in the N-terminal tail of CD38 converted its orientation to type III, with the catalytic domain facing the cytosol and it was fully active in producing intracellular cADPR. Changing the serine residues to aspartate, which is functionally equivalent to phosphorylation, had a similar effect. The mutated CD38 was expressed intracellularly and was un-glycosylated. The membrane topology could also be modulated by changing the highly conserved di-cysteine. The results indicate that the net charge of the N-terminal segment is important in determining the membrane topology of CD38 and that the type III orientation can be a functional form of CD38 for Ca²⁺-signaling. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

  20. CD38 is expressed on inflammatory cells of the intestine and promotes intestinal inflammation.

    PubMed

    Schneider, Michael; Schumacher, Valéa; Lischke, Timo; Lücke, Karsten; Meyer-Schwesinger, Catherine; Velden, Joachim; Koch-Nolte, Friedrich; Mittrücker, Hans-Willi

    2015-01-01

    The enzyme CD38 is expressed on a variety of hematopoietic and non-hematopoietic cells and is involved in diverse processes such as generation of calcium-mobilizing metabolites, cell activation, and chemotaxis. Here, we show that under homeostatic conditions CD38 is highly expressed on immune cells of the colon mucosa of C57BL/6 mice. Myeloid cells recruited to this tissue upon inflammation also express enhanced levels of CD38. To determine the role of CD38 in intestinal inflammation, we applied the dextran sulfate sodium (DSS) colitis model. Whereas wild-type mice developed severe colitis, CD38-/- mice had only mild disease following DSS-treatment. Histologic examination of the colon mucosa revealed pronounced inflammatory damage with dense infiltrates containing numerous granulocytes and macrophages in wild-type animals, while these findings were significantly attenuated in CD38-/- mice. Despite attenuated histological findings, the mRNA expression of inflammatory cytokines and chemokines was only marginally lower in the colons of CD38-/- mice as compared to wild-type mice. In conclusion, our results identify a function for CD38 in the control of inflammatory processes in the colon.

  1. Contribution of NADPH Oxidase to Membrane CD38 Internalization and Activation in Coronary Arterial Myocytes

    PubMed Central

    Xu, Ming; Li, Xiao-Xue; Ritter, Joseph K.; Abais, Justine M.; Zhang, Yang; Li, Pin-Lan

    2013-01-01

    The CD38-ADP-ribosylcyclase-mediated Ca2+ signaling pathway importantly contributes to the vasomotor response in different arteries. Although there is evidence indicating that the activation of CD38-ADP-ribosylcyclase is associated with CD38 internalization, the molecular mechanism mediating CD38 internalization and consequent activation in response to a variety of physiological and pathological stimuli remains poorly understood. Recent studies have shown that CD38 may sense redox signals and is thereby activated to produce cellular response and that the NADPH oxidase isoform, NOX1, is a major resource to produce superoxide (O2·−) in coronary arterial myocytes (CAMs) in response to muscarinic receptor agonist, which uses CD38-ADP-ribosylcyclase signaling pathway to exert its action in these CAMs. These findings led us hypothesize that NOX1-derived O2·− serves in an autocrine fashion to enhance CD38 internalization, leading to redox activation of CD38-ADP-ribosylcyclase activity in mouse CAMs. To test this hypothesis, confocal microscopy, flow cytometry and a membrane protein biotinylation assay were used in the present study. We first demonstrated that CD38 internalization induced by endothelin-1 (ET-1) was inhibited by silencing of NOX1 gene, but not NOX4 gene. Correspondingly, NOX1 gene silencing abolished ET-1-induced O2·− production and increased CD38-ADP-ribosylcyclase activity in CAMs, while activation of NOX1 by overexpression of Rac1 or Vav2 or administration of exogenous O2·− significantly increased CD38 internalization in CAMs. Lastly, ET-1 was found to markedly increase membrane raft clustering as shown by increased colocalization of cholera toxin-B with CD38 and NOX1. Taken together, these results provide direct evidence that Rac1-NOX1-dependent O2·− production mediates CD38 internalization in CAMs, which may represent an important mechanism linking receptor activation with CD38 activity in these cells. PMID:23940720

  2. Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

    SciTech Connect

    Fahrer, Joerg; Wagner, Silvia; Buerkle, Alexander; Koenigsrainer, Alfred

    2009-08-14

    Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.

  3. Anti-CD38 autoimmunity in patients with chronic autoimmune thyroiditis or Graves' disease

    PubMed Central

    Antonelli, A; Fallahi, P; Nesti, C; Pupilli, C; Marchetti, P; Takasawa, S; Okamoto, H; Ferrannini, E

    2001-01-01

    Autoantibodies directed against human CD38 (an enzyme catalysing the interconversion of NAD+ and cyclic ADP-ribose) have been demonstrated recently in patients with type 2 diabetes. We tested 220 consecutive Caucasian patients with autoimmune chronic thyroiditis, 104 patients with Graves' disease, 220 subjects from the general population (control I) and 78 healthy control subjects not affected by thyroid autoimmune disorders (control II) for the presence of anti-CD38 autoimmunity. Using Western blot analysis and optical densitometry, a specific band corresponding to human recombinant CD38 was identified in the serum of several subjects. By defining anti-CD38 positivity as a standardized optical reading >3 s.d. higher than the mean value of control I, 10·4% of patients with thyroiditis and 7·7% of Graves' patients were anti-CD38 positive (P = 0·0009 versus 1·8% of control I). Similarly, 13·1% of patients with thyroiditis and 10·5% of Graves' patients had a standardized optical reading >3 s.d. higher than the mean value of the subjects not affected by thyroid autoimmune disorders (P = 0·002 versus 1·2% of control II). Anti-CD38 autoimmunity did not differ between euthyroid, hyperthyroid or hypothyroid patients or between patients with or without thyroid hypoechogenicity. Anti-CD38 autoantibodies were associated with higher levels of circulating antithyroid-peroxidase antibodies (P = 0·03) and they were more frequent in Graves' patients with ophthalmopathy (P < 0·05). Anti-CD38 autoantibodies are a new autoimmune marker in chronic autoimmune thyroiditis and Graves' disease. The specific role of CD38 and its autoantibodies in the modulation of thyroid cell function or growth remains to be investigated. PMID:11737057

  4. CD38+CD8+ and CD38+CD4+ T Cells and IFN Gamma (+874) Polymorphism Are Associated with a Poor Virological Outcome.

    PubMed

    de Carvalho, Paulo Germano; de Oliveira Rodrigues, Raphael; Ribeiro da Silva, Silvia Fernandes; Ribeiro, Ilana Farias; de Miranda Lucena, Herene Barros; Martins, Lilian Roberta Costa; Rabenhorst, Silvia Helena; de Arruda, Érico Antônio Gomes; Nagao-Dias, Aparecida Tiemi

    2016-05-01

    The main objective of the work was to evaluate the use of CD38 on T lymphocytes, IFNγ (+874 A/T), and IL-10 (-1082 A/G) polymorphisms in HIV-infected patients under antiretroviral (ARV) therapy. Sixty-one patients were selected at the outpatient clinic for HIV infection at the Hospital São José de Doenças Infecciosas, Fortaleza, Ceará, Brazil. The patients were classified into two groups, according to viral load after one year of ARV therapy. In the aviremic group (group I), a reduction of 35.5% of CD38+CD4+ T cells was observed (p = 0.02) and 49.3% of CD38+CD8+ T cells (p = 0.001). In the viremic group (group II), a reduction of 37.2% of CD38+CD4+ T cells (p = 0.067), and 21.4% of CD38+CD8+ T cells (p = 0.60) occurred. No association was found between IL-10 (-1082) polymorphism and the type of response to ARV therapy. Regarding the gene polymorphism on IFNγ (+874 T/A), 73.34% of group I and 33.3% of group II presented the AA genotype. The relative risk of the individuals carrying AA genotype or the A allele and not being able to suppress the viral load level after one year of ARV therapy was 3.44 (1.25-9.45; p = 0.014) or 2.35 (1.05-5.26; p = 0.027), respectively. Our data suggested that an augmented frequency of activated CD38+CD8+ T cells as well as the presence of the A allele of IFNγ polymorphism could contribute to a reduced virological suppression in patients under antiretroviral therapy.

  5. Genetic modification of primary chronic lymphocytic leukemia cells with a lentivirus expressing CD38

    PubMed Central

    Pearce, Laurence; Morgan, Liam; Lin, Thet Thet; Hewamana, Saman; Matthews, R. James; Deaglio, Silvia; Rowntree, Clare; Fegan, Christopher; Pepper, Christopher; Brennan, Paul

    2010-01-01

    Studies of the role of individual genes in chronic lymphocytic leukemia (CLL) have been hampered by the inability to consistently transfect primary tumor cells. Here, we describe a highly efficient method of genetically modifying primary CLL cells using a VSVG pseudotyped lentiviral vector. We transduced CD38 negative CLL cells with a lentiviral vector encoding CD38 which caused increased surface CD38 expression in all the samples tested (n=17) with no evidence of plasmacytoid differentiation. The mean percentage of positive cells expressing CD38 was 87%±8.5% and the mean cell viability 74%±17%. This high level of transduction of all the CLL cell samples tested demonstrates the utility of this technique which should prove applicable for the introduction and analysis of other genes in these non-dividing cells. PMID:20207849

  6. CD38-mediated Ca(2+) signaling contributes to glucagon-induced hepatic gluconeogenesis.

    PubMed

    Rah, So-Young; Kim, Uh-Hyun

    2015-06-03

    CD38 is a multifunctional enzyme for the synthesis of Ca(2+) second messengers. Glucagon promotes hepatic glucose production through Ca(2+) signaling in the fasting condition. In this study, we investigated the role of CD38 in the glucagon signaling of hepatocytes. Here, we show that glucagon induces cyclic ADP-ribose (cADPR) production and sustained Ca(2+) increases via CD38 in hepatocytes. 8-Br-cADPR, an antagonistic cADPR analog, completely blocked glucagon-induced Ca(2+) increases and phosphorylation of cAMP response element-binding protein (CREB). Moreover, glucagon-induced sustained Ca(2+) signals and translocation of CREB-regulated transcription coactivator 2 to the nucleus were absent and glucagon-induced glucose production and expression of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (Pck1) are remarkably reduced in hepatocytes from CD38(-/-) mice. Furthermore, in the fasting condition, CD38(-/-) mice have decreased blood glucose and hepatic expression of G6Pase and Pck1 compared to wild type mice. Our data suggest that CD38/cADPR-mediated Ca(2+) signals play a key role in glucagon-induced gluconeogenesis in hepatocytes, and that the signal pathway has significant clinical implications in metabolic diseases, including type 2 diabetes.

  7. Catalysis-based inhibitors of the calcium signaling function of CD38.

    PubMed

    Kwong, Anna Ka Yee; Chen, Zhe; Zhang, HongMin; Leung, Fung Ping; Lam, Connie Mo Ching; Ting, Kai Yiu; Zhang, Liangren; Hao, Quan; Zhang, Li-He; Lee, Hon Cheung

    2012-01-10

    CD38 is a signaling enzyme responsible for catalyzing the synthesis of cyclic ADP ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate; both are universal Ca(2+) messenger molecules. Ablation of the CD38 gene in mice causes multiple physiological defects, including impaired oxytocin release, that result in altered social behavior. A series of catalysis-based inhibitors of CD38 were designed and synthesized, starting with arabinosyl-2'-fluoro-2'-deoxynicotinamide mononucleotide. Structure-function relationships were analyzed to assess the structural determinants important for inhibiting the NADase activity of CD38. X-ray crystallography was used to reveal the covalent intermediates that were formed with the catalytic residue, Glu226. Metabolically stable analogues that were resistant to inactivation by phosphatase and esterase were synthesized and shown to be effective in inhibiting intracellular cADPR production in human HL-60 cells during induction of differentiation by retinoic acid. The inhibition was species-independent, and the analogues were similarly effective in blocking the cyclization reaction of CD38 in rat ventricular tissue extracts, as well as inhibiting the α-agonist-induced constriction in rat mesentery arteries. These compounds thus represent the first generally applicable and catalysis-based inhibitors of the Ca(2+) signaling function of CD38.

  8. CD38-mediated Ca2+ signaling contributes to glucagon-induced hepatic gluconeogenesis

    PubMed Central

    Rah, So-Young; Kim, Uh-Hyun

    2015-01-01

    CD38 is a multifunctional enzyme for the synthesis of Ca2+ second messengers. Glucagon promotes hepatic glucose production through Ca2+ signaling in the fasting condition. In this study, we investigated the role of CD38 in the glucagon signaling of hepatocytes. Here, we show that glucagon induces cyclic ADP-ribose (cADPR) production and sustained Ca2+ increases via CD38 in hepatocytes. 8-Br-cADPR, an antagonistic cADPR analog, completely blocked glucagon-induced Ca2+ increases and phosphorylation of cAMP response element-binding protein (CREB). Moreover, glucagon-induced sustained Ca2+ signals and translocation of CREB-regulated transcription coactivator 2 to the nucleus were absent and glucagon-induced glucose production and expression of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (Pck1) are remarkably reduced in hepatocytes from CD38−/− mice. Furthermore, in the fasting condition, CD38−/− mice have decreased blood glucose and hepatic expression of G6Pase and Pck1 compared to wild type mice. Our data suggest that CD38/cADPR-mediated Ca2+ signals play a key role in glucagon-induced gluconeogenesis in hepatocytes, and that the signal pathway has significant clinical implications in metabolic diseases, including type 2 diabetes. PMID:26038839

  9. Mobilization of CD34+CD38- hematopoietic stem cells after priming in acute myeloid leukemia

    PubMed Central

    Plesa, Adriana; Chelghoum, Youcef; Mattei, Eve; Labussière, Hélène; Elhamri, Mohamed; Cannas, Giovanna; Morisset, Stéphane; Tagoug, Inès; Michallet, Mauricette; Dumontet, Charles; Thomas, Xavier

    2013-01-01

    AIM: To evaluate quantitatively and qualitatively the different CD34+ cell subsets after priming by chemotherapy granulocyte colony-stimulating factor (± G-CSF) in patients with acute myeloid leukemia. METHODS: Peripheral blood and bone marrow samples were harvested in 8 acute myeloid leukemia patients during and after induction chemotherapy. The CD34/CD38 cell profile was analyzed by multi-parameter flow cytometry. Adhesion profile was made using CXC chemokine receptor 4 (CXCR4) (CD184), VLA-4 (CD49d/CD29) and CD47. RESULTS: Chemotherapy ± G-CSF mobilized immature cells (CD34+CD38− population), while the more mature cells (CD34+CD38low and CD34+CD38+ populations) decreased progressively after treatment. Circulating CD34+ cells tended to be more sensitive to chemotherapy after priming with G-CSF. CD34+ cell mobilization was correlated with a gradual increase in CXCR4 and CD47 expression, suggesting a role in cell protection and the capacity of homing back to the marrow. CONCLUSION: Chemotherapy ± G-CSF mobilizes into the circulation CD34+ bone marrow cells, of which, the immature CD34+CD38– cell population. Further manipulations of these interactions may be a means with which to control the trafficking of leukemia stem cells to improve patients’ outcomes. PMID:24179607

  10. Signaling properties of CD38 in the mouse immune system: enzyme-dependent and -independent roles in immunity.

    PubMed

    Lund, Frances E

    2006-01-01

    The 5th international CD38 meeting, held in Torino, Italy, spanned a range of topics from the role of CD38 as a signaling receptor in lymphocytic tumors to the importance of CD38-derived metabolites in NAD(+) metabolism, calcium signaling, and immune function. This meeting was particularly exciting as data were presented demonstrating that collaborative experiments between enzymologists, biochemists, cell biologists, immunologists, and clinicians have started to unravel the secrets of CD38 biology. It is now clear that all of the products of the CD38 enzyme reaction regulate calcium signal transduction in cell types as diverse as sea urchin oocytes and mammalian lymphocytes. It is also apparent that CD38 plays important immunomodulatory role(s), however there is still much debate on how CD38 mediates its immunoregulatory functions and whether the enzymatic products generated by CD38 are important for immunity. The data presented at this meeting have begun to resolve some of these controversies. First, CD38 regulates the function of leukocytes by enzyme-dependent and enzyme-independent mechanisms. Second, CD38 regulates inflammatory responses by modulating the activity of the responding leukocytes and by altering the activity of non-hematopoietic cells in the inflamed tissue. Finally, crosstalk between CD38 and other NAD(+) utilizing enzymes such as ART2, SIRT1, and PARP-1 impacts NAD(+) homeostasis, inflammation, and immunity. Thus, immunity is regulated by CD38 in multiple and unexpected ways and the new research challenge will be to determine whether we can exploit the complex biology of CD38 to therapeutically regulate the immune system.

  11. Genetic Ablation of CD38 Protects against Western Diet-Induced Exercise Intolerance and Metabolic Inflexibility.

    PubMed

    Chiang, Shian-Huey; Harrington, W Wallace; Luo, Guizhen; Milliken, Naphtali O; Ulrich, John C; Chen, Jing; Rajpal, Deepak K; Qian, Ying; Carpenter, Tiffany; Murray, Rusty; Geske, Robert S; Stimpson, Stephen A; Kramer, Henning F; Haffner, Curt D; Becherer, J David; Preugschat, Frank; Billin, Andrew N

    2015-01-01

    Nicotinamide adenine dinucleotide (NAD+) is a key cofactor required for essential metabolic oxidation-reduction reactions. It also regulates various cellular activities, including gene expression, signaling, DNA repair and calcium homeostasis. Intracellular NAD+ levels are tightly regulated and often respond rapidly to nutritional and environmental changes. Numerous studies indicate that elevating NAD+ may be therapeutically beneficial in the context of numerous diseases. However, the role of NAD+ on skeletal muscle exercise performance is poorly understood. CD38, a multi-functional membrane receptor and enzyme, consumes NAD+ to generate products such as cyclic-ADP-ribose. CD38 knockout mice show elevated tissue and blood NAD+ level. Chronic feeding of high-fat, high-sucrose diet to wild type mice leads to exercise intolerance and reduced metabolic flexibility. Loss of CD38 by genetic mutation protects mice from diet-induced metabolic deficit. These animal model results suggest that elevation of tissue NAD+ through genetic ablation of CD38 can profoundly alter energy homeostasis in animals that are maintained on a calorically-excessive Western diet. PMID:26287487

  12. Genetic Ablation of CD38 Protects against Western Diet-Induced Exercise Intolerance and Metabolic Inflexibility

    PubMed Central

    Chiang, Shian-Huey; Harrington, W. Wallace; Luo, Guizhen; Milliken, Naphtali O.; Ulrich, John C.; Chen, Jing; Rajpal, Deepak K.; Qian, Ying; Carpenter, Tiffany; Murray, Rusty; Geske, Robert S.; Stimpson, Stephen A.; Kramer, Henning F.; Haffner, Curt D.; Becherer, J. David; Preugschat, Frank; Billin, Andrew N.

    2015-01-01

    Nicotinamide adenine dinucleotide (NAD+) is a key cofactor required for essential metabolic oxidation-reduction reactions. It also regulates various cellular activities, including gene expression, signaling, DNA repair and calcium homeostasis. Intracellular NAD+ levels are tightly regulated and often respond rapidly to nutritional and environmental changes. Numerous studies indicate that elevating NAD+ may be therapeutically beneficial in the context of numerous diseases. However, the role of NAD+ on skeletal muscle exercise performance is poorly understood. CD38, a multi-functional membrane receptor and enzyme, consumes NAD+ to generate products such as cyclic-ADP-ribose. CD38 knockout mice show elevated tissue and blood NAD+ level. Chronic feeding of high-fat, high-sucrose diet to wild type mice leads to exercise intolerance and reduced metabolic flexibility. Loss of CD38 by genetic mutation protects mice from diet-induced metabolic deficit. These animal model results suggest that elevation of tissue NAD+ through genetic ablation of CD38 can profoundly alter energy homeostasis in animals that are maintained on a calorically-excessive Western diet. PMID:26287487

  13. Regulation of SIRT 1 mediated NAD dependent deacetylation: A novel role for the multifunctional enzyme CD38

    SciTech Connect

    Aksoy, Pinar; Escande, Carlos; White, Thomas A.; Thompson, Michael; Soares, Sandra; Benech, Juan Claudio; Chini, Eduardo N. . E-mail: chini.eduardo@mayo.edu

    2006-10-13

    The SIRT 1 enzyme is a NAD dependent deacetylase implicated in ageing, cell protection, and energy metabolism in mammalian cells. How the endogenous activity of SIRT 1 is modulated is not known. The enzyme CD38 is a multifunctional enzyme capable of synthesis of the second messenger, cADPR, NAADP, and ADPR. However, the major enzymatic activity of CD38 is the hydrolysis of NAD. Of particular interest is the fact that CD38 is present on the inner nuclear membrane. Here, we investigate the modulation of the SIRT 1 activity by CD38. We propose that by modulating availability of NAD to the SIRT1 enzyme, CD38 may regulate SIRT1 enzymatic activity. We observed that in CD38 knockout mice, tissue levels of NAD are significantly increased. We also observed that incubation of purified recombinant SIRT1 enzyme with CD38 or nuclear extracts of wild-type mice led to a significant inhibition of its activity. In contrast, incubation of SIRT1 with cellular extract from CD38 knockout mice was without effect. Furthermore, the endogenous activity of SIRT1 was several time higher in nuclear extracts from CD38 knockout mice when compared to wild-type nuclear extracts. Finally, the in vivo deacetylation of the SIRT1 substrate P53 is increased in CD38 knockout mice tissue. Our data support the novel concept that nuclear CD38 is a major regulator of cellular/nuclear NAD level, and SIRT1 activity. These findings have strong implications for understanding the basic mechanisms that modulate intracellular NAD levels, energy homeostasis, as well as ageing and cellular protection modulated by the SIRT enzymes.

  14. Pre-clinical evaluation of CD38 chimeric antigen receptor engineered T cells for the treatment of multiple myeloma.

    PubMed

    Drent, Esther; Groen, Richard W J; Noort, Willy A; Themeli, Maria; Lammerts van Bueren, Jeroen J; Parren, Paul W H I; Kuball, Jürgen; Sebestyen, Zsolt; Yuan, Huipin; de Bruijn, Joost; van de Donk, Niels W C J; Martens, Anton C M; Lokhorst, Henk M; Mutis, Tuna

    2016-05-01

    Adoptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody sequences to generate second-generation retroviral CD38-chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite becoming CD38-negative during culture. CD38-chimeric antigen receptor-transduced T cells also displayed significant anti-tumor effects in a xenotransplant model, in which multiple myeloma tumors were grown in a human bone marrow-like microenvironment. CD38-chimeric antigen receptor-transduced T cells also appeared to lyse the CD38(+) fractions of CD34(+) hematopoietic progenitor cells, monocytes, natural killer cells, and to a lesser extent T and B cells but did not inhibit the outgrowth of progenitor cells into various myeloid lineages and, furthermore, were effectively controllable with a caspase-9-based suicide gene. These results signify the potential importance of CD38-chimeric antigen receptor-transduced T cells as therapeutic tools for CD38(+) malignancies and warrant further efforts to diminish the undesired effects of this immunotherapy using appropriate strategies. PMID:26858358

  15. Pre-clinical evaluation of CD38 chimeric antigen receptor engineered T cells for the treatment of multiple myeloma

    PubMed Central

    Drent, Esther; Groen, Richard W.J.; Noort, Willy A.; Themeli, Maria; Lammerts van Bueren, Jeroen J.; Parren, Paul W.H.I.; Kuball, Jürgen; Sebestyen, Zsolt; Yuan, Huipin; de Bruijn, Joost; van de Donk, Niels W.C.J.; Martens, Anton C.M.; Lokhorst, Henk M.; Mutis, Tuna

    2016-01-01

    Adoptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody sequences to generate second-generation retroviral CD38-chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite becoming CD38-negative during culture. CD38-chimeric antigen receptor-transduced T cells also displayed significant anti-tumor effects in a xenotransplant model, in which multiple myeloma tumors were grown in a human bone marrow-like microenvironment. CD38-chimeric antigen receptor-transduced T cells also appeared to lyse the CD38+ fractions of CD34+ hematopoietic progenitor cells, monocytes, natural killer cells, and to a lesser extent T and B cells but did not inhibit the outgrowth of progenitor cells into various myeloid lineages and, furthermore, were effectively controllable with a caspase-9-based suicide gene. These results signify the potential importance of CD38-chimeric antigen receptor-transduced T cells as therapeutic tools for CD38+ malignancies and warrant further efforts to diminish the undesired effects of this immunotherapy using appropriate strategies. PMID:26858358

  16. Inhibition of the intrinsic NAD+ glycohydrolase activity of CD38 by carbocyclic NAD analogues.

    PubMed Central

    Wall, K A; Klis, M; Kornet, J; Coyle, D; Amé, J C; Jacobson, M K; Slama, J T

    1998-01-01

    Carba-NAD and pseudocarba-NAD are carbocyclic analogues of NAD+ in which a 2,3-dihydroxycyclopentane methanol replaces the beta-d-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ [Slama and Simmons (1988) Biochemistry 27, 183-193]. These carbocyclic NAD+ analogues, related to each other as diastereomers, have been tested as inhibitors of the intrinsic NAD+ glycohydrolase activity of human CD38, dog spleen NAD+ glycohydrolase, mouse CD38 and Aplysia californica cADP-ribose synthetase. Pseudocarba-NAD, the carbocyclic dinucleotide in which l-2,3-dihydroxycyclopentane methanol replaces the d-ribose of the nicotinamide riboside moiety of NAD+, was found to be the more potent inhibitor. Pseudocarba-NAD was shown to inhibit the intrinsic NAD+ glycohydrolase activity of human CD38 competitively, with Ki=148 microM determined for the recombinant extracellular protein domain and Ki=180 microM determined for the native protein expressed as a cell-surface enzyme on cultured Jurkat cells. Pseudocarba-NAD was shown to be a non-competitive inhibitor of the purified dog spleen NAD+ glycohydrolase, with Kis=47 miroM and Kii=198 microM. Neither pseudocarba-NAD nor carba-NAD inhibited mouse CD38 or Aplysia californica cADP-ribose synthetase significantly at concentrations up to 1 mM. The results underscore significant species differences in the sensitivity of these enzymes to inhibition, and indicate that pseudocarba-NAD will be useful as an inhibitor of the enzymic activity of human but not mouse CD38 in studies using cultured cells. PMID:9794804

  17. Contribution of Nrf2 to Atherogenic Phenotype Switching of Coronary Arterial Smooth Muscle Cells Lacking CD38 Gene

    PubMed Central

    Xu, Ming; Li, Xiao-Xue; Wang, Lei; Wang, Mi; Zhang, Yang; Li, Pin-Lan

    2015-01-01

    Background/Aims Recent studies have indicated that CD38 gene deficiency results in dedifferentiation or transdifferentiation of arterial smooth muscle cells upon atherogenic stimulations. However, the molecular mechanisms mediating this vascular smooth muscle (SMC) phenotypic switching remain unknown. Methods & Results In the present study, we first characterized the phenotypic change in the primary cultures of coronary arterial myocytes (CAMs) from CD38−/− mice. It was shown that CD38 deficiency decreased the expression of contractile marker calponin, SM22α and α-SMA but increased the expression of SMC dedifferentiation marker, vimentin, which was accompanied by enhanced cell proliferation. This phenotypic change in CD38−/− CAMs was enhanced by 7-ketocholesterol (7-Ket), an atherogenic stimulus. We further found that the CD38 deficiency decreased the expression and activity of nuclear factor E2-related factor 2 (Nrf2), a basic leucine zipper (bZIP) transcription factor sensitive to redox regulation. Similar to CD38 deletion, Nrf2 gene silencing increased CAM dedifferentiation upon 7-Ket stimulation. In contrast, the overexpression of Nrf2 gene abolished 7-Ket-induced dedifferentiation in CD38−/− CAMs. Given the sensitivity of Nrf2 to oxidative stress, we determined the role of redox signaling in the regulation of Nrf2 expression and activity associated with CD38 effect in CAM phenotype changes. It was demonstrated that in CD38−/− CAMs, 7-Ket failed to stimulate the production of O2−., while in CD38+/+ CAMs 7-Ket induced marked O2−. production and enhancement of Nrf2 activity, which was substantially attenuated by NOX4 gene silencing. Finally, we demonstrated that 7-Ket-induced and NOX4-dependent O2−. production was inhibited by 8-Br-cADPR, an antagonist of cADPR or NED-19, an antagonist of NAADP as product of CD38 ADP-ribosylcyclase, which significantly inhibited the level of cytosolic Ca2+ and the activation of Nrf2 under 7-Ket. Conclusion

  18. Global transcriptional response of Clostridium difficile carrying the CD38 prophage.

    PubMed

    Sekulovic, Ognjen; Fortier, Louis-Charles

    2015-02-01

    Clostridium difficile is one of the most dangerous pathogens in hospital settings. Most strains of C. difficile carry one or more prophages, and some of them, like CD38-2 and CD119, can influence the expression of toxin genes. However, little is known about the global host response in the presence of a given prophage. In order to fill this knowledge gap, we used high-throughput RNA sequencing (RNA-seq) to conduct a genome-wide transcriptomic analysis of the epidemic C. difficile strain R20291 carrying the CD38-2 prophage. A total of 39 bacterial genes were differentially expressed in the R20291 lysogen, 26 of them being downregulated. Several of the regulated genes encode transcriptional regulators and phosphotransferase system (PTS) subunits involved in glucose, fructose, and glucitol/sorbitol uptake and metabolism. CD38-2 also upregulated the expression of a group of regulatory genes located in phi-027, a resident prophage common to most ribotype 027 isolates. The most differentially expressed gene was that encoding the conserved phase-variable cell wall protein CwpV, which was upregulated 20-fold in the lysogen. Quantitative PCR and immunofluorescence showed that the increased cwpV expression results from a greater proportion of cells actively transcribing the gene. Indeed, 95% of f lysogenic cells express cwpV, as opposed to only 5% of wild-type cells. Furthermore, the higher proportion of cells expressing cwpV results from a higher frequency of recombination of the genetic switch controlling phase variation, which we confirmed to be dependent on the host-encoded recombinase RecV. In summary, CD38-2 interferes with phase variation of the surface protein CwpV and the expression of metabolic genes. PMID:25501487

  19. Clinical efficacy and management of monoclonal antibodies targeting CD38 and SLAMF7 in multiple myeloma.

    PubMed

    van de Donk, Niels W C J; Moreau, Philippe; Plesner, Torben; Palumbo, Antonio; Gay, Francesca; Laubach, Jacob P; Malavasi, Fabio; Avet-Loiseau, Hervé; Mateos, Maria-Victoria; Sonneveld, Pieter; Lokhorst, Henk M; Richardson, Paul G

    2016-02-11

    Immunotherapeutic strategies are emerging as promising therapeutic approaches in multiple myeloma (MM), with several monoclonal antibodies in advanced stages of clinical development. Of these agents, CD38-targeting antibodies have marked single agent activity in extensively pretreated MM, and preliminary results from studies with relapsed/refractory patients have shown enhanced therapeutic efficacy when daratumumab and isatuximab are combined with other agents. Furthermore, although elotuzumab (anti-SLAMF7) has no single agent activity in advanced MM, randomized trials in relapsed/refractory MM have demonstrated significantly improved progression-free survival when elotuzumab is added to lenalidomide-dexamethasone or bortezomib-dexamethasone. Importantly, there has been no significant additive toxicity when these monoclonal antibodies are combined with other anti-MM agents, other than infusion-related reactions specific to the therapeutic antibody. Prevention and management of infusion reactions is important to avoid drug discontinuation, which may in turn lead to reduced efficacy of anti-MM therapy. Therapeutic antibodies interfere with several laboratory tests. First, interference of therapeutic antibodies with immunofixation and serum protein electrophoresis assays may lead to underestimation of complete response. Strategies to mitigate interference, based on shifting the therapeutic antibody band, are in development. Furthermore, daratumumab, and probably also other CD38-targeting antibodies, interfere with blood compatibility testing and thereby complicate the safe release of blood products. Neutralization of the therapeutic CD38 antibody or CD38 denaturation on reagent red blood cells mitigates daratumumab interference with transfusion laboratory serologic tests. Finally, therapeutic antibodies may complicate flow cytometric evaluation of normal and neoplastic plasma cells, since the therapeutic antibody can affect the availability of the epitope for binding

  20. A preclinical model of CD38-pretargeted radioimmunotherapy for plasma cell malignancies

    PubMed Central

    Green, Damian J.; Orgun, Nural N.; Jones, Jon C.; Hylarides, Mark D.; Pagel, John M.; Hamlin, Donald K.; Wilbur, D.S.; Lin, Yukang; Fisher, Darrell R.; Kenoyer, Aimee L.; Frayo, Shani L.; Gopal, Ajay K.; Orozco, Johnnie J.; Gooley, Theodore A.; Wood, Brent L.; Bensinger, William I.; Press, Oliver W.

    2014-01-01

    The vast majority of patients with plasma cell neoplasms die of progressive disease despite high response rates to novel agents. Malignant plasma cells are very radiosensitive, but the potential role of radioimmunotherapy (RIT) in the management of plasmacytomas and multiple myeloma (MM) has undergone only limited evaluation. Furthermore, CD38 has not been explored as a RIT target despite its uniform high expression on plasma cell malignancies. In this report, both conventional RIT (directly radiolabeled antibody) and streptavidin-biotin pretargeted RIT (PRIT) directed against the CD38 antigen, were assessed as approaches to deliver radiation doses sufficient for MM cell eradication. PRIT demonstrated biodistributions that were markedly superior to conventional RIT. Tumor-to-blood ratios as high as 638:1 were seen 24hr after PRIT, while ratios never exceeded 1:1 with conventional RIT. 90Yttrium absorbed dose estimates demonstrated excellent target-to-normal organ ratios (6:1 for the kidney, lung, liver; 10:1 for the whole body). Objective remissions were observed within 7 days in 100% of the mice treated with doses ranging from 800 µCi to 1200 µCi of anti-CD38 pretargeted 90Y-DOTA-biotin, including 100% complete remissions (no detectable tumor in treated mice compared to tumors that were 2982±2834% of initial tumor volume in control animals) by day 23. Furthermore, 100% of animals bearing NCI-H929 multiple myeloma tumor xenografts treated with 800 µCi of anti-CD38 pretargeted 90Y-DOTA-biotin achieved long-term myeloma-free survival (>70 days) compared to none (0%) of the control animals. PMID:24371230

  1. Dynamic conformations of the CD38-mediated NAD cyclization captured in a single crystal

    PubMed Central

    Zhang, HongMin; Graeff, Richard; Chen, Zhe; Zhang, LiangRen; Zhang, LiHe; Lee, HonCheung; Hao, Quan

    2010-01-01

    The extracellular domain of human CD38 is a multifunctional enzyme involved in the metabolism of two Ca2+ messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). When NAD is used as a substrate, CD38 predominantly hydrolyzes it to ADP-ribose with a trace mount of cADPR produced through cyclization of the substrate. However, a mutation of a key residue at the active site, E146A, inhibits the hydrolysis activity of CD38 but greatly increases its cyclization activity. To understand the role of the residue, E146, in the catalytic process, we determined the crystal structure of the E146A mutant protein with a substrate analogue, ara-2’F-NAD. The structure captured the enzymatic reaction intermediates in six different conformations in a crystallographic asymmetric unit. The structural results indicate a folding-back process for the adenine ring of the substrate and provides the first multiple snap shots of the process. Our approach of utilizing multiple molecules in the crystallographic asymmetric unit should be generally applicable for capturing the dynamic nature of enzymatic catalysis. PMID:21134381

  2. All-trans Retinoic Acid Upregulates Reduced CD38 Transcription in Lymphoblastoid Cell Lines from Autism Spectrum Disorder

    PubMed Central

    Riebold, Mathias; Mankuta, David; Lerer, Elad; Israel, Salomon; Zhong, Songfa; Nemanov, Luba; Monakhov, Mikhail V; Levi, Shlomit; Yirmiya, Nurit; Yaari, Maya; Malavasi, Fabio; Ebstein, Richard P

    2011-01-01

    Deficits in social behavior in mice lacking the CD38 gene have been attributed to impaired secretion of oxytocin. In humans, similar deficits in social behavior are associated with autistic spectrum disorder (ASD), for which genetic variants of CD38 have been pinpointed as provisional risk factors. We sought to explore, in an in vitro model, the feasibility of the theory that restoring the level of CD38 in ASD patients could be of potential clinical benefit. CD38 transcription is highly sensitive to several cytokines and vitamins. One of these, all-trans retinoic acid (ATRA), a known inducer of CD38, was added during cell culture and tested on a large sample of N = 120 lymphoblastoid cell (LBC) lines from ASD patients and their parents. Analysis of CD38 mRNA levels shows that ATRA has an upmodulatory potential on LBC derived from ASD patients as well as from their parents. The next crucial issue addressed in our study was the relationship between levels of CD38 expression and psychological parameters. The results obtained indicate a positive correlation between CD38 expression levels and patient scores on the Vineland Adaptive Behavior Scale. In addition, analysis of the role of genetic polymorphisms in the dynamics of the molecule revealed that the genotype of a single-nucleotide polymorphism (rs6449182; C>G variation) in the CpG island of intron 1, harboring the retinoic-acid response element, exerts differential roles in CD38 expression in ASD and in parental LBC. In conclusion, our results provide an empirical basis for the development of a pharmacological ASD treatment strategy based on retinoids. PMID:21528155

  3. Ectonucleotidase CD38 Demarcates Regulatory, Memory-Like CD8+ T Cells with IFN-γ-Mediated Suppressor Activities

    PubMed Central

    Bollinger, Thomas; Orinska, Zane; Bulfone-Paus, Silvia

    2012-01-01

    Regulatory CD8+ T cells are critical for self-tolerance and restricting excessive immune responses. The variety of immune functions they fulfill, the heterogeneity of their phenotype, and the mechanism of action are still poorly understood. Here we describe that regulatory CD8+ T cells exhibiting immunosuppressive actions in vitro and in vivo are recognized as CD38high T cells and present in naive mice. CD38 is a glycosylated membrane protein with ectonucleotidase properties. CD8+CD38high (CD44+CD122+CD62Lhigh) lymphocytes suppress CD4+ effector T-cell proliferation in an antigen-non specific manner via IFN-γ. While direct cell-to-cell contact is needed for this suppressor activity, it is independent of membrane-bound TGF-β and granzyme B release. IL-15 potentiates the suppressive activity of CD8+CD38high T cells and controls their survival and expansion. In humans CD8+CD38high T cells inhibit CD4+ effector T cell proliferation. In vivo, CD8+CD38high, but not CD8+CD38− T cells mitigate murine experimental autoimmune encephalomyelitis (EAE) by reducing the clinical score and delaying disease occurrence. EAE suppression is enhanced by pre-treatment of CD8+CD38high T cells with IL-15. These findings add evidence that the expression of ectoenzyme receptor family members positively correlates with suppressor functions and identifies CD8+CD38high T cells as potential inhibitors of excessive immune responses. PMID:23028866

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

    SciTech Connect

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

    1986-05-01

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

  5. Transient receptor potential melastatin-2 and temperature participate in the process of CD38-regulated oxytocin secretion.

    PubMed

    Liu, Hong-Xiang; Ma, Shuang; Nan, Yong; Yang, Wan-Hua

    2016-08-17

    In recent studies, oxytocin showed potential for the treatment of mental diseases. CD38 is essential for oxytocin release, and hence plays a critical role in social behavior. CD38 catalyzes β-NAD into cyclic ADP ribose (cADPR), which could elevate the intracellular Ca by Ca-permeable channels for oxytocin secretion. The temperature-sensitive cation channel, transient receptor potential melastatin-2 (TRPM2), is a cation-nonselective cation and has been shown to affect oxytocin indirectly. The aim of the present study was to verify the participation of temperature and TRPM2 in CD38-regulated oxytocin release. The crude membranes were prepared to isolate the nerve terminals from the posterior pituitary. At 34°C, 37°C, and 39°C, agonists (β-NAD, ADPR, cADPR) and antagonists (8-Br-cADPR, 2-APB) were used to stimulate the nerve terminals. Oxytocin releases were investigated by enzyme-linked immunosorbent assay. In addition, the expression of TRPM2 and CD38 in the hypothalamus and pituitary was detected by western blotting and quantitative PCR. CD38 agonists (β-NAD, cADPR) and antagonist (8-Br-cADPR) could increase or reduce the oxytocin release, respectively. TRPM2 agonist (ADPR) and antagonist (2-APB) alone could also regulate oxytocin release. Furthermore, temperature could increase agonist stimulation and attenuate the antagonist inhibition on oxytocin release. In addition, CD38 and TRPM2 were expressed in the hypothalamus and pituitary at both the mRNA and the protein level. TRPM2 in pituitary nerve terminals plays a role in oxytocin release. Temperature- enhanced oxytocin release by CD38 and TRPM2. TRPM2 might be involved in the process of CD38-regulated oxytocin release.

  6. Anti-CD38 Antibody Therapy: Windows of Opportunity Yielded by the Functional Characteristics of the Target Molecule

    PubMed Central

    Chillemi, Antonella; Zaccarello, Gianluca; Quarona, Valeria; Ferracin, Manuela; Ghimenti, Chiara; Massaia, Massimo; Horenstein, Alberto L; Malavasi, Fabio

    2013-01-01

    In vivo use of monoclonal antibodies (mAbs) has become a mainstay of routine clinical practice in the treatment of various human diseases. A number of molecules can serve as targets, according to the condition being treated. Now entering human clinical trials, CD38 molecule is a particularly attractive target because of its peculiar pattern of expression and its twin role as receptor and ectoenzyme. This review provides a range of analytical perspectives on the current progress in and challenges to anti-CD38 mAb therapy. We present a synopsis of the evidence available on CD38, particularly in myeloma and chronic lymphocytic leukemia (CLL). Our aim is to make the data from basic science helpful and accessible to a diverse clinical audience and, at the same time, to improve its potential for in vivo use. The topics covered include tissue distribution and signal implementation by mAb ligation and the possibility of increasing cell density on target cells by exploiting information about the molecule’s regulation in combination with drugs approved for in vivo use. Also analyzed is the behavior of CD38 as an enzyme: CD38 is a component of a pathway leading to the production of adenosine in the tumor microenvironment, thus inducing local anergy. Consequently, not only might CD38 be a prime target for mAb-mediated therapy, but its functional block may contribute to general improvement in cancer immunotherapy and outcomes. PMID:23615966

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

    SciTech Connect

    Liang, B.T.

    1989-06-01

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

  8. Practical Considerations for the Use of Daratumumab, a Novel CD38 Monoclonal Antibody, in Myeloma.

    PubMed

    Moreau, Philippe; van de Donk, Niels W C J; San Miguel, Jesus; Lokhorst, Henk; Nahi, Hareth; Ben-Yehuda, Dina; Cavo, Michele; Cook, Gordon; Delforge, Michel; Einsele, Hermann; Zweegman, Sonja; Ludwig, Heinz; Driessen, Christoph; Palumbo, Antonio; Facon, Thierry; Plesner, Torben; Dimopoulos, Meletios; Sondergeld, Pia; Sonneveld, Pieter; Mateos, María-Victoria

    2016-05-01

    Monoclonal antibodies (mAbs) are a recent addition to multiple myeloma (MM) therapies and a number of mAbs directed at myeloma cell surface molecules are in development. Daratumumab is a CD38 mAb that has demonstrated substantial activity and good tolerability in four phase I, phase I/II and phase II studies as monotherapy, as well as in combination with current standard treatments in MM. The positive results obtained in the relapsed/refractory setting in patients with advanced-stage disease and in a small number of patients with newly diagnosed disease provide the rationale for the investigation of the agent in a number of ongoing phase III trials. mAbs are generally better tolerated than conventional chemotherapy; however, their use requires other special considerations. Such factors include those common to all mAbs, namely infusion-related reactions, but also factors that are observed with mAbs used in myeloma, such as interference with response assessment, or factors that are related to CD38 mAbs such as daratumumab, for instance blood typing interference. Our review provides an overview of the results from the daratumumab clinical trials conducted to date, as well as practical management considerations for the use of daratumumab based on our experience with the agent. PMID:27113582

  9. CD38 and interleukin 6 gene polymorphism in egyptians with diffuse large B-cell lymphoma (DLBCL).

    PubMed

    Talaat, Roba M; Abdel-Aziz, Amal M; El-Maadawy, Eman A; Abdel-Bary, Naser

    2015-01-01

    Given the importance of understanding the genetic variations involved in the pathogenesis of non-Hodgkin's lymphoma (NHL), this pilot study was designed to investigate the impact of CD38 (184C/G; rs6449182) and IL-6 (-174 G/C; rs1800795) gene polymorphism on susceptibility of Egyptians to diffuse large B cell lymphoma (DLBCL); major types of NHL. To the best of our knowledge, this study is the first one that examines CD38 polymorphism in the NHL. Genotyping polymorphism is performed using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) for CD38 and Mutagenically separated PCR (MS-PCR) for IL-6 in 100 Egyptian NHL patients with DLBCL subtype and 119 normal controls. The serum level of IL-6 was measured using Enzyme-linked immunosorbent assay (ELISA). CD38 (184C/G) genotype is significantly increased in NHL patients (p < 0.01), while the GG genotype is significantly increased in controls (p < 0.05). Only two genotypes were found (GG and GC) in IL-6 (-174), no CC in our NHL patients and only one case in the controls. Insignificant change in IL-6 (-174 G/C) genotypes was recorded. Significantly increased serum IL-6 (p < 0.05) was positively correlated (r = 0.17; p < 0.05) with the disease. Taken together, our data stressed the importance of CD38 gene polymorphism in developing DLBCL. Our pilot study indicates that CD38 (184) CG genotype might play a role in DLBCL susceptibility in Egyptians. Additional prospective studies on larger population are needed to confirm our findings.

  10. Bacterial terpene cyclases.

    PubMed

    Dickschat, Jeroen S

    2016-01-01

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

  11. CD38 Expression in a Subset of Memory T Cells Is Independent of Cell Cycling as a Correlate of HIV Disease Progression

    PubMed Central

    Würsch, Daniela; Ormsby, Christopher E.; Romero-Rodríguez, Dámaris P.; Olvera-García, Gustavo; Zúñiga, Joaquín; Jiang, Wei; Pérez-Patrigeon, Santiago

    2016-01-01

    In order to determine if the expression of the activation marker CD38 can correlate with HIV disease progression independently of cycling, we performed a cluster-based multivariate correlation analysis of total circulating CD4+ T cell counts and viral loads with frequencies of CD38 and Ki67 expression on CD4+ lymphocytes from patients with untreated HIV infection, stratified in maturation subpopulations, and subpopulation subsets defined by the expression of CXCR5, CXCR3, and CCR4. The frequencies of the activated phenotypes %CD38+ Ki67− and %CD38+ Ki67+ of the CXCR5− CXCR3− CCR4+ (“pre-Th2”) central memory (TCM) cell subset clustered together, comprising a significant negative correlate of total circulating CD4+ T cell counts and a positive correlate of viral load in multivariate analysis. Frequency of cycling-uncoupled CD38 expression in “pre-Th2” TCM cells was a negative correlate of total circulating CD4+ T cell counts in univariate analysis, which was not the case of their %CD38+ Ki67+. CXCR5+ CXCR3− CCR4−  TCM cells were underrepresented in patients, and their absolute counts correlated negatively with their %CD38+ Ki67− but not with their % CD38+ Ki67+. Our results may imply that CD38 expression either reflects or participates in pathogenic mechanisms of HIV disease independently of cell cycling. PMID:27064238

  12. CD38 Expression in a Subset of Memory T Cells Is Independent of Cell Cycling as a Correlate of HIV Disease Progression.

    PubMed

    Würsch, Daniela; Ormsby, Christopher E; Romero-Rodríguez, Dámaris P; Olvera-García, Gustavo; Zúñiga, Joaquín; Jiang, Wei; Pérez-Patrigeon, Santiago; Espinosa, Enrique

    2016-01-01

    In order to determine if the expression of the activation marker CD38 can correlate with HIV disease progression independently of cycling, we performed a cluster-based multivariate correlation analysis of total circulating CD4(+) T cell counts and viral loads with frequencies of CD38 and Ki67 expression on CD4(+) lymphocytes from patients with untreated HIV infection, stratified in maturation subpopulations, and subpopulation subsets defined by the expression of CXCR5, CXCR3, and CCR4. The frequencies of the activated phenotypes %CD38(+) Ki67(-) and %CD38(+) Ki67(+) of the CXCR5(-) CXCR3(-) CCR4(+) ("pre-Th2") central memory (T(CM)) cell subset clustered together, comprising a significant negative correlate of total circulating CD4(+) T cell counts and a positive correlate of viral load in multivariate analysis. Frequency of cycling-uncoupled CD38 expression in "pre-Th2" T(CM) cells was a negative correlate of total circulating CD4(+) T cell counts in univariate analysis, which was not the case of their %CD38(+) Ki67(+). CXCR5(+) CXCR3(-) CCR4(-) T(CM) cells were underrepresented in patients, and their absolute counts correlated negatively with their %CD38(+) Ki67(-) but not with their % CD38(+) Ki67(+). Our results may imply that CD38 expression either reflects or participates in pathogenic mechanisms of HIV disease independently of cell cycling.

  13. NAD(P)H oxidase-dependent intracellular and extracellular O2·- production in coronary arterial myocytes from CD38 knockout mice

    PubMed Central

    Xu, Ming; Zhang, Yang; Xia, Min; Li, Xiao-Xue; Ritter, Joseph K; Zhang, Fan; Li, Pin-Lan

    2011-01-01

    Activation of NAD(P)H oxidase has been reported to produce superoxide (O2 ·-) extracellularly as an autocrine/paracrine regulator or intracellularly as a signaling messenger in a variety of mammalian cells. However, it remains unknown how the activity of NAD(P)H oxidase is regulated in arterial myocytes. Recently, CD38-associated ADP-ribosylcyclase has been reported to use NAD(P)H oxidase product, NAD+ or NADP+ to produce cyclic ADP-ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP), which mediates intracellular Ca2+ signaling. The present study was designed to test a hypothesis that CD38/cADPR pathway as a downstream event exerts feedback regulatory action on the NAD(P)H oxidase activity in production of extra- or intracellular O2 ·-in mouse coronary arterial myocytes (CAMs). By fluorescent microscopic imaging, we simultaneously monitored extra- and intracellular O2 ·-production in wild-type (CD38+/+) and CD38 knockout (CD38-/-) CAMs in response to oxotremorine (OXO), a muscarinic type 1 (M1) receptor agonist. It was found that CD38 deficiency prevented OXO-induced intracellular but not extracellular O2 ·-production in CAMs. Consistently, the OXO-induced intracellular O2 ·-production was markedly inhibited by CD38 shRNA or CD38 inhibitor nicotinamide in CD38+/+ CAMs. Further, Nox4 siRNA inhibited OXO-induced intracellular but not extracellular O2 ·- production, whereas Nox1 siRNA attenuated both intracellular and extracellular O2 ·-production in CD38+/+ CAMs. Direct delivery of exogenous cADPR into CAMs markedly elevated intracellular Ca2+ concentration and restored intracellular O2 ·-production in CD38-/- CAMs. Functionally, CD38 deficiency or Nox1 siRNA and Nox4 siRNA prevented OXO-induced contraction in isolated perfused coronary arteries in CD38 WT mice. These results provide direct evidence that CD38/cADPR pathway importantly controls Nox4-mediated intracellular O2 ·-production and that CD38-dependent intracellular O2

  14. α-Radioimmunotherapy with 213Bi-anti-CD38 immunoconjugates is effective in a mouse model of human multiple myeloma

    PubMed Central

    Blechert, Birgit; Gaertner, Florian C.; Gilbertz, Klaus-Peter; Fernandez, Vanesa; Bassermann, Florian; Endell, Jan; Boxhammer, Rainer; Leclair, Stephane; Vallon, Mario; Aichler, Michaela; Feuchtinger, Annette; Bruchertseifer, Frank; Morgenstern, Alfred; Essler, Markus

    2015-01-01

    In spite of development of molecular therapeutics, multiple myeloma (MM) is fatal in most cases. CD38 is a promising target for selective treatment of MM. We tested radioimmunoconjugates consisting of the α-emitter 213Bi coupled to an anti-CD38 MAb in preclinical treatment of MM. Efficacy of 213Bi-anti-CD38-MAb was assayed towards different MM cell lines with regard to induction of DNA double-strand breaks, induction of apoptosis and initiation of cell cycle arrest. Moreover, mice bearing luciferase-expressing MM xenografts were treated with 213Bi-anti-CD38-MAb. Therapeutic efficacy was monitored by bioluminescence imaging, overall survival and histology. 213Bi-anti-CD38-MAb treatment induced DNA damage which did not result in activation of the G2 DNA-damage-response checkpoint, but instead in mitotic arrest and subsequent mitotic catastrophe. The anti-tumor effect of 213Bi-anti-CD38-MAb correlated with the expression level of CD38 in each MM cell line. In myeloma xenografts, treatment with 213Bi-anti-CD38-MAb suppressed tumor growth via induction of apoptosis in tumor tissue and significantly prolonged survival compared to controls. The major organ systems did not show any signs of 213Bi-induced toxicity. Preclinical treatment of MM with 213Bi-anti-CD38-MAb turned out as an effective therapeutic option. PMID:25576914

  15. Genetic variation in CD38 and breastfeeding experience interact to impact infants’ attention to social eye cues

    PubMed Central

    Krol, Kathleen M.; Monakhov, Mikhail; Lai, Poh San; Ebstein, Richard P.; Grossmann, Tobias

    2015-01-01

    Attending to emotional information conveyed by the eyes is an important social skill in humans. The current study examined this skill in early development by measuring attention to eyes while viewing emotional faces in 7-mo-old infants. In particular, we investigated individual differences in infant attention to eyes in the context of genetic variation (CD38 rs3796863 polymorphism) and experiential variation (exclusive breastfeeding duration) related to the oxytocin system. Our results revealed that, whereas infants at this age show a robust fear bias (increased attention to fearful eyes), their attention to angry and happy eyes varies as a function of exclusive breastfeeding experience and genetic variation in CD38. Specifically, extended exclusive breastfeeding duration selectively enhanced looking preference to happy eyes and decreased looking to angry eyes. Importantly, however, this interaction was impacted by CD38 variation, such that only the looking preferences of infants homozygous for the C allele of rs3796863 were affected by breastfeeding experience. This genotype has been associated with reduced release of oxytocin and higher rates of autism. In contrast, infants with the CA/AA genotype showed similar looking preferences regardless of breastfeeding exposure. Thus, differences in the sensitivity to emotional eyes may be linked to an interaction between the endogenous (CD38) and exogenous (breastfeeding) availability of oxytocin. These findings underline the importance of maternal care and the oxytocin system in contributing to the early development of responding to social eye cues. PMID:26371313

  16. Activation of immobilized, biotinylated choleragen AI protein by a 19-kilodalton guanine nucleotide-binding protein.

    PubMed

    Noda, M; Tsai, S C; Adamik, R; Bobak, D A; Moss, J; Vaughan, M

    1989-09-19

    Cholera toxin catalyzes the ADP-ribosylation that results in activation of the stimulatory guanine nucleotide-binding protein of the adenylyl cyclase system, known as Gs. The toxin also ADP-ribosylates other proteins and simple guanidino compounds and auto-ADP-ribosylates its AI protein (CTA1). All of the ADP-ribosyltransferase activities of CTAI are enhanced by 19-21-kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors, or ARFs. CTAI contains a single cysteine located near the carboxy terminus. CTAI was immobilized through this cysteine by reaction with iodoacetyl-N-biotinyl-hexylenediamine and binding of the resulting biotinylated protein to avidin-agarose. Immobilized CTAI catalyzed the ARF-stimulated ADP-ribosylation of agmatine. The reaction was enhanced by detergents and phospholipid, but the fold stimulation by purified sARF-II from bovine brain was considerably less than that observed with free CTA. ADP-ribosylation of Gsa by immobilized CTAI, which was somewhat enhanced by sARF-II, was much less than predicted on the basis of the NAD:agmatine ADP-ribosyltransferase activity. Immobilized CTAI catalyzed its own auto-ADP-ribosylation as well as the ADP-ribosylation of the immobilized avidin and CTA2, with relatively little stimulation by sARF-II. ADP-ribosylation of CTA2 by free CTAI is minimal. These observations are consistent with the conclusion that the cysteine near the carboxy terminus of the toxin is not critical for ADP-ribosyltransferase activity or for its regulation by sARF-II. Biotinylation and immobilization of the toxin through this cysteine may, however, limit accessibility to Gsa or SARF-II, or perhaps otherwise reduce interaction with these proteins whether as substrates or activator.

  17. Depletion of NADP(H) due to CD38 activation triggers endothelial dysfunction in the postischemic heart.

    PubMed

    Reyes, Levy A; Boslett, James; Varadharaj, Saradhadevi; De Pascali, Francesco; Hemann, Craig; Druhan, Lawrence J; Ambrosio, Giuseppe; El-Mahdy, Mohamed; Zweier, Jay L

    2015-09-15

    In the postischemic heart, coronary vasodilation is impaired due to loss of endothelial nitric oxide synthase (eNOS) function. Although the eNOS cofactor tetrahydrobiopterin (BH4) is depleted, its repletion only partially restores eNOS-mediated coronary vasodilation, indicating that other critical factors trigger endothelial dysfunction. Therefore, studies were performed to characterize the unidentified factor(s) that trigger endothelial dysfunction in the postischemic heart. We observed that depletion of the eNOS substrate NADPH occurs in the postischemic heart with near total depletion from the endothelium, triggering impaired eNOS function and limiting BH4 rescue through NADPH-dependent salvage pathways. In isolated rat hearts subjected to 30 min of ischemia and reperfusion (I/R), depletion of the NADP(H) pool occurred and was most marked in the endothelium, with >85% depletion. Repletion of NADPH after I/R increased NOS-dependent coronary flow well above that with BH4 alone. With combined NADPH and BH4 repletion, full restoration of NOS-dependent coronary flow occurred. Profound endothelial NADPH depletion was identified to be due to marked activation of the NAD(P)ase-activity of CD38 and could be prevented by inhibition or specific knockdown of this protein. Depletion of the NADPH precursor, NADP(+), coincided with formation of 2'-phospho-ADP ribose, a CD38-derived signaling molecule. Inhibition of CD38 prevented NADP(H) depletion and preserved endothelium-dependent relaxation and NO generation with increased recovery of contractile function and decreased infarction in the postischemic heart. Thus, CD38 activation is an important cause of postischemic endothelial dysfunction and presents a novel therapeutic target for prevention of this dysfunction in unstable coronary syndromes.

  18. Targeting Attenuated Interferon-α to Myeloma Cells with a CD38 Antibody Induces Potent Tumor Regression with Reduced Off-Target Activity.

    PubMed

    Pogue, Sarah L; Taura, Tetsuya; Bi, Mingying; Yun, Yong; Sho, Angela; Mikesell, Glen; Behrens, Collette; Sokolovsky, Maya; Hallak, Hussein; Rosenstock, Moti; Sanchez, Eric; Chen, Haiming; Berenson, James; Doyle, Anthony; Nock, Steffen; Wilson, David S

    2016-01-01

    Interferon-α (IFNα) has been prescribed to effectively treat multiple myeloma (MM) and other malignancies for decades. Its use has waned in recent years, however, due to significant toxicity and a narrow therapeutic index (TI). We sought to improve IFNα's TI by, first, attaching it to an anti-CD38 antibody, thereby directly targeting it to MM cells, and, second, by introducing an attenuating mutation into the IFNα portion of the fusion protein rendering it relatively inactive on normal, CD38 negative cells. This anti-CD38-IFNα(attenuated) immunocytokine, or CD38-Attenukine™, exhibits 10,000-fold increased specificity for CD38 positive cells in vitro compared to native IFNα and, significantly, is ~6,000-fold less toxic to normal bone marrow cells in vitro than native IFNα. Moreover, the attenuating mutation significantly decreases IFNα biomarker activity in cynomolgus macaques indicating that this approach may yield a better safety profile in humans than native IFNα or a non-attenuated IFNα immunocytokine. In human xenograft MM tumor models, anti-CD38-IFNα(attenuated) exerts potent anti-tumor activity in mice, inducing complete tumor regression in most cases. Furthermore, anti-CD38-IFNα(attenuated) is more efficacious than standard MM treatments (lenalidomide, bortezomib, dexamethasone) and exhibits strong synergy with lenalidomide and with bortezomib in xenograft models. Our findings suggest that tumor-targeted attenuated cytokines such as IFNα can promote robust tumor killing while minimizing systemic toxicity. PMID:27611189

  19. Targeting Attenuated Interferon-α to Myeloma Cells with a CD38 Antibody Induces Potent Tumor Regression with Reduced Off-Target Activity

    PubMed Central

    Pogue, Sarah L.; Taura, Tetsuya; Bi, Mingying; Yun, Yong; Sho, Angela; Mikesell, Glen; Behrens, Collette; Sokolovsky, Maya; Hallak, Hussein; Rosenstock, Moti; Sanchez, Eric; Chen, Haiming; Berenson, James; Doyle, Anthony; Nock, Steffen; Wilson, David S.

    2016-01-01

    Interferon-α (IFNα) has been prescribed to effectively treat multiple myeloma (MM) and other malignancies for decades. Its use has waned in recent years, however, due to significant toxicity and a narrow therapeutic index (TI). We sought to improve IFNα’s TI by, first, attaching it to an anti-CD38 antibody, thereby directly targeting it to MM cells, and, second, by introducing an attenuating mutation into the IFNα portion of the fusion protein rendering it relatively inactive on normal, CD38 negative cells. This anti-CD38-IFNα(attenuated) immunocytokine, or CD38-Attenukine™, exhibits 10,000-fold increased specificity for CD38 positive cells in vitro compared to native IFNα and, significantly, is ~6,000-fold less toxic to normal bone marrow cells in vitro than native IFNα. Moreover, the attenuating mutation significantly decreases IFNα biomarker activity in cynomolgus macaques indicating that this approach may yield a better safety profile in humans than native IFNα or a non-attenuated IFNα immunocytokine. In human xenograft MM tumor models, anti-CD38-IFNα(attenuated) exerts potent anti-tumor activity in mice, inducing complete tumor regression in most cases. Furthermore, anti-CD38-IFNα(attenuated) is more efficacious than standard MM treatments (lenalidomide, bortezomib, dexamethasone) and exhibits strong synergy with lenalidomide and with bortezomib in xenograft models. Our findings suggest that tumor-targeted attenuated cytokines such as IFNα can promote robust tumor killing while minimizing systemic toxicity. PMID:27611189

  20. CD38 Deficiency Protects the Heart from Ischemia/Reperfusion Injury through Activating SIRT1/FOXOs-Mediated Antioxidative Stress Pathway

    PubMed Central

    Guan, Xiao-Hui; Liu, Xiao-Hong; Hong, Xuan; Zhao, Ning; Xiao, Yun-Fei; Wang, Ling-Fang; Qian, Yi-Song; Deng, Ke-Yu; Ji, Guangju; Fu, Mingui

    2016-01-01

    Ischemia/reperfusion (I/R) injury induces irreversible oxidative stress damage to the cardiac muscle. We previously observed that CD38 deficiency remarkably protects mouse embryonic fibroblasts (MEFs) from oxidative stress-induced injury. However, whether CD38 deficiency protects from I/R injury in the heart is not explored. Here, we showed that the hearts of CD38 deficient mice or wild type mice supplied with exogenous NAD were significantly protected from ischemia/reperfusion injury, seen as reduction of the myocardial infarct sizes when the mice were subjected to 30 min ischemia followed by 24 hours of reperfusion. Consistently, the protection of CD38 deficiency on hypoxia/reoxygenation (H/R) injury was confirmed with a CD38 knockdown H9c2 stable cell line. Furthermore, we observed that knockdown of CD38 remarkably inhibited ROS generation and intracellular Ca2+ overloading induced by H/R in H9c2 cells. The FOXO1 and FOXO3 expressions were significantly elevated by H/R injury in CD38 knockdown cells compared with normal H9c2 cells. The cell immunofluorescence assay showed that FOXO1 nuclear translocation was significantly increased in CD38 knockdown H9c2 cells. In addition, we demonstrated that the increase of FOXO1 nuclear translocation was associated with the increased expressions of antioxidant catalase and SOD2 and the attenuated expression of the ROS generation enzyme NOX4. In conclusion, our results provide new evidence that CD38 deficiency protects the heart from I/R injury through activating SIRT1/FOXOs-mediated antioxidative stress pathway. PMID:27547294

  1. CD38 Deficiency Protects the Heart from Ischemia/Reperfusion Injury through Activating SIRT1/FOXOs-Mediated Antioxidative Stress Pathway.

    PubMed

    Guan, Xiao-Hui; Liu, Xiao-Hong; Hong, Xuan; Zhao, Ning; Xiao, Yun-Fei; Wang, Ling-Fang; Tang, Ling; Jiang, Kai; Qian, Yi-Song; Deng, Ke-Yu; Ji, Guangju; Fu, Mingui; Xin, Hong-Bo

    2016-01-01

    Ischemia/reperfusion (I/R) injury induces irreversible oxidative stress damage to the cardiac muscle. We previously observed that CD38 deficiency remarkably protects mouse embryonic fibroblasts (MEFs) from oxidative stress-induced injury. However, whether CD38 deficiency protects from I/R injury in the heart is not explored. Here, we showed that the hearts of CD38 deficient mice or wild type mice supplied with exogenous NAD were significantly protected from ischemia/reperfusion injury, seen as reduction of the myocardial infarct sizes when the mice were subjected to 30 min ischemia followed by 24 hours of reperfusion. Consistently, the protection of CD38 deficiency on hypoxia/reoxygenation (H/R) injury was confirmed with a CD38 knockdown H9c2 stable cell line. Furthermore, we observed that knockdown of CD38 remarkably inhibited ROS generation and intracellular Ca(2+) overloading induced by H/R in H9c2 cells. The FOXO1 and FOXO3 expressions were significantly elevated by H/R injury in CD38 knockdown cells compared with normal H9c2 cells. The cell immunofluorescence assay showed that FOXO1 nuclear translocation was significantly increased in CD38 knockdown H9c2 cells. In addition, we demonstrated that the increase of FOXO1 nuclear translocation was associated with the increased expressions of antioxidant catalase and SOD2 and the attenuated expression of the ROS generation enzyme NOX4. In conclusion, our results provide new evidence that CD38 deficiency protects the heart from I/R injury through activating SIRT1/FOXOs-mediated antioxidative stress pathway. PMID:27547294

  2. Sam68 Is Required for DNA Damage Responses via Regulating Poly(ADP-ribosyl)ation

    PubMed Central

    Hodgson, Andrea; Wier, Eric M.; Wen, Matthew G.; Kamenyeva, Olena; Xia, Xue; Koo, Lily Y.

    2016-01-01

    The rapid and robust synthesis of polymers of adenosine diphosphate (ADP)-ribose (PAR) chains, primarily catalyzed by poly(ADP-ribose) polymerase 1 (PARP1), is crucial for cellular responses to DNA damage. However, the precise mechanisms through which PARP1 is activated and PAR is robustly synthesized are not fully understood. Here, we identified Src-associated substrate during mitosis of 68 kDa (Sam68) as a novel signaling molecule in DNA damage responses (DDRs). In the absence of Sam68, DNA damage-triggered PAR production and PAR-dependent DNA repair signaling were dramatically diminished. With serial cellular and biochemical assays, we demonstrated that Sam68 is recruited to and significantly overlaps with PARP1 at DNA lesions and that the interaction between Sam68 and PARP1 is crucial for DNA damage-initiated and PARP1-conferred PAR production. Utilizing cell lines and knockout mice, we illustrated that Sam68-deleted cells and animals are hypersensitive to genotoxicity caused by DNA-damaging agents. Together, our findings suggest that Sam68 plays a crucial role in DDR via regulating DNA damage-initiated PAR production. PMID:27635653

  3. Cholix Toxin, a Novel ADP-ribosylating Factor from Vibrio cholerae

    SciTech Connect

    Jorgensen, Rene; Purdy, Alexandra E.; Fieldhouse, Robert J.; Kimber, Matthew S.; Bartlett, Douglas H.; Merrill, A. Rod

    2008-07-15

    The ADP-ribosyltransferases are a class of enzymes that display activity in a variety of bacterial pathogens responsible for causing diseases in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report the characterization of a novel toxin from Vibrio cholerae, which we call cholix toxin. The toxin is active against mammalian cells (IC50 = 4.6 {+-} 0.4 ng/ml) and crustaceans (Artemia nauplii LD50 = 10 {+-} 2 {mu}g/ml). Here we show that this toxin is the third member of the diphthamide-specific class of ADP-ribose transferases and that it possesses specific ADP-ribose transferase activity against ribosomal eukaryotic elongation factor 2. We also describe the high resolution crystal structures of the multidomain toxin and its catalytic domain at 2.1- and 1.25-{angstrom} resolution, respectively. The new structural data show that cholix toxin possesses the necessary molecular features required for infection of eukaryotes by receptor-mediated endocytosis, translocation to the host cytoplasm, and inhibition of protein synthesis by specific modification of elongation factor 2. The crystal structures also provide important insight into the structural basis for activation of toxin ADP-ribosyltransferase activity. These results indicate that cholix toxin may be an important virulence factor of Vibrio cholerae that likely plays a significant role in the survival of the organism in an aquatic environment.

  4. Sam68 Is Required for DNA Damage Responses via Regulating Poly(ADP-ribosyl)ation.

    PubMed

    Sun, Xin; Fu, Kai; Hodgson, Andrea; Wier, Eric M; Wen, Matthew G; Kamenyeva, Olena; Xia, Xue; Koo, Lily Y; Wan, Fengyi

    2016-09-01

    The rapid and robust synthesis of polymers of adenosine diphosphate (ADP)-ribose (PAR) chains, primarily catalyzed by poly(ADP-ribose) polymerase 1 (PARP1), is crucial for cellular responses to DNA damage. However, the precise mechanisms through which PARP1 is activated and PAR is robustly synthesized are not fully understood. Here, we identified Src-associated substrate during mitosis of 68 kDa (Sam68) as a novel signaling molecule in DNA damage responses (DDRs). In the absence of Sam68, DNA damage-triggered PAR production and PAR-dependent DNA repair signaling were dramatically diminished. With serial cellular and biochemical assays, we demonstrated that Sam68 is recruited to and significantly overlaps with PARP1 at DNA lesions and that the interaction between Sam68 and PARP1 is crucial for DNA damage-initiated and PARP1-conferred PAR production. Utilizing cell lines and knockout mice, we illustrated that Sam68-deleted cells and animals are hypersensitive to genotoxicity caused by DNA-damaging agents. Together, our findings suggest that Sam68 plays a crucial role in DDR via regulating DNA damage-initiated PAR production. PMID:27635653

  5. Protein ADP-ribosylation and the cellular response to DNA strand breaks.

    PubMed

    Caldecott, K W

    2014-07-01

    DNA strand breaks arise continuously in cells and can lead to chromosome rearrangements and genome instability or cell death. The commonest DNA breaks are DNA single-strand breaks, which arise at a frequency of tens-of-thousands per cell each day and which can block the progression of RNA/DNA polymerases and disrupt gene transcription and genome duplication. If not rapidly repaired, SSBs can be converted into DNA double-strand breaks (DSBs) during genome duplication, eliciting a complex series of DNA damage responses that attempt to protect cells from irreversible replication fork collapse. DSBs are the most cytotoxic and clastogenic type of DNA breaks, and can also arise independently of DNA replication, albeit at a frequency several orders of magnitude lower than SSBs. Here, I discuss the evidence that DNA single- and double -strand break repair pathways, and cellular tolerance mechanisms for protecting replication forks during genome duplication, utilize signalling by protein ADP-ribosyltransferases to protect cells from the harmful impact of DNA strand breakage.

  6. An age-related numerical and functional deficit in CD19(+) CD24(hi) CD38(hi) B cells is associated with an increase in systemic autoimmunity.

    PubMed

    Duggal, Niharika A; Upton, Jane; Phillips, Anna C; Sapey, Elizabeth; Lord, Janet M

    2013-10-01

    Autoimmunity increases with aging indicative of reduced immune tolerance, but the mechanisms involved are poorly defined. In recent years, subsets of B cells with immunoregulatory properties have been identified in murine models of autoimmune disorders, and these cells downregulate immune responses via secretion of IL10. In humans, immature transitional B cells with a CD19(+) CD24(hi) CD38(hi) phenotype have been reported to regulate immune responses via IL10 production. We found the frequency and numbers of CD19(+) CD24(hi) CD38(hi) cells were reduced in the PBMC pool with age. IL10 expression and secretion following activation via either CD40, or Toll-like receptors was also impaired in CD19(+) CD24(hi) CD38(hi) B cells from healthy older donors. When investigating the mechanisms involved, we found that CD19(+) CD24(hi) CD38(hi) B-cell function was compromised by age-related effects on both T cells and B cells: specifically, CD40 ligand expression was lower in CD4 T cells from older donors following CD3 stimulation, and signalling through CD40 was impaired in CD19(+) CD24(hi) CD38(hi) B cells from elders as evidenced by reduced phosphorylation (Y705) and activation of STAT3. However, there was no age-associated change in expression of costimulatory molecules CD80 and CD86 on CD19(+) CD24(hi) CD38(hi) cells, suggesting IL10-dependent immune suppression is impaired, but contact-dependent suppressive capacity is intact with age. Finally, we found a negative correlation between CD19(+) CD24(hi) CD38(hi) B-cell IL10 production and autoantibody (Rheumatoid factor) levels in older adults. We therefore propose that an age-related decline in CD19(+) CD24(hi) CD38(hi) B cell number and function may contribute towards the increased autoimmunity and reduced immune tolerance seen with aging.

  7. Reduced Plasmodium Parasite Burden Associates with CD38+ CD4+ T Cells Displaying Cytolytic Potential and Impaired IFN-γ Production

    PubMed Central

    Burel, Julie G.; Apte, Simon H.; Groves, Penny L.; Klein, Kerenaftali; McCarthy, James S.; Doolan, Denise L.

    2016-01-01

    Using a unique resource of samples from a controlled human malaria infection (CHMI) study, we identified a novel population of CD4+ T cells whose frequency in the peripheral blood was inversely correlated with parasite burden following P. falciparum infection. These CD4+ T cells expressed the multifunctional ectoenzyme CD38 and had unique features that distinguished them from other CD4+ T cells. Specifically, their phenotype was associated with proliferation, activation and cytotoxic potential as well as significantly impaired production of IFN-γ and other cytokines and reduced basal levels of activated STAT1. A CD38+ CD4+ T cell population with similar features was identified in healthy uninfected individuals, at lower frequency. CD38+ CD4+ T cells could be generated in vitro from CD38- CD4+ T cells after antigenic or mitogenic stimulation. This is the first report of a population of CD38+ CD4+ T cells with a cytotoxic phenotype and markedly impaired IFN-γ capacity in humans. The expansion of this CD38+ CD4+ T population following infection and its significant association with reduced blood-stage parasite burden is consistent with an important functional role for these cells in protective immunity to malaria in humans. Their ubiquitous presence in humans suggests that they may have a broad role in host-pathogen defense. Trial Registration ClinicalTrials.gov clinical trial numbers ACTRN12612000814875, ACTRN12613000565741 and ACTRN12613001040752 PMID:27662621

  8. Computational study on the conformations of CD38 and inclusion complexes of some lower-size large-ring cyclodextrins

    NASA Astrophysics Data System (ADS)

    Ivanov, Petko; Atanassov, Emanouil; Jaime, Carlos

    2014-01-01

    The conformations of CD38 were examined by conformational search with molecular dynamics simulations using the Glycam04 force field. The results were compared with previous ones for CD26, the largest cyclodextrin for which crystal data are available. Principal component analysis (PCA) was applied for post-processing of the simulation trajectories. Limited number of modes determine the overall deformations of the macroring of CD38. The longer perimeter of the macroring allowed the formation of a form not observed so far - a three-turn helix shaped as a short tube. In analogy with CD26, significant participation was monitored for conformations of CD38 with one-turn spirals at the opposite sides of the macroring linked together from the 'bottom' and from the 'top' with extended bridge spacers. Computationally were examined for the first time inclusion complexes of some lower-size LR-CDs, namely complexes of CDn (n = 13, 14, 26) with adamantane and of CD14 with 1-hydroxyadamantane. The macroring conformation of CD13 was not altered by the inclusion of the substrate molecule which acquired preferred positioning not in the middle of the cavity but rather close to the glucose residues at one of the sides. The same positioning of the small molecule in the cavity of the more flexible CD14 macroring enhanced the appearance of bent onto two conformation of this cyclodextrin. The most interesting behaviour presented the complex of CD26 with adamantane in which case the small molecule acts as a 'nucleation center' for the formation of a second helical turn about the substrate molecule.

  9. Concentrative uptake of cyclic ADP-ribose generated by BST-1+ stroma stimulates proliferation of human hematopoietic progenitors.

    PubMed

    Podestà, Marina; Benvenuto, Federica; Pitto, Anna; Figari, Osvaldo; Bacigalupo, Andrea; Bruzzone, Santina; Guida, Lucrezia; Franco, Luisa; Paleari, Laura; Bodrato, Nicoletta; Usai, Cesare; De Flora, Antonio; Zocchi, Elena

    2005-02-18

    Cyclic ADP-ribose (cADPR) is an intracellular calcium mobilizer generated from NAD(+) by the ADP-ribosyl cyclases CD38 and BST-1. cADPR, both exogenously added and paracrinally produced by a CD38(+) feeder layer, has recently been demonstrated to stimulate the in vitro proliferation of human hemopoietic progenitors (HP) and also the in vivo expansion of hemopoietic stem cells. The low density of BST-1 expression on bone marrow (BM) stromal cells and the low specific activity of the enzyme made it unclear whether cADPR generation by a BST-1(+) stroma could stimulate HP proliferation in the BM microenvironment. We developed and characterized two BST-1(+) stromal cell lines, expressing an ectocellular cyclase activity similar to that of BST-1(+) human mesenchymal stem cells, the precursors of BM stromal cells. Long term co-culture of cord blood-derived HP over these BST-1(+) feeders determined their expansion. Influx of paracrinally generated cADPR into clonogenic HP was mediated by a concentrative, nitrobenzylthioinosine- and dipyridamole-inhibitable nucleoside transporter, this providing a possible explanation to the effectiveness of the hormone-like concentrations of the cyclic nucleotide measured in the medium conditioned by BST-1(+) feeders. These results suggest that the BST-1-catalyzed generation of extracellular cADPR, followed by the concentrative uptake of the cyclic nucleotide by HP, may be physiologically relevant in normal hemopoiesis.

  10. A non-canonical adenosinergic pathway led by CD38 in human melanoma cells induces suppression of T cell proliferation.

    PubMed

    Morandi, Fabio; Morandi, Barbara; Horenstein, Alberto L; Chillemi, Antonella; Quarona, Valeria; Zaccarello, Gianluca; Carrega, Paolo; Ferlazzo, Guido; Mingari, Maria Cristina; Moretta, Lorenzo; Pistoia, Vito; Malavasi, Fabio

    2015-09-22

    Nucleotide-metabolizing ectoenzymes are endowed with an extracellular catalytic domain, which is involved in regulating the extracellular nucleotide/nucleoside balance. The tumor microenvironment contains high levels of adenosine (ADO) generated by this enzymatic network, thus promoting tumor growth by inhibiting anti-tumor immune responses. ADO inhibition in melanoma murine models limits tumor metastases and restores anti-tumor immune responses. This work investigates the expression and function of ectoenzymes in primary human melanoma cell lines. All of latter cells expressed CD38, CD39, CD73, and CD203a/PC-1, and produced ADO from AMP and NAD(+ )T cell proliferation. Accordingly, phosphorylation of S6 ribosomal protein, p38 and Stat1 was lower in activated memory cells than in naïve CD4(+) T lymphocytes. Melanoma cells also inhibited proliferation of naïve, memory and -to a lesser extent- of effector CD8(+) T cells. These different inhibitory effects correlated with distinct patterns of expression of the ADO receptor A2a and A2b. These results show that primary human melanoma cell lines suppress in vitro T cell proliferation through an adenosinergic pathway in which CD38 and CD73 play a prominent role. PMID:26329660

  11. Cyclic ADP-ribose generation by CD38 improves human hemopoietic stem cell engraftment into NOD/SCID mice.

    PubMed

    Podestà, Marina; Pitto, Anna; Figari, Osvaldo; Bacigalupo, Andrea; Bruzzone, Santina; Guida, Lucrezia; Franco, Luisa; De Flora, Antonio; Zocchi, Elena

    2003-02-01

    Cyclic ADP-ribose (cADPR) is a potent and universal intracellular calcium mobilizer, recently shown to behave as a new hemopoietic cytokine stimulating the in vitro proliferation of both committed and uncommitted human hemopoietic progenitors (HP). Here, we investigated the effects of cADPR on engraftment of hemopoietic stem cells (HSC) into irradiated NOD/SCID mice. Two different protocols were used: i) a 24 h in vitro priming of cord blood-derived mononuclear cells (MNC) with micromolar cADPR, followed by their infusion into irradiated mice (both primary and secondary transplants); and ii) co-infusion of MNC with CD38-transfected, cADPR-generating, irradiated murine 3T3 fibroblasts. We demonstrated a dual effect of cADPR on human HP in vivo: i) enhanced proliferation of committed progenitors, responsible for improvement of short-term engraftment; ii) expansion of HSC, with increased long-term human engraftment into secondary recipients and a significantly higher expansion factor of CD34+ progenitors in mice co-infused with MNC and CD38+ 3T3 fibroblasts. These results hold promise for the possible therapeutic use of cADPR, and of cADPR-producing stroma, to achieve long-term expansion of human HSC, that is, those HP capable of self-renewal and responsible for repopulation of the bone marrow.

  12. Daratumumab depletes CD38+ immune regulatory cells, promotes T-cell expansion, and skews T-cell repertoire in multiple myeloma

    PubMed Central

    Krejcik, Jakub; Casneuf, Tineke; Nijhof, Inger S.; Verbist, Bie; Bald, Jaime; Plesner, Torben; Syed, Khaja; Liu, Kevin; van de Donk, Niels W. C. J.; Weiss, Brendan M.; Ahmadi, Tahamtan; Lokhorst, Henk M.; Mutis, Tuna

    2016-01-01

    Daratumumab targets CD38-expressing myeloma cells through a variety of immune-mediated mechanisms (complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cellular phagocytosis) and direct apoptosis with crosslinking. These mechanisms may also target nonplasma cells that express CD38, which prompted evaluation of daratumumab’s effects on CD38-positive immune subpopulations. Peripheral blood (PB) and bone marrow (BM) from patients with relapsed/refractory myeloma from 2 daratumumab monotherapy studies were analyzed before and during therapy and at relapse. Regulatory B cells and myeloid-derived suppressor cells, previously shown to express CD38, were evaluated for immunosuppressive activity and daratumumab sensitivity in the myeloma setting. A novel subpopulation of regulatory T cells (Tregs) expressing CD38 was identified. These Tregs were more immunosuppressive in vitro than CD38-negative Tregs and were reduced in daratumumab-treated patients. In parallel, daratumumab induced robust increases in helper and cytotoxic T-cell absolute counts. In PB and BM, daratumumab induced significant increases in CD8+:CD4+ and CD8+:Treg ratios, and increased memory T cells while decreasing naïve T cells. The majority of patients demonstrated these broad T-cell changes, although patients with a partial response or better showed greater maximum effector and helper T-cell increases, elevated antiviral and alloreactive functional responses, and significantly greater increases in T-cell clonality as measured by T-cell receptor (TCR) sequencing. Increased TCR clonality positively correlated with increased CD8+ PB T-cell counts. Depletion of CD38+ immunosuppressive cells, which is associated with an increase in T-helper cells, cytotoxic T cells, T-cell functional response, and TCR clonality, represents possible additional mechanisms of action for daratumumab and deserves further exploration. PMID:27222480

  13. Impact of changes in antigen level on CD38/PD-1 co-expression on HIV-specific CD8 T cells in chronic, untreated HIV-1 infection.

    PubMed

    Vollbrecht, Thomas; Brackmann, Heike; Henrich, Nadja; Roeling, Joerg; Seybold, Ulrich; Bogner, Johannes R; Goebel, Frank D; Draenert, Rika

    2010-03-01

    Excessive immune activation is a hallmark of chronic uncontrolled HIV infection. During the past years, growing evidence suggests that immune inhibitory signals also play an important role in progressive disease. However, the relationship between positive and negative immune signals on HIV-specific CD8 T cells has not been studied in detail so far in chronic HIV-1 infection. In this study, the expression of markers of positive (CD38) and negative (PD-1) immune signals on virus-specific CD8 T cells in chronic, untreated HIV-1 infection was evaluated using intracellular cytokine staining. Viral escape mutations were assessed by autologous virus sequence analysis and subsequent peptide titration assays. Single-epitope CD8 T-cell responses toward Gag, Pol, and Nef were compared in 12 HIV-1 controllers (viral load <5,000 cp/ml) and 12 HIV-1 progressors (viral load >50,000 cp/ml) and a highly significant increase of CD38/PD-1 co-expression on virus-specific CD8 T cells in progressors was found (P < 0.0001). The level of CD38/PD-1 co-expression was independent of epitope specificity. Longitudinal follow-up revealed a clear drop in CD38/PD-1 co-expression on virus-specific CD8 T cells after the suppression of antigen following either viral escape mutation or the initiation of HAART (P = 0.004). Antigen persistence with a fluctuating viral load revealed stable levels of CD38/PD-1 co-expression whereas significant rises in viral load were accompanied or even preceded by substantial increases in CD38/PD-1 co-expression. The CD38/PD-1 phenotype clearly distinguishes HIV-specific CD8 T-cell responses between controllers and progressors. Whether it plays a causative role in disease progression remains debatable. J. Med. Virol. 82:358-370, 2010. (c) 2010 Wiley-Liss, Inc.

  14. Interest in Determining the CD34+ CD38− Phenotype in the Diagnosis and Prognosis of Acute Leukemia in Abidjan – Côte d’Ivoire

    PubMed Central

    Sawadogo, Duni; Tolo, Aissata; Kassi, Hermance; Sangare, Mahawa; Inwoley, Andre

    2013-01-01

    Background In Côte d’Ivoire, acute leukemias account for 12.5% of hematological malignancies. Acute leukemias are due to an anomaly of the stem cell characterized among other things by the expression of CD34+ CD38− surface markers. This CD34+ CD38− phenotype as well as other factors such as tumor syndrome, high leukocytosis and blasts are considered as important factors of poor prognosis. We therefore proposed to investigate the prognostic value of the expression of CD34+ CD38− markers in acute leukemias in Abidjan. Methods We selected 23 patients aged 33 years on whom we performed Complete Blood Count, bone marrow aspiration and immunophenotyping. To search for myeloperoxydase, smears of blood or bone marrow were stained with benzidine and revealed by the use of Hydrogen peroxide. Acute leukemias were then identified and distributed using the score proposed by the European Group for the Immunological characterization of Leukemias. The definitive diagnosis was made by combining morphological characters that serve as the basis for the French-American-British classification as well as cytochemical and immunophenotypic characters. Results According to the cytological and immunophenotypic classifications, the acute lymphoid leukemia 2 and B IV predominated. 52.2% (12/33) of patients were CD34+ CD38−. This phenotype was found in almost all cytological immunophenotypic types. The medullary invasion by blasts (reflection of the tumor mass) of the total sample of CD34+, CD34+ CD38− patients and those not expressing CD34+ was respectively 79.4%, 81.25%, 83.3% and 74.8%. Conclusion There was therefore no correlation between medullary blasts and the expression of CD34+ CD38−. To the factors we selected it would have been necessary to associate the study of cytogenetic and molecular anomalies to better understand the role of CD34+ CD38− phenotype, concerning prognosis. PMID:23667721

  15. Nilotinib enhances the efficacy of conventional chemotherapeutic drugs in CD34⁺CD38⁻ stem cells and ABC transporter overexpressing leukemia cells.

    PubMed

    Wang, Fang; Wang, Xiao-Kun; Shi, Cheng-Jun; Zhang, Hui; Hu, Ya-Peng; Chen, Yi-Fan; Fu, Li-Wu

    2014-03-19

    Incomplete chemotherapeutic eradication of leukemic CD34⁺CD38⁻ stem cells is likely to result in disease relapse. The purpose of this study was to evaluate the effect of nilotinib on eradicating leukemia stem cells and enhancing the efficacy of chemotherapeutic agents. Our results showed that ABCB1 and ABCG2 were preferentially expressed in leukemic CD34⁺CD38⁻ cells. Nilotinib significantly enhanced the cytotoxicity of doxorubicin and mitoxantrone in CD34⁺CD38⁻ cells and led to increased apoptosis. Moreover, nilotinib strongly reversed multidrug resistance and increased the intracellular accumulation of rhodamine 123 in primary leukemic blasts overexpressing ABCB1 and/or ABCG2. Studies with ABC transporter-overexpressing carcinoma cell models confirmed that nilotinib effectively reversed ABCB1- and ABCG2-mediated drug resistance, while showed no significant reversal effect on ABCC1- and ABCC4-mediated drug resistance. Results from cytotoxicity assays showed that CD34⁺CD38⁻ cells exhibited moderate resistance (2.41-fold) to nilotinib, compared with parental K562 cells. Furthermore, nilotinib was less effective in blocking the phosphorylation of Bcr-Abl and CrkL (a substrate of Bcr-Abl kinase) in CD34⁺CD38⁻ cells. Taken together, these data suggest that nilotinib particularly targets CD34⁺CD38⁻ stem cells and MDR leukemia cells, and effectively enhances the efficacy of chemotherapeutic drugs by blocking the efflux function of ABC transporters.

  16. Mechanism of cholera toxin activation by a guanine nucleotide-dependent 19 kDa protein.

    PubMed

    Noda, M; Tsai, S C; Adamik, R; Moss, J; Vaughan, M

    1990-05-16

    Cholera toxin causes the devastating diarrheal syndrome characteristic of cholera by catalyzing the ADP-ribosylation of Gs alpha, a GTP-binding regulatory protein, resulting in activation of adenylyl cyclase. ADP-ribosylation of Gs alpha is enhanced by 19 kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors or ARFs. We investigated the effects of agents known to alter toxin-catalyzed activation of adenylyl cyclase on the stimulation of toxin- and toxin subunit-catalyzed ADP-ribosylation of Gs alpha and other substrates by an ADP-ribosylation factor purified from a soluble fraction of bovine brain (sARF II). In the presence of GTP, sARF II enhanced activity of both the toxin catalytic unit and a reduced and alkylated fragment ('A1'), as a result of an increase in substrate affinity with no significant effects on Vmax. Activation of toxin was independent of Gs alpha and was stimulated 4-fold by sodium dodecyl sulfate, but abolished by Triton X-100. sARF II therefore serves as a direct allosteric activator of the A1 protein and may thus amplify the pathological effects of cholera toxin.

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

    PubMed Central

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

    2001-01-01

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

  18. Long-Lived Plasma Cells Are Contained within the CD19(-)CD38(hi)CD138(+) Subset in Human Bone Marrow.

    PubMed

    Halliley, Jessica L; Tipton, Christopher M; Liesveld, Jane; Rosenberg, Alexander F; Darce, Jaime; Gregoretti, Ivan V; Popova, Lana; Kaminiski, Denise; Fucile, Christopher F; Albizua, Igor; Kyu, Shuya; Chiang, Kuang-Yueh; Bradley, Kyle T; Burack, Richard; Slifka, Mark; Hammarlund, Erika; Wu, Hao; Zhao, Liping; Walsh, Edward E; Falsey, Ann R; Randall, Troy D; Cheung, Wan Cheung; Sanz, Iñaki; Lee, F Eun-Hyung

    2015-07-21

    Antibody responses to viral infections are sustained for decades by long-lived plasma cells (LLPCs). However, LLPCs have yet to be characterized in humans. Here we used CD19, CD38, and CD138 to identify four PC subsets in human bone marrow (BM). We found that the CD19(-)CD38(hi)CD138(+) subset was morphologically distinct, differentially expressed PC-associated genes, and exclusively contained PCs specific for viral antigens to which the subjects had not been exposed for more than 40 years. Protein sequences of measles- and mumps-specific circulating antibodies were encoded for by CD19(-)CD38(hi)CD138(+) PCs in the BM. Finally, we found that CD19(-)CD38(hi)CD138(+) PCs had a distinct RNA transcriptome signature and human immunoglobulin heavy chain (VH) repertoire that was relatively uncoupled from other BM PC subsets and probably represents the B cell response's "historical record" of antigenic exposure. Thus, our studies define human LLPCs and provide a mechanism for the life-long maintenance of anti-viral antibodies in the serum.

  19. Percentage of Peripheral CD19+CD24hiCD38hi Regulatory B Cells in Neonatal Sepsis Patients and Its Functional Implication

    PubMed Central

    Pan, Xiao; Ji, Zuoquan; Xue, Jiang

    2016-01-01

    Background As a major cause of mortality in neonates, neonatal sepsis is often accompanied by immune dysfunctions, which are frequently caused by dysregulated T cell sub-populations. The role of regulatory B cells in neonatal sepsis, however, remains unknown. Therefore, this study investigated the percentage and functional variation of CD19+CD24hiCD38hi regulatory B cells in peripheral blood of neonatal sepsis patients in an attempt to elucidate the role of these regulatory B cells in pathogenesis of sepsis. Material/Methods Flow cytometry was used to quantify the percentage of CD19+CD24hiCD38hi regulatory B cells from peripheral blood samples. The correlation between B cell percentage and C reactive protein (CRP) level was analyzed. Secretion level of interleukin-10 (IL-10) and effects on the proliferation of naïve CD4+ T cells were further analyzed. Results The percentage of CD19+CD24hiCD38hi regulatory B cells in neonatal sepsis patients was significantly higher compared to healthy controls (p<0.05), and was positively correlated with serum CRP level. The percentage of IL-10+ CD19+CD24hiCD38hi regulatory B cells was also higher in sepsis patients, and also had more potent inhibition on naïve CD4+ T cells (p<0.01). Conclusions The elevation of CD19+CD24hiCD38hi regulatory B cells in neonatal sepsis can inhibit body immune function and thus may participate in the pathogenesis of sepsis. PMID:27389933

  20. Digitonin effects on photoreceptor adenylate cyclase.

    PubMed

    Bitensky, M W; Gorman, R E; Miller, W H

    1972-03-24

    Adenylate cyclase is described in a number of photoreceptor membranes. Vertebrate rod outer segments contain light-regulated cyclase, and light regulation is abolished by digitonin. Disruption of microvilli in cone and rhabdomphotoreceptors is also associated with loss of light regulation and retention of full enzymic activity. The data suggest that inhibitory constraint provides regulation in cyclase systems and that disruption of membrane structure uncouples catalytic and regulatory elements.

  1. Evidence for a role of the oxytocin system, indexed by genetic variation in CD38, in the social bonding effects of expressed gratitude

    PubMed Central

    2014-01-01

    Oxytocin is thought to play a central role in promoting close social bonds via influence on social interactions. The current investigation targeted interactions involving expressed gratitude between members of romantic relationships because recent evidence suggests gratitude and its expression provides behavioral and psychological ‘glue’ to bind individuals closer together. Specifically, we took a genetic approach to test the hypothesis that social interactions involving expressed gratitude would be associated with variation in a gene, CD38, which has been shown to affect oxytocin secretion. A polymorphism (rs6449182) that affects CD38 expression was significantly associated with global relationship satisfaction, perceived partner responsiveness and positive emotions (particularly love) after lab-based interactions, observed behavioral expression of gratitude toward a romantic partner in the lab, and frequency of expressed gratitude in daily life. A separate polymorphism in CD38 (rs3796863) previously associated with plasma oxytocin levels and social engagement was also associated with perceived responsiveness in the benefactor after an expression of gratitude. The combined influence of the two polymorphisms was associated with a broad range of gratitude-related behaviors and feelings. The consistent pattern of findings suggests that the oxytocin system is associated with solidifying the glue that binds adults into meaningful and important relationships. PMID:24396004

  2. Evidence for a role of the oxytocin system, indexed by genetic variation in CD38, in the social bonding effects of expressed gratitude.

    PubMed

    Algoe, Sara B; Way, Baldwin M

    2014-12-01

    Oxytocin is thought to play a central role in promoting close social bonds via influence on social interactions. The current investigation targeted interactions involving expressed gratitude between members of romantic relationships because recent evidence suggests gratitude and its expression provides behavioral and psychological 'glue' to bind individuals closer together. Specifically, we took a genetic approach to test the hypothesis that social interactions involving expressed gratitude would be associated with variation in a gene, CD38, which has been shown to affect oxytocin secretion. A polymorphism (rs6449182) that affects CD38 expression was significantly associated with global relationship satisfaction, perceived partner responsiveness and positive emotions (particularly love) after lab-based interactions, observed behavioral expression of gratitude toward a romantic partner in the lab, and frequency of expressed gratitude in daily life. A separate polymorphism in CD38 (rs3796863) previously associated with plasma oxytocin levels and social engagement was also associated with perceived responsiveness in the benefactor after an expression of gratitude. The combined influence of the two polymorphisms was associated with a broad range of gratitude-related behaviors and feelings. The consistent pattern of findings suggests that the oxytocin system is associated with solidifying the glue that binds adults into meaningful and important relationships.

  3. Increased frequency of circulating Tc22/Th22 cells and polyfunctional CD38(-) T cells in HIV-exposed uninfected subjects.

    PubMed

    Oliveira, Luanda M S; Lima, Josenilson F; Cervantes, Cesar A C; Casseb, Jorge S; Mendonça, Marcelo; Duarte, Alberto J S; Sato, Maria N

    2015-09-08

    Some individuals are resistant to HIV-1 infection despite repeated exposure to the virus, suggesting the presence of a complex antiviral response. Innate factors like IL-22 exert gut mucosal protection and polyfunctional T cells have been associated with low progression in HIV infection; therefore, we evaluated the frequencies of CD4+ and CD8+ T cell-secreting cytokines, including Tc22/Th22 cells and polyfunctional T cells in HIV-1-exposed uninfected individuals (EUs), their HIV-1-infected partners and healthy controls. EUs exhibited an increased frequency of p15 Gag CD4+ IL-22+ secreting T cells, whereas HIV-infected partners demonstrated a high frequency of CD4+ IL-17+ T cells in response to p24. Similar responses of Th22 and Tc22 cells to Gag peptides and Staphylococcal enterotoxin B (SEB) stimulation were detected in the serodiscordant couples. However, polyfunctionality in HIV subjects was associated with an HIV Gag response of CD38+ T cells, whereas polyfunctionality for EUs was induced upon SEB stimulation by CD38- T cells. EUs demonstrated the presence of Tc22/Th22 cells and polyfunctional CD38- T cells with a low activation profile. These data suggest that SEB-induced polyfunctional CD4+ and CD8+ T cells together with Tc22/Th22 cells in EU individuals can provide an immunological advantage in the response to pathogens such as HIV-1.

  4. Cyclic ADP-Ribose and Heat Regulate Oxytocin Release via CD38 and TRPM2 in the Hypothalamus during Social or Psychological Stress in Mice.

    PubMed

    Zhong, Jing; Amina, Sarwat; Liang, Mingkun; Akther, Shirin; Yuhi, Teruko; Nishimura, Tomoko; Tsuji, Chiharu; Tsuji, Takahiro; Liu, Hong-Xiang; Hashii, Minako; Furuhara, Kazumi; Yokoyama, Shigeru; Yamamoto, Yasuhiko; Okamoto, Hiroshi; Zhao, Yong Juan; Lee, Hon Cheung; Tominaga, Makoto; Lopatina, Olga; Higashida, Haruhiro

    2016-01-01

    Hypothalamic oxytocin (OT) is released into the brain by cyclic ADP-ribose (cADPR) with or without depolarizing stimulation. Previously, we showed that the intracellular free calcium concentration ([Ca(2+)]i) that seems to trigger OT release can be elevated by β-NAD(+), cADPR, and ADP in mouse oxytocinergic neurons. As these β-NAD(+) metabolites activate warm-sensitive TRPM2 cation channels, when the incubation temperature is increased, the [Ca(2+)]i in hypothalamic neurons is elevated. However, it has not been determined whether OT release is facilitated by heat in vitro or hyperthermia in vivo in combination with cADPR. Furthermore, it has not been examined whether CD38 and TRPM2 exert their functions on OT release during stress or stress-induced hyperthermia in relation to the anxiolytic roles and social behaviors of OT under stress conditions. Here, we report that OT release from the isolated hypothalami of male mice in culture was enhanced by extracellular application of cADPR or increasing the incubation temperature from 35°C to 38.5°C, and simultaneous stimulation showed a greater effect. This release was inhibited by a cADPR-dependent ryanodine receptor inhibitor and a nonspecific TRPM2 inhibitor. The facilitated release by heat and cADPR was suppressed in the hypothalamus isolated from CD38 knockout mice and CD38- or TRPM2-knockdown mice. In the course of these experiments, we noted that OT release differed markedly between individual mice under stress with group housing. That is, when male mice received cage-switch stress and eliminated due to their social subclass, significantly higher levels of OT release were found in subordinates compared with ordinates. In mice exposed to anxiety stress in an open field, the cerebrospinal fluid (CSF) OT level increased transiently at 5 min after exposure, and the rectal temperature also increased from 36.6°C to 37.8°C. OT levels in the CSF of mice with lipopolysaccharide-induced fever (+0.8°C) were higher than

  5. Cyclic ADP-Ribose and Heat Regulate Oxytocin Release via CD38 and TRPM2 in the Hypothalamus during Social or Psychological Stress in Mice

    PubMed Central

    Zhong, Jing; Amina, Sarwat; Liang, Mingkun; Akther, Shirin; Yuhi, Teruko; Nishimura, Tomoko; Tsuji, Chiharu; Tsuji, Takahiro; Liu, Hong-Xiang; Hashii, Minako; Furuhara, Kazumi; Yokoyama, Shigeru; Yamamoto, Yasuhiko; Okamoto, Hiroshi; Zhao, Yong Juan; Lee, Hon Cheung; Tominaga, Makoto; Lopatina, Olga; Higashida, Haruhiro

    2016-01-01

    Hypothalamic oxytocin (OT) is released into the brain by cyclic ADP-ribose (cADPR) with or without depolarizing stimulation. Previously, we showed that the intracellular free calcium concentration ([Ca2+]i) that seems to trigger OT release can be elevated by β-NAD+, cADPR, and ADP in mouse oxytocinergic neurons. As these β-NAD+ metabolites activate warm-sensitive TRPM2 cation channels, when the incubation temperature is increased, the [Ca2+]i in hypothalamic neurons is elevated. However, it has not been determined whether OT release is facilitated by heat in vitro or hyperthermia in vivo in combination with cADPR. Furthermore, it has not been examined whether CD38 and TRPM2 exert their functions on OT release during stress or stress-induced hyperthermia in relation to the anxiolytic roles and social behaviors of OT under stress conditions. Here, we report that OT release from the isolated hypothalami of male mice in culture was enhanced by extracellular application of cADPR or increasing the incubation temperature from 35°C to 38.5°C, and simultaneous stimulation showed a greater effect. This release was inhibited by a cADPR-dependent ryanodine receptor inhibitor and a nonspecific TRPM2 inhibitor. The facilitated release by heat and cADPR was suppressed in the hypothalamus isolated from CD38 knockout mice and CD38- or TRPM2-knockdown mice. In the course of these experiments, we noted that OT release differed markedly between individual mice under stress with group housing. That is, when male mice received cage-switch stress and eliminated due to their social subclass, significantly higher levels of OT release were found in subordinates compared with ordinates. In mice exposed to anxiety stress in an open field, the cerebrospinal fluid (CSF) OT level increased transiently at 5 min after exposure, and the rectal temperature also increased from 36.6°C to 37.8°C. OT levels in the CSF of mice with lipopolysaccharide-induced fever (+0.8°C) were higher than those

  6. Development of an electrochemical biosensor for the detection of an ADP-ribosylating toxin, exo A from Pseudomonas aeruginosa

    NASA Astrophysics Data System (ADS)

    Enríquez, Y.; Negrón, Y.; Navarreto, M.; Guadalupe, A. R.

    2013-03-01

    A free radical copolymerization of Styrene (Sty) and acrylic acid N-hydroxysuccinimide ester (NAS) has been done in a range of 10:90 to 90:10 (Sty:NAS) molar ratios. The FT-IR spectra for all seven copolymers showed the absorption peaks for the carbonyl signals of the ester and the amide in NAS (1773 cm-1 and 1727 cm-1 respectively), the styrene aromatic signal (1494 cm-1) and the disappearance of the absorption peak for the vinyl group in both monomers (1629 cm-1). HPLC-UV results showed an increment in the average molecular weight with an increase in the molar ratio of the styrene monomer, from 1528.51 g/mol for 10:90 Sty:NAS to 7141.67 g/mol for 90:10 Sty:NAS. These copolymers will be used to generate films on carbon surfaces to anchor a β-NAD+ electroactive analog. Also, a Ferrocene-labeled NAAD (Fc-NAAD) was prepared by attaching Ferrocene Succinimide (Fc-NHS) to the primary amine in the adenine moiety of the cofactor. Osteryoung Squatre Wave Voltammetry (OSWV) of the new Fc-NAAD showed an anodic peak in 320 mV and the cyclic voltammetry (CV) showed chemical reversibility and electrochemical quasi-reversibility.

  7. Are genetic variations in OXTR, AVPR1A, and CD38 genes important to social integration? Results from two large U.S. cohorts

    PubMed Central

    Chang, Shun-Chiao; Glymour, M Maria; Rewak, Marissa; Cornelis, Marilyn; Walter, Stefan; Koenen, Karestan C; Kawachi, Ichiro; Liang, Liming; Tchetgen, Eric Tchetgen; Kubzansky, Laura D.

    2013-01-01

    Some evidence suggests that genetic polymorphisms in oxytocin pathway genes influence various social behaviors, but findings thus far have been mixed. Many studies have been based in small samples and there is possibility of publication bias. Using data from 2 large U.S. prospective cohorts with over 11,000 individuals, we investigated 88 SNPs in OXTR, AVPR1A, and CD38, in relation to social integration (measured as social connectedness in both binary and continuous forms and being continuously married). After correction for multiple testing only one SNP in CD38 (rs12644506) was significantly associated with social integration and that SNP predicted when using a dichotomized indicator of social connectedness (adjusted p=0.02), but not a continuous measure of social connectedness or the continuously married outcome. A significant gender-heterogeneous effect was identified in one OXTR SNP on dichotomized social connectedness; specifically, rs4686302 T allele was nominally associated with social connectedness in men, whereas the association direction was opposite in women (adjusted gender heterogeneity p=0.02). Furthermore, the rs53576 A allele was significantly associated with social connectedness only in women, and the effect magnitude was stronger in a dominant genetic model (adjusted p=0.003). In summary, our findings suggested that common genetic variants of OXTR, CD38, and AVPR1A are not associated with social integration as measured in this study using the simplified Berkman-Syme Social Network Index, but these findings and other work hint that effects may be modified by gender or other social experiences. Further work considering genetic pathways in relation to social integration may be more fruitful if these additional factors can be more comprehensively evaluated. PMID:24209975

  8. High proportion of CD95(+) and CD38(+) in cultured CD8(+) T cells predicts acute rejection and infection, respectively, in kidney recipients.

    PubMed

    Mancebo, Esther; Castro, María José; Allende, Luís M; Talayero, Paloma; Brunet, Mercè; Millán, Olga; Guirado, Luís; López-Hoyos, Marcos; San Segundo, David; Rodrigo, Emilio; Muñoz, Pedro; Boix Giner, Francisco; Llorente Viñas, Santiago; Muro-Amador, Manuel; Paz-Artal, Estela

    2016-02-01

    The aim of this study was to find noninvasive T-cell markers able to predict rejection or infection risk after kidney transplantation. We prospectively examined T-lymphocyte subsets after cell culture stimulation (according to CD38, CD69, CD95, CD40L, and CD25 expression) in 79 first graft recipients from four centers, before and after transplantation. Patients were followed up for one year. Patients who rejected within month-1 (n=10) showed high pre-transplantation and week-1 post-transplantation percentages of CD95(+), in CD4(+) and CD8(+) T-cells (P<0.001 for all comparisons). These biomarkers conferred independent risk for early rejection (HR:5.05, P=0.061 and HR:75.31, P=0.004; respectively). The cut-off values were able to accurately discriminate between rejectors and non-rejectors and Kaplan-Meier curves showed significantly different free-of-rejection time rates (P<0.005). Patients who rejected after the month-1 (n=4) had a higher percentage of post-transplantation CD69(+) in CD8(+) T-cells than non-rejectors (P=0.002). Finally, patients with infection (n=41) previously showed higher percentage of CD38(+) in CD8(+) T-cells at all post-transplantation times evaluated, being this increase more marked in viral infections. A cut-off of 59% CD38(+) in CD8(+) T-cells at week-1, week-2 and month-2 reached 100% sensitivity for the detection of subsequent viral infections. In conclusion, predictive biomarkers of rejection and infection risk after transplantation were detected that could be useful for the personalized care of kidney recipients.

  9. CD56brightCD16- NK Cells Produce Adenosine through a CD38-Mediated Pathway and Act as Regulatory Cells Inhibiting Autologous CD4+ T Cell Proliferation.

    PubMed

    Morandi, Fabio; Horenstein, Alberto L; Chillemi, Antonella; Quarona, Valeria; Chiesa, Sabrina; Imperatori, Andrea; Zanellato, Silvia; Mortara, Lorenzo; Gattorno, Marco; Pistoia, Vito; Malavasi, Fabio

    2015-08-01

    Recent studies suggested that human CD56(bright)CD16(-) NK cells may play a role in the regulation of the immune response. Since the mechanism(s) involved have not yet been elucidated, in the present study we have investigated the role of nucleotide-metabolizing enzymes that regulate the extracellular balance of nucleotides/nucleosides and produce the immunosuppressive molecule adenosine (ADO). Peripheral blood CD56(dim)CD16(+) and CD56(bright)CD16(-) NK cells expressed similar levels of CD38. CD39, CD73, and CD157 expression was higher in CD56(bright)CD16(-) than in CD56(dim)CD16(+) NK cells. CD57 was mostly expressed by CD56(dim)CD16(+) NK cells. CD203a/PC-1 expression was restricted to CD56(bright)CD16(-) NK cells. CD56(bright)CD16(-) NK cells produce ADO and inhibit autologous CD4(+) T cell proliferation. Such inhibition was 1) reverted pretreating CD56(bright)CD16(-) NK cells with a CD38 inhibitor and 2) increased pretreating CD56(bright)CD16(-) NK cells with a nucleoside transporter inhibitor, which increase extracellular ADO concentration. CD56(bright)CD16(-) NK cells isolated from the synovial fluid of juvenile idiopathic arthritis patients failed to inhibit autologous CD4(+) T cell proliferation. Such functional impairment could be related to 1) the observed reduced CD38/CD73 expression, 2) a peculiar ADO production kinetics, and 3) a different expression of ADO receptors. In contrast, CD56(bright)CD16(-) NK cells isolated from inflammatory pleural effusions display a potent regulatory activity. In conclusion, CD56(bright)CD16(-) NK cells act as "regulatory cells" through ADO produced by an ectoenzymes network, with a pivotal role of CD38. This function may be relevant for the modulation of the immune response in physiological and pathological conditions, and it could be impaired during autoimmune/inflammatory diseases. PMID:26091716

  10. Insights into the Mechanism of Bovine CD38/NAD+Glycohydrolase from the X-Ray Structures of Its Michaelis Complex and Covalently-Trapped Intermediates

    PubMed Central

    Egea, Pascal F.; Muller-Steffner, Hélène; Kuhn, Isabelle; Cakir-Kiefer, Céline; Oppenheimer, Norman J.; Stroud, Robert M.; Kellenberger, Esther; Schuber, Francis

    2012-01-01

    Bovine CD38/NAD+glycohydrolase (bCD38) catalyses the hydrolysis of NAD+ into nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose (cADPR). We solved the crystal structures of the mono N-glycosylated forms of the ecto-domain of bCD38 or the catalytic residue mutant Glu218Gln in their apo state or bound to aFNAD or rFNAD, two 2′-fluorinated analogs of NAD+. Both compounds behave as mechanism-based inhibitors, allowing the trapping of a reaction intermediate covalently linked to Glu218. Compared to the non-covalent (Michaelis) complex, the ligands adopt a more folded conformation in the covalent complexes. Altogether these crystallographic snapshots along the reaction pathway reveal the drastic conformational rearrangements undergone by the ligand during catalysis with the repositioning of its adenine ring from a solvent-exposed position stacked against Trp168 to a more buried position stacked against Trp181. This adenine flipping between conserved tryptophans is a prerequisite for the proper positioning of the N1 of the adenine ring to perform the nucleophilic attack on the C1′ of the ribofuranoside ring ultimately yielding cADPR. In all structures, however, the adenine ring adopts the most thermodynamically favorable anti conformation, explaining why cyclization, which requires a syn conformation, remains a rare alternate event in the reactions catalyzed by bCD38 (cADPR represents only 1% of the reaction products). In the Michaelis complex, the substrate is bound in a constrained conformation; the enzyme uses this ground-state destabilization, in addition to a hydrophobic environment and desolvation of the nicotinamide-ribosyl bond, to destabilize the scissile bond leading to the formation of a ribooxocarbenium ion intermediate. The Glu218 side chain stabilizes this reaction intermediate and plays another important role during catalysis by polarizing the 2′-OH of the substrate NAD+. Based on our structural analysis and data on active site mutants

  11. 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

    PubMed Central

    Beste, Kerstin Y.; Spangler, Corinna M.; Burhenne, Heike; Koch, Karl-Wilhelm; Shen, Yuequan; Tang, Wei-Jen; Kaever, Volkhard; Seifert, Roland

    2013-01-01

    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. PMID:23922959

  12. Low oxygen tension favored expansion and hematopoietic reconstitution of CD34(+) CD38(-) cells expanded from human cord blood-derived CD34(+) Cells.

    PubMed

    Wang, Ziyan; Du, Zheng; Cai, Haibo; Ye, Zhaoyang; Fan, Jinli; Tan, Wen-Song

    2016-07-01

    Oxygen tension is an important factor that regulates hematopoietic stem cells (HSCs) in both in vivo hematopoietic microenvironment and ex vivo culture system. Although the effect of oxygen tension on ex vivo expansion of HSCs was extensively studied, there were no clear descriptions on physiological function and gene expression analysis of HSCs under different oxygen tensions. In this study, the effects of oxygen tension on ex vivo expansion characteristics of human umbilical cord blood (UCB)-derived CD34(+) cells are evaluated. Moreover, the physiological function of expanded CD34(+) cells was assessed by secondary expansion ability ex vivo and hematopoietic reconstitution ability in vivo. Also, genetic profiling was applied to analyze the expression of genes related to cell function. It was found that low oxygen tension favored expansion of CD34(+) CD38(-) cells. Additionally, CD34(+) cells expanded under low oxygen tension showed better secondary expansion ability and reconstitution ability than those under atmospheric oxygen concentration. Finally, the genetic profiling of CD34(+) CD38(-) cells cultured under low oxygen tension was more akin to freshly isolated cells. These results collectively demonstrate that low oxygen tension was able to better maintain both self-renewal and hematopoietic reconstitution potential and may lay an experimental basis for clinical transplantation of HSCs.

  13. T. vaginalis Infection Is Associated with Increased IL-8 and TNFr1 Levels but with the Absence of CD38 and HLADR Activation in the Cervix of ESN

    PubMed Central

    Brady, Kirsten E.; Plants, Jill; Landay, Alan L.; Ghassemi, Mahmood; Golub, Elizabeth T.; Spear, Greg T.

    2015-01-01

    Introduction Trichomonas vaginalis infection is associated with an increased risk of HIV infection in exposed-seronegative women (ESN) despite their unique immune quiescent profile. It is important to understand possible mechanisms, such as recruitment of activated T cells, by which T. vaginalis could facilitate HIV infection in this population. Methods We conducted a cross-sectional study exploring the relationships between T. vaginalis infection, inflammatory markers and T cell activation in the cervix of ESN. During scheduled study visits, participants completed a behavioral questionnaire and physical exam, including sexually transmitted infection (STI) screening and collection of endocervical sponge and cytobrush specimens. T cell and monocyte phenotypes were measured in cervical cytobrush specimens using multi-parameter flow cytometry. Cervical sponge specimens were used to measure cytokines (IL-6, IL-8,IL-10, IP-10, RANTES) using Luminex immunoassays and the immune activation marker soluble TNF receptor 1 using ELISA. Results Specimens of 65 women were tested. Twenty-one of these women were infected with T. vaginalis. T. vaginalis infection was associated with significantly increased concentrations of IL-8 (1275pg/ml vs. 566pg/ml, p=.02) and sTNFr1 (430 pg/ml vs. 264 pg/ml, p=.005). However, T. vaginalis infection was not associated with increased percent expression of CCR5+ T cells nor increased CD38 and HLADR activation compared to uninfected women. It was also not associated with increased expression of CCR5+ monocytes. Conclusions Among ESN T. vaginalis infection is associated with increased levels of genital pro-inflammatory/immune activation markers IL-8 and TNFr1, but was not associated with an increased percentage of activated endocervical T cells along the CD38 and HLADR pathways. Thus, while T.vaginalis infection may result in some reversal of the immune quiescent profile of ESN, enhanced recruitment of activated CD38 and HLADR expressing CD4+ cells

  14. Bifunctional Homodimeric Triokinase/FMN Cyclase

    PubMed Central

    Rodrigues, Joaquim Rui; Couto, Ana; Cabezas, Alicia; Pinto, Rosa María; Ribeiro, João Meireles; Canales, José; Costas, María Jesús; Cameselle, José Carlos

    2014-01-01

    Mammalian triokinase, which phosphorylates exogenous dihydroxyacetone and fructose-derived glyceraldehyde, is neither molecularly identified nor firmly associated to an encoding gene. Human FMN cyclase, which splits FAD and other ribonucleoside diphosphate-X compounds to ribonucleoside monophosphate and cyclic X-phosphodiester, is identical to a DAK-encoded dihydroxyacetone kinase. This bifunctional protein was identified as triokinase. It was modeled as a homodimer of two-domain (K and L) subunits. Active centers lie between K1 and L2 or K2 and L1: dihydroxyacetone binds K and ATP binds L in different subunits too distant (≈14 Å) for phosphoryl transfer. FAD docked to the ATP site with ribityl 4′-OH in a possible near-attack conformation for cyclase activity. Reciprocal inhibition between kinase and cyclase reactants confirmed substrate site locations. The differential roles of protein domains were supported by their individual expression: K was inactive, and L displayed cyclase but not kinase activity. The importance of domain mobility for the kinase activity of dimeric triokinase was highlighted by molecular dynamics simulations: ATP approached dihydroxyacetone at distances below 5 Å in near-attack conformation. Based upon structure, docking, and molecular dynamics simulations, relevant residues were mutated to alanine, and kcat and Km were assayed whenever kinase and/or cyclase activity was conserved. The results supported the roles of Thr112 (hydrogen bonding of ATP adenine to K in the closed active center), His221 (covalent anchoring of dihydroxyacetone to K), Asp401 and Asp403 (metal coordination to L), and Asp556 (hydrogen bonding of ATP or FAD ribose to L domain). Interestingly, the His221 point mutant acted specifically as a cyclase without kinase activity. PMID:24569995

  15. Removal of inhibitory substances with recombinant fibronectin-CH-296 plates enhances the retroviral transduction efficiency of CD34(+)CD38(-) bone marrow cells.

    PubMed

    Chono, H; Yoshioka, H; Ueno, M; Kato, I

    2001-09-01

    In retroviral gene transduction, the efficiency of viral infection was reduced by the proteoglycans and some other materials secreted by the producer lines. In order to remove these inhibitors we have developed the rFN-CH-296-facilitated protocol. Because the rFN-CH-296 molecule has strong ability to bind a retroviral vector, rFN-CH-296 bound plates are utilized to wash out the unbound putative inhibitors present in a virus supernatant. The gene transduction efficiencies of human CD34(+)CD38(-) BMCs with a GALV-pseudotyped vector and the rFN-CH-296-facilitated protocol were compared with the protocol without a coating plate with CH-296, the mean gene transduction efficiencies being found to be 95.5 and 71.1%, respectively.

  16. Another Look at the Origin of the Narrow Metal Lines in the Ultraviolet Spectrum of the White Dwarf CD-38 10980

    NASA Astrophysics Data System (ADS)

    Dupuis, Jean; Chayer, P.

    2013-01-01

    The origin of a system of metal lines observed in addition to interstellar lines in the hot DA star CD-38 10980 has been subject to debates. The interpretation of the high dispersion ultraviolet spectra, originally obtained by IUE, led to evidence in favor of the circumstellar origin of the observed silicon and carbon lines although the alternate view arguing for a photospheric origin of the same lines was still defended by some groups. This circumstellar interpretation was supported by the evidence of a significant velocity shift of these lines relative to what was then assumed to be the radial velocity of the white dwarf. In order to explore and possibly settle this question, we have performed an analysis of the HST STIS and GHRS spectra of this white dwarf. These spectra should, in principle, have a more reliable and accurate calibration of the wavelength solution and lead to improved velocity measurements of the detected metal lines. Our analysis suggests that there was an unaccounted zero-point offset in the IUE spectra which led to spurious velocity shifts. Our new results show that when we adjust the velocity of the interstellar lines detected in the IUE spectrum to match those of the interstellar lines measured in the GHRS and STIS spectra, we get an excellent agreement of the velocity of the metal lines with the extensive radial velocity measurements made by Maxted et al. (2000) for this white dwarf. This, in our opinion, is definitely confirming the photospheric origin of the metal lines in CD-38 10980. The measured abundances of silicon and carbon are in excellent agreement with the predictions of the radiative levitation theory.

  17. Upregulation of CD19⁺CD24(hi)CD38(hi) regulatory B cells is associated with a reduced risk of acute lung injury in elderly pneumonia patients.

    PubMed

    Song, Haihan; Xi, Jianjun; Li, Guang-Gang; Xu, Shumin; Wang, Chunmei; Cheng, Tingting; Li, Hongqiang; Zhang, Ying; Liu, Xiandong; Bai, Jianwen

    2016-04-01

    Acute lung injury (ALI) is a common complication in elderly pneumonia patients who have a rapid progression, and is accompanied by a high mortality rate. Because the treatment options of ALI are limited to supportive care, identifying pneumonia patients who are at higher risk of ALI development is the emphasis of many studies. Here, we approach this problem from an immunological perspective by examining CD19(+)CD24(hi)CD38(hi) B cells, an important participant in acute and chronic inflammation. We find that elderly pneumonia patients have elevated CD19(+)CD24(hi)CD38(hi) B cell frequency compared to healthy individuals. This B cell population may express a higher level of IL-10, which has been was shown to suppress CD4(+) T cell-mediated proinflammatory cytokine interferon gamma (IFNg) and tumor necrosis factor alpha (TNFa) production, through an IL-10-dependent mechanism. We also observe that the frequency of CD19(+)CD24(hi)CD38(hi) B cell is positively correlated with the frequency of CD4(+)CD25(+)Foxp3(+)Tregs in peripheral blood. Moreover, consistent with CD19(+)CD24(hi)CD38(hi) B cell's anti-inflammatory role, we find that pneumonia patients who later developed ALI have reduced level of CD19(+)CD24(hi)CD38(hi) B cells. Together, our results demonstrated that CD19(+)CD24(hi)CD38(hi) B cells in pneumonia patients possess regulatory function in vivo, and are associated with a reduced ALI risk.

  18. Reduced CD5(+) CD24(hi) CD38(hi) and interleukin-10(+) regulatory B cells in active anti-neutrophil cytoplasmic autoantibody-associated vasculitis permit increased circulating autoantibodies.

    PubMed

    Aybar, L T; McGregor, J G; Hogan, S L; Hu, Y; Mendoza, C E; Brant, E J; Poulton, C J; Henderson, C D; Falk, R J; Bunch, D O

    2015-05-01

    Pathogenesis of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis is B cell-dependent, although how particular B cell subsets modulate immunopathogenesis remains unknown. Although their phenotype remains controversial, regulatory B cells (Bregs ), play a role in immunological tolerance via interleukin (IL)-10. Putative CD19(+) CD24(hi) CD38(hi) and CD19(+) CD24(hi) CD27(+) Bregs were evaluated in addition to their CD5(+) subsets in 69 patients with ANCA-associated vasculitis (AAV). B cell IL-10 was verified by flow cytometry following culture with CD40 ligand and cytosine-phosphate-guanosine (CpG) DNA. Patients with active disease had decreased levels of CD5(+) CD24(hi) CD38(hi) B cells and IL-10(+) B cells compared to patients in remission and healthy controls (HCs). As IL-10(+) and CD5(+) CD24(hi) CD38(hi) B cells normalized in remission within an individual, ANCA titres decreased. The CD5(+) subset of CD24(hi) CD38(hi) B cells decreases in active disease and rebounds during remission similarly to IL-10-producing B cells. Moreover, CD5(+) B cells are enriched in the ability to produce IL-10 compared to CD5(neg) B cells. Together these results suggest that CD5 may identify functional IL-10-producing Bregs . The malfunction of Bregs during active disease due to reduced IL-10 expression may thus permit ANCA production.

  19. Reduced CD5+CD24hiCD38hi and interleukin-10+ regulatory B cells in active anti-neutrophil cytoplasmic autoantibody-associated vasculitis permit increased circulating autoantibodies

    PubMed Central

    Aybar, L T; McGregor, J G; Hogan, S L; Hu, Y; Mendoza, C E; Brant, E J; Poulton, C J; Henderson, C D; Falk, R J; Bunch, D O

    2015-01-01

    Pathogenesis of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis is B cell-dependent, although how particular B cell subsets modulate immunopathogenesis remains unknown. Although their phenotype remains controversial, regulatory B cells (Bregs), play a role in immunological tolerance via interleukin (IL)-10. Putative CD19+CD24hiCD38hi and CD19+CD24hiCD27+ Bregs were evaluated in addition to their CD5+ subsets in 69 patients with ANCA-associated vasculitis (AAV). B cell IL-10 was verified by flow cytometry following culture with CD40 ligand and cytosine–phosphate–guanosine (CpG) DNA. Patients with active disease had decreased levels of CD5+CD24hiCD38hi B cells and IL-10+ B cells compared to patients in remission and healthy controls (HCs). As IL-10+ and CD5+CD24hiCD38hi B cells normalized in remission within an individual, ANCA titres decreased. The CD5+ subset of CD24hiCD38hi B cells decreases in active disease and rebounds during remission similarly to IL-10-producing B cells. Moreover, CD5+ B cells are enriched in the ability to produce IL-10 compared to CD5neg B cells. Together these results suggest that CD5 may identify functional IL-10-producing Bregs. The malfunction of Bregs during active disease due to reduced IL-10 expression may thus permit ANCA production. PMID:25376552

  20. Reduced CD5(+) CD24(hi) CD38(hi) and interleukin-10(+) regulatory B cells in active anti-neutrophil cytoplasmic autoantibody-associated vasculitis permit increased circulating autoantibodies.

    PubMed

    Aybar, L T; McGregor, J G; Hogan, S L; Hu, Y; Mendoza, C E; Brant, E J; Poulton, C J; Henderson, C D; Falk, R J; Bunch, D O

    2015-05-01

    Pathogenesis of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis is B cell-dependent, although how particular B cell subsets modulate immunopathogenesis remains unknown. Although their phenotype remains controversial, regulatory B cells (Bregs ), play a role in immunological tolerance via interleukin (IL)-10. Putative CD19(+) CD24(hi) CD38(hi) and CD19(+) CD24(hi) CD27(+) Bregs were evaluated in addition to their CD5(+) subsets in 69 patients with ANCA-associated vasculitis (AAV). B cell IL-10 was verified by flow cytometry following culture with CD40 ligand and cytosine-phosphate-guanosine (CpG) DNA. Patients with active disease had decreased levels of CD5(+) CD24(hi) CD38(hi) B cells and IL-10(+) B cells compared to patients in remission and healthy controls (HCs). As IL-10(+) and CD5(+) CD24(hi) CD38(hi) B cells normalized in remission within an individual, ANCA titres decreased. The CD5(+) subset of CD24(hi) CD38(hi) B cells decreases in active disease and rebounds during remission similarly to IL-10-producing B cells. Moreover, CD5(+) B cells are enriched in the ability to produce IL-10 compared to CD5(neg) B cells. Together these results suggest that CD5 may identify functional IL-10-producing Bregs . The malfunction of Bregs during active disease due to reduced IL-10 expression may thus permit ANCA production. PMID:25376552

  1. The expression of molecule CD28 and CD38 on CD4⁺/CD8⁺ T lymphocytes in thymus and spleen elicited by Schistosoma japonicum infection in mice model.

    PubMed

    Li, Na; Ji, Peng-yu; Song, Lan-gui; Lei, Jun-xia; Lv, Zhi-yue; Wu, Zhong-dao; Shao, Xiao; Sun, Xi

    2015-08-01

    Schistosomiasis caused by human schistosomes such as Schistosoma japonicum (S. japonicum) is considered as an immune-related disease. It was demonstrated that specific cytokine antibodies' response elicited by S. japonicum infection was gradually downregulated with the progress of the disease, resulting in a Th1/Th2 polarization and suppression of immune response. CD28 (cluster of differentiation 28) is one of the proteins expressed on T cells that provide co-stimulatory signals required for T cell activation and survival, and CD38 is an activating marker of T lymphocyte with high expression in many acute or chronic infections. The immune signature of CD28null T cells in the peripheral circulation associates with chronic inflammation in many diseases, such as HIV and CMV infection. In the thymus, CD28 expression on developing thymocytes appears to play a role for their selection, and it synergizes with CD38 to induce apoptosis of DP (double-positive) thymocytes. Few reports about CD28 and CD38 have been published in schistosomiasis. Here, we investigated the dynamic patterns of the expression of molecules CD28 and CD38 on CD4(+)/CD8(+) T lymphocytes of the thymus and spleen in mice model with S. japonicum infection. Our data indicated that at an early period of infection, the frequency of CD8(+)CD28(-) T cell in the spleen decreased significantly, but higher at chronic infection than that in control. However, it demonstrated an increasing trend in the thymus with the progression of infection. The frequency of CD4(+)CD28(-) T cells increased from acute infection in the thymus, while from chronic infection in the spleen. The expression of CD38 on CD8(+) T cells began to increase at 4 weeks post infection both in the thymus and spleen; its elevated expression on CD4(+) T cells emerged at 6 weeks post infection in the thymus and at 10 weeks post infection in the spleen. Praziquantel (PZQ) treatment could partially restore the frequency of CD28(+) T cell of CD4(+) T

  2. Toll-like receptor 7 cooperates with IL-4 in activated B cells through antigen receptor or CD38 and induces class switch recombination and IgG1 production.

    PubMed

    Tsukamoto, Yumiko; Nagai, Yoshinori; Kariyone, Ai; Shibata, Takuma; Kaisho, Tsuneyasu; Akira, Shizuo; Miyake, Kensuke; Takatsu, Kiyoshi

    2009-04-01

    IL-4 and 8-mercaptoguanosine (8-SGuo) stimulation of CD38-activated B cells induces mu to gamma1 class switch recombination (CSR) at the DNA level leading to a high level of IgG1 production. Although some of signaling events initiated by IL-4 in activated B cells have been characterized, the involvement of TLR/MyD88 and Btk pathway in IL-4-dependent mu to gamma1 CSR has not been thoroughly evaluated. In this study, we characterized receptors for 8-SGuo and differential roles of 8-SGuo and IL-4 in the induction and mu to gamma1 CSR and IgG1 production. The role of TLR7 and MyD88 in 8-SGuo-induced AID expression and mu to gamma1 CSR was documented, as 8-SGuo did not act on CD38-stimulated splenic B cells from Tlr7(-/-) and Myd88(-/-) mice. CD38-activated B cells from Btk-deficient mice failed to respond to TLR7 ligands for the AID expression and CSR, indicating that Btk is also indispensable for the system. Stimulation of CD38-activated B cells with 8-SGuo induced significant AID expression and DNA double strand breaks, but IL-4 stimulation by itself did not trigger mu to gamma1 CSR. Intriguingly, the mu to gamma1 CSR in the B cells stimulated with CD38 and 8-SGuo totally depends on IL-4 stimulation. Similar results were obtained in the activated B cells through BCR and loxoribine, a well-known TLR7 ligand, in place of 8-SGuo. In vivo administration of TLR7 ligand and anti-CD38 antibody induced the generation of CD138(+) IgG1(+) cells. These results indicate that TLR7 is a receptor for 8-SGuo and plays an essential role in the AID and Blimp-1 expression; however it is not enough to complete mu to gamma1 CSR in CD38-activated B cells. IL-4 may be required for the induction of DNA repair system together with AID for the completion of CSR.

  3. Renal Transplant Recipients Treated with Calcineurin-Inhibitors Lack Circulating Immature Transitional CD19+CD24hiCD38hi Regulatory B-Lymphocytes

    PubMed Central

    Tebbe, Bastian; Wilde, Benjamin; Ye, Zeng; Wang, Junyu; Wang, Xinning; Jian, Fu; Dolff, Sebastian; Schedlowski, Manfred; Hoyer, Peter F.; Kribben, Andreas; Witzke, Oliver; Hoerning, André

    2016-01-01

    Background CD19+CD24hiCD38hi transitional immature B-lymphocytes have been demonstrated to play an important role in regulating the alloimmune response in transplant recipients. Here, we analyzed the effect of calcineurin inhibition on these peripherally circulating regulatory B-cells (Breg) in renal transplant recipients receiving cyclosporine A (CsA) or tacrolimus. Methods PBMCs from healthy subjects (HS) (n = 16) and renal transplant recipients (n = 46) were isolated. Flow cytometry was performed for CD19, CD24, CD38 and IL-10 either after isolation or after 72 hours of co-culture in presence of PMA/Ionomycin and TLR9-ligand in presence or absence of increasing concentrations of tacrolimus or CsA. Results The amount of CD19+ B-cells among lymphocytes was ∼9.1% in HS, ∼3.6% in CsA (n = 11, p<0.05) and ∼6.4% in TAC (n = 35, p<0.05) treated patients. Among B-cells, a distinct subset of Breg was found to be 4.7% in HS, 1.4% in tacrolimus treated patients and almost blunted in patients receiving CsA. Similarily, ∼4% of B-cells in HS and even fewer in CsA or tacrolimus treated patients produced IL-10 (0.5% and 1.5%, p<0.05) and this was confirmed both in non-transplanted CsA-treated healthy subjects and in in vitro co-culture experiments. Among 29 patients with <1% of Breg, 9 cases (31%) displayed an allograft rejection in contrast to only one case of rejection (6%) among 17 patients with >1%. Conclusion Calcineurin inhibitors reduce number and IL-10 production of Bregs in the peripheral circulation of both renal transplant recipients and non-transplanted healthy subjects. CNI induced Breg reduction is not restricted to a solid organ transplant setting and is not mediated by co-medication with steroids or MPA. A low proportion of Breg cells is associated with an elevated frequency of allograft rejection events. PMID:27045291

  4. Diterpene Cyclases and the Nature of the Isoprene Fold

    PubMed Central

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

    2013-01-01

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

  5. Soluble Adenylyl Cyclase in Health and Disease

    PubMed Central

    Schmid, Andreas; Meili, Dimirela; Salathe, Matthias

    2014-01-01

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

  6. Membrane guanylyl cyclase receptors: an update

    PubMed Central

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

    2007-01-01

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

  7. Adenylyl cyclases in the digestive system.

    PubMed

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

    2014-06-01

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

  8. Progesterone Levels Associate with a Novel Population of CCR5+CD38+ CD4 T Cells Resident in the Genital Mucosa with Lymphoid Trafficking Potential.

    PubMed

    Swaims-Kohlmeier, Alison; Haaland, Richard E; Haddad, Lisa B; Sheth, Anandi N; Evans-Strickfaden, Tammy; Lupo, L Davis; Cordes, Sarah; Aguirre, Alfredo J; Lupoli, Kathryn A; Chen, Cheng-Yen; Ofotukun, Igho; Hart, Clyde E; Kohlmeier, Jacob E

    2016-07-01

    The female genital tract (FGT) provides a means of entry to pathogens, including HIV, yet immune cell populations at this barrier between host and environment are not well defined. We initiated a study of healthy women to characterize resident T cell populations in the lower FGT from lavage and patient-matched peripheral blood to investigate potential mechanisms of HIV sexual transmission. Surprisingly, we observed FGT CD4 T cell populations were primarily CCR7(hi), consistent with a central memory or recirculating memory T cell phenotype. In addition, roughly half of these CCR7(hi) CD4 T cells expressed CD69, consistent with resident memory T cells, whereas the remaining CCR7(hi) CD4 T cells lacked CD69 expression, consistent with recirculating memory CD4 T cells that traffic between peripheral tissues and lymphoid sites. HIV susceptibility markers CCR5 and CD38 were increased on FGT CCR7(hi) CD4 T cells compared with blood, yet migration to the lymphoid homing chemokines CCL19 and CCL21 was maintained. Infection with GFP-HIV showed that FGT CCR7(hi) memory CD4 T cells are susceptible HIV targets, and productive infection of CCR7(hi) memory T cells did not alter chemotaxis to CCL19 and CCL21. Variations of resident CCR7(hi) FGT CD4 T cell populations were detected during the luteal phase of the menstrual cycle, and longitudinal analysis showed the frequency of this population positively correlated to progesterone levels. These data provide evidence women may acquire HIV through local infection of migratory CCR7(hi) CD4 T cells, and progesterone levels predict opportunities for HIV to access these novel target cells.

  9. Progesterone Levels Associate with a Novel Population of CCR5+CD38+ CD4 T Cells Resident in the Genital Mucosa with Lymphoid Trafficking Potential.

    PubMed

    Swaims-Kohlmeier, Alison; Haaland, Richard E; Haddad, Lisa B; Sheth, Anandi N; Evans-Strickfaden, Tammy; Lupo, L Davis; Cordes, Sarah; Aguirre, Alfredo J; Lupoli, Kathryn A; Chen, Cheng-Yen; Ofotukun, Igho; Hart, Clyde E; Kohlmeier, Jacob E

    2016-07-01

    The female genital tract (FGT) provides a means of entry to pathogens, including HIV, yet immune cell populations at this barrier between host and environment are not well defined. We initiated a study of healthy women to characterize resident T cell populations in the lower FGT from lavage and patient-matched peripheral blood to investigate potential mechanisms of HIV sexual transmission. Surprisingly, we observed FGT CD4 T cell populations were primarily CCR7(hi), consistent with a central memory or recirculating memory T cell phenotype. In addition, roughly half of these CCR7(hi) CD4 T cells expressed CD69, consistent with resident memory T cells, whereas the remaining CCR7(hi) CD4 T cells lacked CD69 expression, consistent with recirculating memory CD4 T cells that traffic between peripheral tissues and lymphoid sites. HIV susceptibility markers CCR5 and CD38 were increased on FGT CCR7(hi) CD4 T cells compared with blood, yet migration to the lymphoid homing chemokines CCL19 and CCL21 was maintained. Infection with GFP-HIV showed that FGT CCR7(hi) memory CD4 T cells are susceptible HIV targets, and productive infection of CCR7(hi) memory T cells did not alter chemotaxis to CCL19 and CCL21. Variations of resident CCR7(hi) FGT CD4 T cell populations were detected during the luteal phase of the menstrual cycle, and longitudinal analysis showed the frequency of this population positively correlated to progesterone levels. These data provide evidence women may acquire HIV through local infection of migratory CCR7(hi) CD4 T cells, and progesterone levels predict opportunities for HIV to access these novel target cells. PMID:27233960

  10. Comparative analysis of plant lycopene cyclases.

    PubMed

    Koc, Ibrahim; Filiz, Ertugrul; Tombuloglu, Huseyin

    2015-10-01

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

  11. Comparative analysis of plant lycopene cyclases.

    PubMed

    Koc, Ibrahim; Filiz, Ertugrul; Tombuloglu, Huseyin

    2015-10-01

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

  12. Effectiveness of combinations of bispecific antibodies for delivering saporin to human acute T-cell lymphoblastic leukaemia cell lines via CD7 and CD38 as cellular target molecules.

    PubMed Central

    Flavell, D. J.; Cooper, S.; Morland, B.; French, R.; Flavell, S. U.

    1992-01-01

    We have investigated the effectiveness of three different F(ab' gamma)2 bispecific antibodies (BsAb) for delivering the ribosome inactivating protein (RIP) saporin via the CD7 or CD38 cell surface molecules to the human T-ALL cell lines HSB-2 and HPB-ALL. Inhibition of 3H-leucine uptake by target cells was used as the parameter of cellular cytotoxicity. Used singly against HSB-2 cells in the presence of varied concentrations of saporin, an anti-CD7 BsAb, (HB2 x DB7-18) and an anti-CD38 BsAb (OKT10 x RabSap), gave 435- and 286-fold increases in saporin toxicity, respectively. For HPB-ALL cells the anti-CD7 BsAb performed poorly giving only an eight-fold increase in toxicity whilst on the same cell line the anti-CD38 BsAb was highly potent giving an 80,000-fold increase in saporin toxicity. A combination of both BsAb used together against HSB-2 cells was ten times more effective, than the best single BsAb HB2 x DB7-18 used alone. Kinetic studies conducted with HSB-2 cells revealed that the BsAb combination also gave an increased rate of protein synthesis inactivation in comparison to either BsAb used alone. These investigations clearly demonstrate a synergistic action when both BsAb are used in combination to target saporin against CD7 and CD38 expressed on the surface of the HSB-2 cell line. PMID:1373293

  13. The triterpene cyclase protein family: a systematic analysis.

    PubMed

    Racolta, Silvia; Juhl, P Benjamin; Sirim, Demet; Pleiss, Jürgen

    2012-08-01

    Triterpene cyclases catalyze a broad range of cyclization reactions to form polycyclic triterpenes. Triterpene cyclases that convert squalene to hopene are named squalene-hopene cyclases (SHC) and triterpene cyclases that convert oxidosqualene are named oxidosqualene cyclases (OSC). Many sequences have been published, but there is only one structure available for each of SHCs and OSCs. Although they catalyze a similar reaction, the sequence similarity between SHCs and OSCs is low. A family classification based on phylogenetic analysis revealed 20 homologous families which are grouped into two superfamilies, SHCs and OSCs. Based on this family assignment, the Triterpene Cyclase Engineering Database (TTCED) was established. It integrates available information on sequence and structure of 639 triterpene cyclases as well as on structurally and functionally relevant amino acids. Family specific multiple sequence alignments were generated to identify the functionally relevant residues. Based on sequence alignments, conserved residues in SHCs and OSCs were analyzed and compared to experimentally confirmed mutational data. Functional schematic models of the central cavities of OSCs and SHCs were derived from structure comparison and sequence conservation analysis. These models demonstrate the high similarity of the substrate binding cavity of SHCs and OSCs and the equivalences of the respective residues. The TTCED is a novel source for comprehensive information on the triterpene cyclase family, including a compilation of previously described mutational data. The schematic models present the conservation analysis in a readily available fashion and facilitate the correlation of residues to a specific function or substrate interaction.

  14. Polymorphism in purified guanylate cyclase from vertebrate rod photoreceptors.

    PubMed Central

    Hayashi, F; Yamazaki, A

    1991-01-01

    Guanylate cyclase from rod photoreceptors of amphibian (toad, Bufo marinus, and frog, Rana catesbeiana) and bovine retinas was solubilized and purified by a single chromatography step on a GTP-agarose column. Silver staining of purified amphibian enzymes in SDS/polyacrylamide gels disclosed a doublet band (110 and 115 kDa), while the bovine enzyme appeared as a singlet band (110 kDa). The identification of these guanylate cyclases was confirmed using three chromatography systems with the purified enzymes. Specific binding to Con A-Sepharose suggested that rod guanylate cyclase is a glycoprotein. Two-dimensional gel electrophoresis of purified toad, frog, and bovine enzymes resolved two, three, and five variants, respectively, that differed in isoelectric point. Two variants of toad guanylate cyclase showed differences in various characterizations. These data suggest multiple mechanisms for regulation of guanylate cyclase activity in vertebrate rod photoreceptors. Images PMID:1675787

  15. Soluble variants of human recombinant glutaminyl cyclase.

    PubMed

    Castaldo, Cristiana; Ciambellotti, Silvia; de Pablo-Latorre, Raquel; Lalli, Daniela; Porcari, Valentina; Turano, Paola

    2013-01-01

    Recombinant human Glutaminyl Cyclase expressed in E. coli is produced as inclusion bodies. Lack of glycosylation is the main origin of its accumulation in insoluble aggregates. Mutation of single isolated hydrophobic amino acids into negative amino acids was not able to circumvent inclusion bodies formation. On the contrary, substitution with carboxyl-terminal residues of two or three aromatic residues belonging to extended hydrophobic patches on the protein surface provided soluble but still active forms of the protein. These mutants could be expressed in isotopically enriched forms for NMR studies and the maximal attainable concentration was sufficient for the acquisition of (1)H-(15)N HSQC spectra that represent the starting point for future drug development projects targeting Alzheimer's disease. PMID:23977104

  16. Adenylate cyclases involvement in pathogenicity, a minireview.

    PubMed

    Costache, Adriana; Bucurenci, Nadia; Onu, Adrian

    2013-01-01

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

  17. Soluble Variants of Human Recombinant Glutaminyl Cyclase

    PubMed Central

    Castaldo, Cristiana; Ciambellotti, Silvia; de Pablo-Latorre, Raquel; Lalli, Daniela; Porcari, Valentina; Turano, Paola

    2013-01-01

    Recombinant human Glutaminyl Cyclase expressed in E. coli is produced as inclusion bodies. Lack of glycosylation is the main origin of its accumulation in insoluble aggregates. Mutation of single isolated hydrophobic amino acids into negative amino acids was not able to circumvent inclusion bodies formation. On the contrary, substitution with carboxyl-terminal residues of two or three aromatic residues belonging to extended hydrophobic patches on the protein surface provided soluble but still active forms of the protein. These mutants could be expressed in isotopically enriched forms for NMR studies and the maximal attainable concentration was sufficient for the acquisition of 1H-15N HSQC spectra that represent the starting point for future drug development projects targeting Alzheimer’s disease. PMID:23977104

  18. Diuretics and the renal adenylate cyclase system

    PubMed Central

    Dawborn, J.K.; Macneil, S.; Martin, T.J.

    1977-01-01

    1 The relationship between the diuretic effectiveness and the effect on the renal adenylate cyclase of three diuretics, acetazolamide, frusemide and ethacrynic acid, was examined. The hypothesis that acetazolamide and parathyroid hormone (PTH), inhibit renal carbonic anhydrase by a cyclic adenosine 3′,5′-monophosphate (cyclic AMP)-dependent mechanism was also tested. 2 In vitro, acetazolamide, frusemide and ethacrynic acid at high concentrations (10-3M) all produced some inhibition of basal and stimulated rat kidney plasma membrane adenylate cyclase. The effect of acetazolamide was much less than that of frusemide and ethacrynic acid. These plasma membrane effects were reproduced in studies of cyclic AMP formation in isolated kidney tubules of rats. 3 Intravenous injections of acetazolamide did not change the total cyclic AMP content of the kidneys of rats killed by microwave irradiation. 4 Acetazolamide produced a diuresis in the rat and a slight inhibition of the antidiuretic effect of Pitressin. Frusemide produced a diuresis and greatly reduced the antidiuretic response to Pitressin. Ethacrynic acid was ineffective as a diuretic in the rat and actually enhanced the antidiuretic response to Pitressin. 5 In investigating the possible influence of diuretics and PTH on the activity and state of phosphorylation of carbonic anhydrase it was found that: there was no correlation between the ability of diuretics to inhibit carbonic anhydrase activity and to inhibit carbonic anhydrase phosphorylation; neither PTH nor cyclic AMP (in the presence of adenosine triphosphate, Mg2+, K+ and incubation at 37°C) inhibited rat cortex homogenate carbonic anhydrase activity. 6 It seems unlikely that any of the tested diuretics exerts its pharmacological effect by means of changes in kidney cyclic AMP metabolism. PMID:202362

  19. Circulating (CD3−CD19+CD20−IgD−CD27highCD38high) Plasmablasts: A Promising Cellular Biomarker for Immune Activity for Anti-PLA2R1 Related Membranous Nephropathy?

    PubMed Central

    Beukinga, Ingrid; Willard-Gallo, Karen; Nortier, Joëlle; Pradier, Olivier

    2016-01-01

    Membranous nephropathy (MN) is a kidney specific autoimmune disease mainly mediated by anti-phospholipase A2 receptor 1 autoantibody (PLA2R1 Ab). The adequate assessment of chimeric anti-CD20 monoclonal antibody, rituximab (RTX), efficacy is still needed to improve clinical outcome of patient with MN. We evaluated the modification of plasmablasts (CD3−CD19+CD20−IgD−CD27highCD38high), a useful biomarker of RTX response in other autoimmune diseases, and memory (CD3−CD19+CD20+IgD−CD27+CD38−) and naive (CD3−CD19+CD20+IgD+CD27−CD38low) B cells by fluorescence-activated cell sorter analysis in PLA2R1 related MN in one patient during the 4 years of follow-up after RTX. RTX induced complete disappearance of CD19+ B cells, plasmablasts, and memory B cells as soon as day 15. Despite severe CD19+ lymphopenia, plasmablasts and memory B cells reemerged early before naive B cells (days 45, 90, and 120, resp.). During the follow-up, plasmablasts decreased more rapidly than memory B cells but still remained elevated as compared to day 0 of RTX. Concomitantly, anti-PLA2R1 Ab increased progressively. Our single case report suggests that, besides monitoring of serum anti-PLA2R1 Ab level, enumeration of circulating plasmablasts and memory B cells represents an attractive and complementary tool to assess immunological activity and efficacy of RTX induced B cells depletion in anti-PLA2R1 Ab related MN. PMID:27493452

  20. Farnesyloxycoumarins, a new class of squalene-hopene cyclase inhibitors.

    PubMed

    Cravotto, Giancarlo; Balliano, Gianni; Robaldo, Bruna; Oliaro-Bosso, Simonetta; Chimichi, Stefano; Boccalini, Marco

    2004-04-19

    A few naturally occurring prenyl- and prenyloxycoumarins and several new related synthetic derivatives were evaluated as inhibitors of squalene-hopene cyclase (SHC), a useful model enzyme, to predict their interactions with oxidosqualene cyclase (OSC). Umbelliprenin-10',11'-monoepoxide (IC(50) 2.5 microM) and the corresponding 6',7'-10',11' diepoxide (IC(50) 1.5 microM) were the most active enzyme inhibitors.

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

    PubMed Central

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

    1988-01-01

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

  2. The Poly(ADP-ribose) Polymerase Enzyme Tankyrase Antagonizes Activity of the β-Catenin Destruction Complex through ADP-ribosylation of Axin and APC2.

    PubMed

    Croy, Heather E; Fuller, Caitlyn N; Giannotti, Jemma; Robinson, Paige; Foley, Andrew V A; Yamulla, Robert J; Cosgriff, Sean; Greaves, Bradford D; von Kleeck, Ryan A; An, Hyun Hyung; Powers, Catherine M; Tran, Julie K; Tocker, Aaron M; Jacob, Kimberly D; Davis, Beckley K; Roberts, David M

    2016-06-10

    Most colon cancer cases are initiated by truncating mutations in the tumor suppressor, adenomatous polyposis coli (APC). APC is a critical negative regulator of the Wnt signaling pathway that participates in a multi-protein "destruction complex" to target the key effector protein β-catenin for ubiquitin-mediated proteolysis. Prior work has established that the poly(ADP-ribose) polymerase (PARP) enzyme Tankyrase (TNKS) antagonizes destruction complex activity by promoting degradation of the scaffold protein Axin, and recent work suggests that TNKS inhibition is a promising cancer therapy. We performed a yeast two-hybrid (Y2H) screen and uncovered TNKS as a putative binding partner of Drosophila APC2, suggesting that TNKS may play multiple roles in destruction complex regulation. We find that TNKS binds a C-terminal RPQPSG motif in Drosophila APC2, and that this motif is conserved in human APC2, but not human APC1. In addition, we find that APC2 can recruit TNKS into the β-catenin destruction complex, placing the APC2/TNKS interaction at the correct intracellular location to regulate β-catenin proteolysis. We further show that TNKS directly PARylates both Drosophila Axin and APC2, but that PARylation does not globally regulate APC2 protein levels as it does for Axin. Moreover, TNKS inhibition in colon cancer cells decreases β-catenin signaling, which we find cannot be explained solely through Axin stabilization. Instead, our findings suggest that TNKS regulates destruction complex activity at the level of both Axin and APC2, providing further mechanistic insight into TNKS inhibition as a potential Wnt pathway cancer therapy. PMID:27068743

  3. Increased poly(ADP-ribosyl)ation in skeletal muscle tissue of pediatric patients with severe burn injury: prevention by propranolol treatment.

    PubMed

    Oláh, Gábor; Finnerty, Celeste C; Sbrana, Elena; Elijah, Itoro; Gerö, Domokos; Herndon, David N; Szabó, Csaba

    2011-07-01

    Activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) has been shown to promote cellular energetic collapse and cellular necrosis in various forms of critical illness. Most of the evidence implicating the PARP pathway in disease processes is derived from preclinical studies. With respect to PARP and burns, studies in rodent and large animal models of burn injury have demonstrated the activation of PARP in various tissues and the beneficial effect of its pharmacological inhibition. The aims of the current study were to measure the activation of PARP in human skeletal muscle biopsies at various stages of severe pediatric burn injury and to identify the cell types where this activation may occur. Another aim of the study was to test the effect of propranolol (an effective treatment of patients with burns) on the activation of PARP in skeletal muscle biopsies. Poly(ADP-ribose) polymerase activation was measured by Western blotting for its product, poly(ADP-ribose) (PAR). The localization of PARP activation was determined by PAR immunohistochemistry. The results showed that PARP becomes activated in the skeletal muscle tissue after burns, with the peak of the activation occurring in the middle stage of the disease (13-18 days after burns). Even at the late stage of the disease (69-369 days after burn), an elevated degree of PARP activation persisted in some of the patients. Immunohistochemical studies localized the staining of PAR primarily to vascular endothelial cells and occasionally to resident mononuclear cells. There was a marked suppression of PARP activation in the skeletal muscle biopsies of patients who received propranolol treatment. We conclude that human burn injury is associated with the activation of PARP. We hypothesize that this response may contribute to the inflammatory responses and cell dysfunction in burns. Some of the clinical benefit of propranolol in burns may be related to its inhibitory effect on PARP activation.

  4. Novel cholix toxin variants, ADP-ribosylating toxins in Vibrio cholerae non-O1/non-O139 strains, and their pathogenicity.

    PubMed

    Awasthi, Sharda Prasad; Asakura, Masahiro; Chowdhury, Nityananda; Neogi, Sucharit Basu; Hinenoya, Atsushi; Golbar, Hossain M; Yamate, Jyoji; Arakawa, Eiji; Tada, Toshiji; Ramamurthy, T; Yamasaki, Shinji

    2013-02-01

    Cholix toxin (ChxA) is a recently discovered exotoxin in Vibrio cholerae which has been characterized as a third member of the eukaryotic elongation factor 2-specific ADP-ribosyltransferase toxins, in addition to exotoxin A of Pseudomonas aeruginosa and diphtheria toxin of Corynebacterium diphtheriae. These toxins consist of three characteristic domains for receptor binding, translocation, and catalysis. However, there is little information about the prevalence of chxA and its genetic variations and pathogenic mechanisms. In this study, we screened the chxA gene in a large number (n = 765) of V. cholerae strains and observed its presence exclusively in non-O1/non-O139 strains (27.0%; 53 of 196) and not in O1 (n = 485) or O139 (n = 84). Sequencing of these 53 chxA genes generated 29 subtypes which were grouped into three clusters designated chxA I, chxA II, and chxA III. chxA I belongs to the prototype, while chxA II and chxA III are newly discovered variants. ChxA II and ChxA III had unique receptor binding and catalytic domains, respectively, in comparison to ChxA I. Recombinant ChxA I (rChxA I) and rChxA II but not rChxA III showed variable cytotoxic effects on different eukaryotic cells. Although rChxA II was more lethal to mice than rChxA I when injected intravenously, no enterotoxicity of any rChxA was observed in a rabbit ileal loop test. Hepatocytes showed coagulation necrosis in rChxA I- or rChxA II-treated mice, seemingly the major target for ChxA. The present study illustrates the potential of ChxA as an important virulence factor in non-O1/non-O139 V. cholerae, which may be associated with extraintestinal infections rather than enterotoxicity.

  5. The ADP-ribosylation domain of Pseudomonas aeruginosa ExoS is required for membrane bleb niche formation and bacterial survival within epithelial cells.

    PubMed

    Angus, Annette A; Evans, David J; Barbieri, Joseph T; Fleiszig, Suzanne M J

    2010-11-01

    Pseudomonas aeruginosa can establish a niche within the plasma membrane of epithelial cells (bleb niches) within which bacteria can survive, replicate, and swim at speeds detectable by real-time phase-contrast imaging. This novel virulence strategy is dependent on the bacterial type three secretion system (T3SS), since mutants lacking the T3SS needle or known T3SS effectors localize to perinuclear vacuoles and fail to replicate. Here, we determined which of the three effectors (ExoS, ExoT, or ExoY) were required for bleb niche formation and intracellular replication. PAO1 strains with mutations in exoS, exoT, exoY, or combinations thereof were compared to wild-type and complemented strains. P. aeruginosa exoS mutants, but not exoT or exoY mutants, lost the capacity for bleb niche formation and intracellular replication. Complementation with exoS rescued both phenotypes, either in the background of an exoS mutant or in a mutant lacking all three known effectors. Complementation with activity domain mutants of exoS revealed that the ADP-ribosyltransferase (ADP-r) activity of ExoS, but not the Rho-GAP activity nor the membrane localization domain (MLD) of ExoS, was required to elicit this phenotype. Membrane bleb niches that contained P. aeruginosa also bound annexin V-enhanced green fluorescent protein (EGFP), a marker of early apoptosis. These data show that P. aeruginosa bleb niches and intracellular survival involve ExoS ADP-r activity and implicate a connection between bleb niche formation and the known role(s) of ExoS-mediated apoptosis and/or Rab GTPase inactivation.

  6. Single molecule detection of PARP1 and PARP2 interaction with DNA strand breaks and their poly(ADP-ribosyl)ation using high-resolution AFM imaging

    PubMed Central

    Sukhanova, Maria V.; Abrakhi, Sanae; Joshi, Vandana; Pastre, David; Kutuzov, Mikhail M.; Anarbaev, Rashid O.; Curmi, Patrick A.; Hamon, Loic; Lavrik, Olga I.

    2016-01-01

    PARP1 and PARP2 are implicated in the synthesis of poly(ADP-ribose) (PAR) after detection of DNA damage. The specificity of PARP1 and PARP2 interaction with long DNA fragments containing single- and/or double-strand breaks (SSBs and DSBs) have been studied using atomic force microscopy (AFM) imaging in combination with biochemical approaches. Our data show that PARP1 localizes mainly on DNA breaks and exhibits a slight preference for nicks over DSBs, although the protein has a moderately high affinity for undamaged DNA. In contrast to PARP1, PARP2 is mainly detected at a single DNA nick site, exhibiting a low level of binding to undamaged DNA and DSBs. The enhancement of binding affinity of PARP2 for DNA containing a single nick was also observed using fluorescence titration. AFM studies reveal that activation of both PARPs leads to the synthesis of highly branched PAR whose size depends strongly on the presence of SSBs and DSBs for PARP1 and of SSBs for PARP2. The initial affinity between the PARP1, PARP2 and the DNA damaged site appears to influence both the size of the PAR synthesized and the time of residence of PARylated PARP1 and PARP2 on DNA damages. PMID:26673720

  7. Single molecule detection of PARP1 and PARP2 interaction with DNA strand breaks and their poly(ADP-ribosyl)ation using high-resolution AFM imaging.

    PubMed

    Sukhanova, Maria V; Abrakhi, Sanae; Joshi, Vandana; Pastre, David; Kutuzov, Mikhail M; Anarbaev, Rashid O; Curmi, Patrick A; Hamon, Loic; Lavrik, Olga I

    2016-04-01

    PARP1 and PARP2 are implicated in the synthesis of poly(ADP-ribose) (PAR) after detection of DNA damage. The specificity of PARP1 and PARP2 interaction with long DNA fragments containing single- and/or double-strand breaks (SSBs and DSBs) have been studied using atomic force microscopy (AFM) imaging in combination with biochemical approaches. Our data show that PARP1 localizes mainly on DNA breaks and exhibits a slight preference for nicks over DSBs, although the protein has a moderately high affinity for undamaged DNA. In contrast to PARP1, PARP2 is mainly detected at a single DNA nick site, exhibiting a low level of binding to undamaged DNA and DSBs. The enhancement of binding affinity of PARP2 for DNA containing a single nick was also observed using fluorescence titration. AFM studies reveal that activation of both PARPs leads to the synthesis of highly branched PAR whose size depends strongly on the presence of SSBs and DSBs for PARP1 and of SSBs for PARP2. The initial affinity between the PARP1, PARP2 and the DNA damaged site appears to influence both the size of the PAR synthesized and the time of residence of PARylated PARP1 and PARP2 on DNA damages.

  8. Inhibition of potentially lethal radiation damage repair in normal and neoplastic human cells by 3-aminobenzamide: an inhibitor of poly(ADP-ribosylation)

    SciTech Connect

    Thraves, P.J.; Mossman, K.L.; Frazier, D.T.; Dritschilo, A.

    1986-08-01

    The effect of 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) synthetase, on potentially lethal damage repair (PLDR) was investigated in normal human fibroblasts and four human tumor cell lines from tumors with varying degrees of radiocurability. The tumor lines selected were: Ewing's sarcoma, a bone tumor considered radiocurable and, human lung adenocarcinoma, osteosarcoma, and melanoma, three tumors considered nonradiocurable. PLDR was measured by comparing cell survival when cells were irradiated in a density-inhibited state and replated at appropriate cell numbers at specified times following irradiation to cell survival when cells were replated immediately following irradiation. 3AB was added to cultures 2 hr prior to irradiation and removed at the time of replating. Different test radiation doses were used for the various cell lines to obtain equivalent levels of cell survival. In the absence of inhibitor, PLDR was similar in all cell lines tested. In the presence of 8 mM 3AB, differential inhibition of PLDR was observed. PLDR was almost completely inhibited in Ewing's sarcoma cells and partially inhibited in normal fibroblast cells and osteosarcoma cells. No inhibition of PLDR was observed in the lung adenocarcinoma or melanoma cells. Except for the osteosarcoma cells, inhibition of PLDR by 3AB correlated well with radiocurability.

  9. Enhancing NAD+ Salvage Pathway Reverts the Toxicity of Primary Astrocytes Expressing Amyotrophic Lateral Sclerosis-linked Mutant Superoxide Dismutase 1 (SOD1).

    PubMed

    Harlan, Benjamin A; Pehar, Mariana; Sharma, Deep R; Beeson, Gyda; Beeson, Craig C; Vargas, Marcelo R

    2016-05-13

    Nicotinamide adenine dinucleotide (NAD(+)) participates in redox reactions and NAD(+)-dependent signaling pathways. Although the redox reactions are critical for efficient mitochondrial metabolism, they are not accompanied by any net consumption of the nucleotide. On the contrary, NAD(+)-dependent signaling processes lead to its degradation. Three distinct families of enzymes consume NAD(+) as substrate: poly(ADP-ribose) polymerases, ADP-ribosyl cyclases (CD38 and CD157), and sirtuins (SIRT1-7). Because all of the above enzymes generate nicotinamide as a byproduct, mammalian cells have evolved an NAD(+) salvage pathway capable of resynthesizing NAD(+) from nicotinamide. Overexpression of the rate-limiting enzyme in this pathway, nicotinamide phosphoribosyltransferase, increases total and mitochondrial NAD(+) levels in astrocytes. Moreover, targeting nicotinamide phosphoribosyltransferase to the mitochondria also enhances NAD(+) salvage pathway in astrocytes. Supplementation with the NAD(+) precursors nicotinamide mononucleotide and nicotinamide riboside also increases NAD(+) levels in astrocytes. Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Superoxide dismutase 1 (SOD1) mutations account for up to 20% of familial ALS and 1-2% of apparently sporadic ALS cases. Primary astrocytes isolated from mutant human superoxide dismutase 1-overexpressing mice as well as human post-mortem ALS spinal cord-derived astrocytes induce motor neuron death in co-culture. Increasing total and mitochondrial NAD(+) content in ALS astrocytes increases oxidative stress resistance and reverts their toxicity toward co-cultured motor neurons. Taken together, our results suggest that enhancing the NAD(+) salvage pathway in astrocytes could be a potential therapeutic target to prevent astrocyte-mediated motor neuron death in ALS. PMID:27002158

  10. Novel hopanoid cyclases from the environment.

    PubMed

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

    2007-09-01

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

  11. Novel hopanoid cyclases from the environment.

    PubMed

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

    2007-09-01

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

  12. Protein kinase C sensitizes olfactory adenylate cyclase

    PubMed Central

    1993-01-01

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

  13. Molecular Physiology of Membrane Guanylyl Cyclase Receptors.

    PubMed

    Kuhn, Michaela

    2016-04-01

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

  14. Activation of Escherichia coli heat-labile enterotoxins by native and recombinant adenosine diphosphate-ribosylation factors, 20-kD guanine nucleotide-binding proteins.

    PubMed Central

    Lee, C M; Chang, P P; Tsai, S C; Adamik, R; Price, S R; Kunz, B C; Moss, J; Twiddy, E M; Holmes, R K

    1991-01-01

    Escherichia coli heat-labile enterotoxins (LT) are responsible in part for "traveler's diarrhea" and related diarrheal illnesses. The family of LTs comprises two serogroups termed LT-I and LT-II; each serogroup includes two or more antigenic variants. The effects of LTs result from ADP ribosylation of Gs alpha, a stimulatory component of adenylyl cyclase; the mechanism of action is identical to that of cholera toxin (CT). The ADP-ribosyltransferase activity of CT is enhanced by 20-kD guanine nucleotide-binding proteins, known as ADP-ribosylation factors or ARFs. These proteins directly activate the CTA1 catalytic unit and stimulate its ADP ribosylation of Gs alpha, other proteins, and simple guanidino compounds (e.g., agmatine). Because of the similarities between CT and LTs, we investigated the effects of purified bovine brain ARF and a recombinant form of bovine ARF synthesized in Escherichia coli on LT activity. ARF enhanced the LT-I-, LT-IIa-, and LT-IIb-catalyzed ADP ribosylation of agmatine, as well as the auto-ADP ribosylation of the toxin catalytic unit. Stimulation of ADP-ribosylagmatine formation by LTs and CT in the presence of ARF was GTP dependent and enhanced by sodium dodecyl sulfate. With agmatine as substrate, LT-IIa and LT-IIb exhibited less than 1% the activity of CT and LT-Ih. CT and LTs catalyzed ADP-ribosyl-Gs alpha formation in a reaction dependent on ARF, GTP, and dimyristoyl phosphatidylcholine/cholate. With Gs alpha as substrate, the ADP-ribosyltransferase activities of the toxins were similar, although CT and LT-Ih appeared to be slightly more active than LT-IIa and LT-IIb. Thus, LT-IIa and LT-IIb appear to differ somewhat from CT and LT-Ih in substrate specificity. Responsiveness to stimulation by ARF, GTP, and phospholipid/detergent as well as the specificity of ADP-ribosyltransferase activity are functions of LTs from serogroups LT-I and LT-II that are shared with CT. Images PMID:1902492

  15. Prokaryotic squalene-hopene cyclases can be converted to citronellal cyclases by single amino acid exchange.

    PubMed

    Siedenburg, Gabriele; Breuer, Michael; Jendrossek, Dieter

    2013-02-01

    Squalene-hopene cyclases (SHCs) are prokaryotic enzymes that catalyse the cyclisation of the linear precursor squalene to pentacyclic hopene. Recently, we discovered that a SHC cloned from Zymomonas mobilis (ZMO-1548 gene product) has the unique property to cyclise the monoterpenoid citronellal to isopulegol. In this study, we performed saturation mutagenesis of three amino acids of the catalytic centre of ZMO-1548 (F428, F486 and W555), which had been previously identified to interact with enzyme-bound substrate. Replacement of F428 by tyrosine increased hopene formation from squalene, but isopulegol-forming activity was strongly reduced or abolished in all muteins of position 428. W555 was essential for hopene formation; however, three muteins (W555Y, W428F or W555T) revealed enhanced cyclisation efficiency with citronellal. The residue at position 486 turned out to be the most important for isopulegol-forming activity. While the presence of phenylalanine or tyrosine favoured cyclisation activity with squalene, several small and/or hydrophobic residues such as cysteine, alanine or isoleucine and others reduced activity with squalene but greatly enhanced isopulegol formation from citronellal. Replacement of the conserved aromatic residue corresponding to F486 to cysteine in other SHCs cloned from Z. mobilis (ZMO-0872), Alicyclobacillus acidocaldarius (SHC(Aac)), Acetobacter pasteurianus (SHC(Apa)), Streptomyces coelicolor (SHC(Sco)) and Bradyrhizobium japonicum (SHC(Bja)) resulted in more or less strong isopulegol-forming activities from citronellal. In conclusion, many SHCs can be converted to citronellal cyclases by mutagenesis of the active centre thus broadening the applicability of this interesting class of biocatalyst. PMID:22526778

  16. Prokaryotic squalene-hopene cyclases can be converted to citronellal cyclases by single amino acid exchange.

    PubMed

    Siedenburg, Gabriele; Breuer, Michael; Jendrossek, Dieter

    2013-02-01

    Squalene-hopene cyclases (SHCs) are prokaryotic enzymes that catalyse the cyclisation of the linear precursor squalene to pentacyclic hopene. Recently, we discovered that a SHC cloned from Zymomonas mobilis (ZMO-1548 gene product) has the unique property to cyclise the monoterpenoid citronellal to isopulegol. In this study, we performed saturation mutagenesis of three amino acids of the catalytic centre of ZMO-1548 (F428, F486 and W555), which had been previously identified to interact with enzyme-bound substrate. Replacement of F428 by tyrosine increased hopene formation from squalene, but isopulegol-forming activity was strongly reduced or abolished in all muteins of position 428. W555 was essential for hopene formation; however, three muteins (W555Y, W428F or W555T) revealed enhanced cyclisation efficiency with citronellal. The residue at position 486 turned out to be the most important for isopulegol-forming activity. While the presence of phenylalanine or tyrosine favoured cyclisation activity with squalene, several small and/or hydrophobic residues such as cysteine, alanine or isoleucine and others reduced activity with squalene but greatly enhanced isopulegol formation from citronellal. Replacement of the conserved aromatic residue corresponding to F486 to cysteine in other SHCs cloned from Z. mobilis (ZMO-0872), Alicyclobacillus acidocaldarius (SHC(Aac)), Acetobacter pasteurianus (SHC(Apa)), Streptomyces coelicolor (SHC(Sco)) and Bradyrhizobium japonicum (SHC(Bja)) resulted in more or less strong isopulegol-forming activities from citronellal. In conclusion, many SHCs can be converted to citronellal cyclases by mutagenesis of the active centre thus broadening the applicability of this interesting class of biocatalyst.

  17. Regulation and organization of adenylyl cyclases and cAMP.

    PubMed Central

    Cooper, Dermot M F

    2003-01-01

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

  18. AKAPs and Adenylyl Cyclase in Cardiovascular Physiology and Pathology

    PubMed Central

    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

  19. High levels of CD34+CD38low/−CD123+ blasts are predictive of an adverse outcome in acute myeloid leukemia: a Groupe Ouest-Est des Leucémies Aiguës et Maladies du Sang (GOELAMS) study

    PubMed Central

    Vergez, François; Green, Alexa S.; Tamburini, Jerome; Sarry, Jean-Emmanuel; Gaillard, Baptiste; Cornillet-Lefebvre, Pascale; Pannetier, Melanie; Neyret, Aymeric; Chapuis, Nicolas; Ifrah, Norbert; Dreyfus, François; Manenti, Stéphane; Demur, Cecile; Delabesse, Eric; Lacombe, Catherine; Mayeux, Patrick; Bouscary, Didier; Recher, Christian; Bardet, Valerie

    2011-01-01

    Background Acute myeloid leukemias arise from a rare population of leukemic cells, known as leukemic stem cells, which initiate the disease and contribute to frequent relapses. Although the phenotype of these cells remains unclear in most patients, these cells are enriched within the CD34+CD38low/− compartment expressing the interleukin-3 alpha chain receptor, CD123. The aim of this study was to determine the prognostic value of the percentage of blasts with the CD34+CD38low/−CD123+ phenotype. Design and Methods The percentage of CD34+CD38low/−CD123+ cells in the blast population was determined at diagnosis using flow cytometry. One hundred and eleven patients under 65 years of age with de novo acute myeloid leukemia and treated with intensive chemotherapy were retrospectively included in the study. Correlations with complete response, disease-free survival and overall survival were evaluated with univariate and multivariate analyses. Results A proportion of CD34+CD38low/−CD123+ cells greater than 15% at diagnosis and an unfavorable karyotype were significantly correlated with a lack of complete response. By logistic regression analysis, a percentage of CD34+CD38low/−CD123+ higher than 15% retained significance with an odds ratio of 0.33 (0.1–0.97; P=0.044). A greater than 1% population of CD34+CD38low/−CD123+ cells negatively affected disease-free survival (0.9 versus 4.7 years; P<0.0001) and overall survival (1.25 years versus median not reached; P<0.0001). A greater than 1% population of CD34+CD38low/−CD123+ cells retained prognostic significance for both parameters after multivariate analysis. Conclusions The percentage of CD34+CD38low/−CD123+ leukemic cells at diagnosis was significantly correlated with response to treatment and survival. This prognostic marker might be easily adopted in clinical practice to rapidly identify patients at risk of treatment failure. PMID:21933861

  20. Virulence of Bordetella bronchiseptica: role of adenylate cyclase-hemolysin.

    PubMed Central

    Gueirard, P; Guiso, N

    1993-01-01

    Bordetella bronchiseptica is a pathogen of laboratory, domestic, and wild animals and sometimes of humans. In the present study some characteristics of the virulence of B. bronchiseptica isolates of different origin were studied. All isolates had similar phenotypes, similar bacteriological characters, and synthesized adenylate cyclase-hemolysin, filamentous hemagglutinin and pertactin but not pertussis toxin. These isolates, however, differed in their ability to express dermonecrotic toxin and to cause a lethal infection, but no correlation was found with the human or animal origin of the isolates. The fact that the most virulent isolate did not express dermonecrotic toxin suggests that this toxin does not play an important role in the virulence of the bacteria in the murine model. After infection with virulent B. bronchiseptica a very early synthesis and a persistence of anti-adenylate cyclase-hemolysin and anti-filamentous hemagglutinin antibodies were observed in the sera of infected mice, suggesting a persistence of the bacteria or of its antigens. B. bronchiseptica adenylate cyclase-hemolysin was purified and was shown to be a major protective antigen against B. bronchiseptica infection. Furthermore, we showed that its immunological and protective properties were different from that of B. pertussis adenylate cyclase-hemolysin, confirming that Bordetella species are immunologically different. Images PMID:8406794

  1. Tocopherol Cyclases-Substrate Specificity and Phylogenetic Relations.

    PubMed

    Dłużewska, Jolanta; Szymańska, Renata; Gabruk, Michal; Kós, Peter B; Nowicka, Beatrycze; Kruk, Jerzy

    2016-01-01

    In the present studies, we focused on substrate specificity of tocopherol cyclase, the key enzyme in the biosynthesis of the tocopherols and plastochromanol-8, the main plant lipid antioxidants, with special emphasis on the preference for tocopherols and plastochromanol-8 precursors, taking advantage of the recombinant enzyme originating from Arabidopsis thaliana and isolated plastoglobules, thylakoids and various model systems like micelles and thylakoids. Plastoglobules and triacylglycerol micelles were the most efficient reaction environment for the cyclase. In various investigated systems, synthesis of γ-tocopherol proceeded considerably faster than that of plastochromanol-8, probably mainly due to different localization of the corresponding substrates in the analyzed lipid structures. Moreover, our study was complemented by bioinformatics analysis of the phylogenetic relations of the cyclases and sequence motifs, crucial for the enzyme activity, were proposed. The analysis revealed also a group of tocopherol cyclase-like proteins in a number of heterotrophic bacterial species, with a conserved region common with photosynthetic organisms, that might be engaged in the catalytic activity of both groups of organisms. PMID:27462710

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

    PubMed

    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.

  3. Influence of bacterial toxins on the GTPase activity of transducin from bovine retinal rod outer segments

    SciTech Connect

    Rybin, V.O.; Gureeva, A.A.

    1986-05-10

    The action of cholera toxin, capable of ADP-ribosylation of the activator N/sub s/ protein, and pertussis toxin, capable of ADP-ribosylation of the inhibitor N/sub i/ protein of the adenylate cyclase complex, on transducin, the GTP-binding protein of the rod outer segments of the retina, was investigated. It was shown that under the action of pertussis and cholera toxins, the GTPase activity of transducin is inhibited. Pertussin toxin inhibits the GTPase of native retinal rod outer segments by 30-40%, while GTPase of homogeneous transducin produces a 70-80% inhibition. The action of toxins on transducin depends on the presence and nature of the guanylic nucleotide with which incubation is performed. On the basis of the data obtained it is suggested that pertussis toxin interacts with pretransducin and with the transducin-GDP complex, while cholera toxin ADP-ribosylates the transducin-GTP complex and does not act on transducin lacking GTP.

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

    PubMed Central

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

    1986-01-01

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

  5. High skeletal muscle adenylate cyclase in malignant hyperthermia.

    PubMed Central

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

    1981-01-01

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

  6. Engineering adenylate cyclases regulated by near-infrared window light

    PubMed Central

    Ryu, Min-Hyung; Kang, In-Hye; Nelson, Mathew D.; Jensen, Tricia M.; Lyuksyutova, Anna I.; Siltberg-Liberles, Jessica; Raizen, David M.; Gomelsky, Mark

    2014-01-01

    Bacteriophytochromes sense light in the near-infrared window, the spectral region where absorption by mammalian tissues is minimal, and their chromophore, biliverdin IXα, is naturally present in animal cells. These properties make bacteriophytochromes particularly attractive for optogenetic applications. However, the lack of understanding of how light-induced conformational changes control output activities has hindered engineering of bacteriophytochrome-based optogenetic tools. Many bacteriophytochromes function as homodimeric enzymes, in which light-induced conformational changes are transferred via α-helical linkers to the rigid output domains. We hypothesized that heterologous output domains requiring homodimerization can be fused to the photosensory modules of bacteriophytochromes to generate light-activated fusions. Here, we tested this hypothesis by engineering adenylate cyclases regulated by light in the near-infrared spectral window using the photosensory module of the Rhodobacter sphaeroides bacteriophytochrome BphG1 and the adenylate cyclase domain from Nostoc sp. CyaB1. We engineered several light-activated fusion proteins that differed from each other by approximately one or two α-helical turns, suggesting that positioning of the output domains in the same phase of the helix is important for light-dependent activity. Extensive mutagenesis of one of these fusions resulted in an adenylate cyclase with a sixfold photodynamic range. Additional mutagenesis produced an enzyme with a more stable photoactivated state. When expressed in cholinergic neurons in Caenorhabditis elegans, the engineered adenylate cyclase affected worm behavior in a light-dependent manner. The insights derived from this study can be applied to the engineering of other homodimeric bacteriophytochromes, which will further expand the optogenetic toolset. PMID:24982160

  7. Dephosphorylation of sperm guanylate cyclase during sea urchin fertilization

    SciTech Connect

    Ward, G.E.

    1985-01-01

    When intact Arbacia punctulata spermatozoa are exposed to solubilized egg jelly, the electrophoretic mobility of an abundant sperm flagellar membrane protein changes from an apparent molecular mass of 160 kDa to 150 kDa. A. punctulata spermatozoa can be labeled in vivo with /sup 32/P-labeled cells it was demonstrated that the mobility shift of the 160-kDa protein is due to dephosphorylation. The peptide resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH/sub 2/) is the component of egg jelly which is responsible for inducing the dephosphorylation. The 160/150-kdal sperm membrane protein has been purified to homogeneity by affinity chromatography on concanavalin A-agarose, and identified as sperm guanylate cyclase. The enzymatic activity of the guanylate cyclase is tightly coupled to its phosphorylation state. Resact has been shown to act as a potent chemoattractant for A. punctulata spermatozoa. The chemotactic response is concentration-dependent, is abolished by pretreatment of the spermatozoa with resact, and shows an absolute requirement for external calcium. This work represents the first demonstration of animal sperm chemotaxis in response to a precisely-defined molecule of egg origin. The results established a new, biologically meaningful function for resact, and may implicate sperm guanylate cyclase and cGMP in flagellar function and the chemotactic response.

  8. Crystal structure of papaya glutaminyl cyclase, an archetype for plant and bacterial glutaminyl cyclases.

    PubMed

    Wintjens, René; Belrhali, Hassan; Clantin, Bernard; Azarkan, Mohamed; Bompard, Coralie; Baeyens-Volant, Danielle; Looze, Yvan; Villeret, Vincent

    2006-03-24

    Glutaminyl cyclases (QCs) (EC 2.3.2.5) catalyze the intramolecular cyclization of protein N-terminal glutamine residues into pyroglutamic acid with the concomitant liberation of ammonia. QCs may be classified in two groups containing, respectively, the mammalian enzymes, and the enzymes from plants, bacteria, and parasites. The crystal structure of the QC from the latex of Carica papaya (PQC) has been determined at 1.7A resolution. The structure was solved by the single wavelength anomalous diffraction technique using sulfur and zinc as anomalous scatterers. The enzyme folds into a five-bladed beta-propeller, with two additional alpha-helices and one beta hairpin. The propeller closure is achieved via an original molecular velcro, which links the last two blades into a large eight stranded beta-sheet. The zinc ion present in the PQC is bound via an octahedral coordination into an elongated cavity located along the pseudo 5-fold axis of the beta-propeller fold. This zinc ion presumably plays a structural role and may contribute to the exceptional stability of PQC, along with an extended hydrophobic packing, the absence of long loops, the three-joint molecular velcro and the overall folding itself. Multiple sequence alignments combined with structural analyses have allowed us to tentatively locate the active site, which is filled in the crystal structure either by a Tris molecule or an acetate ion. These analyses are further supported by the experimental evidence that Tris is a competitive inhibitor of PQC. The active site is located at the C-terminal entrance of the PQC central tunnel. W83, W110, W169, Q24, E69, N155, K225, F22 and F67 are highly conserved residues in the C-terminal entrance, and their putative role in catalysis is discussed. The PQC structure is representative of the plants, bacterial and parasite enzymes and contrasts with that of mammalian enzymes, that may possibly share a conserved scaffold of the bacterial aminopeptidase.

  9. Effect of Stress-Free Therapy on immune system: Induction of Interleukin 10 expression in lymphocytes through activation of CD19+ CD24hi CD38hi regulatory B Cells

    PubMed Central

    Nakajima, Takuma; Ishimaru, Keisou; Ozaki-Shimada, Atsuko; Kihara, Kazuhiko; Namiki, Yoshihisa; Otani, Satoru

    2015-01-01

    Background and aims: Mild thermal treatment with “Pinpoint Plantar Long-wavelength Infrared Light Irradiation (PP-LILI)” named as Stress-Free Therapy® increases peripheral-deep body temperature and blood flow, and improves multiple disorders including hyperpiesia, type II diabetes and cardiovascular patients. Immunomodulatory effects of PP-LILI were investigated. Materials and methods: Seven healthy individuals and 4 people with underlying medical condition (UMC) participated in this study. Participants were given PP-LILI stimuli twice a week over 3 weeks and followed with placebo stimuli over 3 weeks. This set of sessions was repeated 3 times. For analyses, fresh peripheral mononuclear cells from participants were stained with fluorescencedye conjugated monoclonal antibodies and changes in populational compositions and IL-10 expression levels were observed by flow cytometry. Results: Distinct expression of IL-10 in lymphocytes was induced by PP-LILI from the second session in the healthy individuals. This induction was terminated during the following placebo sessions. PP-LILI induced activation of CD19+ CD24hi CD38hi regulatory B cells in every session prior to induce the IL-10 in major lymphocytes. Activated regulatory B cells in the individuals with UMC decreased as same levels of healthy individuals after second PP-LILI session and re-activated with the stimuli. Significant population changes in neither regulatory T cells nor proinflammatory IL-17A expressing CD4+ T cells were observed. Conclusions: PP-LILI is a potent immunomodulatory inducer that activates regulatory B cells and consequent IL-10 expression in lymphocytes. Moreover, its stimulatory intervals down-regulate the higher activation of regulatory B cells and lymphocyte's IL-10 expression occurred by UMC to the healthy people's level. PMID:26557732

  10. Particular activation phenotype of T cells expressing HLA-DR but not CD38 in GALT from HIV-controllers is associated with immune regulation and delayed progression to AIDS.

    PubMed

    Gonzalez, Sandra M; Taborda, Natalia A; Correa, Luis A; Castro, Gustavo A; Hernandez, Juan C; Montoya, Carlos J; Rugeles, Maria T

    2016-06-01

    The spontaneous control of HIV replication in HIV-controllers underlines the importance of these subjects for exploring factors related to delayed progression. Several studies have revealed fewer immune alterations and effector mechanisms related to viral control, mainly in peripheral blood, in these individuals compared to normal progressors. However, immune characterization of gut-associated lymphoid tissue (GALT), the major target of infection, has not been thoroughly explored in these subjects. We evaluated the following parameters in GALT samples from 11 HIV-controllers and 15 HIV-progressors: (i) frequency and activation phenotype of T cells; (ii) expression of transcription factors associated with immune response profiles; and (iii) frequency of apoptotic cells. Interestingly, HIV-controllers exhibited a particular activation phenotype, with predominance of T cells expressing HLA-DR but not CD38 in GALT. This phenotype, previously associated with better control of infection, was correlated with low viral load and higher CD4(+) T cell count. Furthermore, a positive correlation of this activation phenotype with higher expression of Foxp3 and RORγT transcription factors suggested a key role for Treg and Th17 cells in the control of the immune activation and in the maintenance of gut mucosal integrity. Although we evaluated apoptosis by measuring expression of cleaved caspase-3 in GALT, we did not find differences between HIV-controllers and HIV-progressors. Taken together, our findings suggest that predominance of HLA-DR(+) T cells, along with lower immune activation and higher expression of transcription factors required for the development of Treg and Th17 cells, is associated with better viral control and delayed progression to AIDS.

  11. A new small molecule inhibitor of soluble guanylate cyclase

    PubMed Central

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

    2015-01-01

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

  12. Expression of a fungal sesquiterpene cyclase gene in transgenic tobacco.

    PubMed

    Hohn, T M; Ohlrogge, J B

    1991-09-01

    The complete coding sequence for the trichodiene synthase gene from Fusarium sporotrichioides was introduced into tobacco (Nicotiana tabacum) under the regulation of the cauliflower mosiac virus 35S promoter. Expression of trichodiene synthase was demonstrated in the leaves of transformed plants. Leaf homogenates incubated with [(3)H]farnesyl pyrophosphate produced trichodiene as a major product. Trichodiene was detected in the leaves of a transformed plant at a level of 5 to 10 nanograms per gram fresh weight. The introduction of a fungal sesquiterpene cyclase gene into tobacco has resulted in the expression of an active enzyme and the accumulation of low levels of its sesquiterpenoid product. PMID:16668409

  13. Origin of asymmetry in adenylyl cyclases: structures of Mycobacterium tuberculosis Rv1900c.

    PubMed

    Sinha, Sangita C; Wetterer, Martina; Sprang, Stephen R; Schultz, Joachim E; Linder, Jürgen U

    2005-02-23

    Rv1900c, a Mycobacterium tuberculosis adenylyl cyclase, is composed of an N-terminal alpha/beta-hydrolase domain and a C-terminal cyclase homology domain. It has an unusual 7% guanylyl cyclase side-activity. A canonical substrate-defining lysine and a catalytic asparagine indispensable for mammalian adenylyl cyclase activity correspond to N342 and H402 in Rv1900c. Mutagenic analysis indicates that these residues are dispensable for activity of Rv1900c. Structures of the cyclase homology domain, solved to 2.4 A both with and without an ATP analog, form isologous, but asymmetric homodimers. The noncanonical N342 and H402 do not interact with the substrate. Subunits of the unliganded open dimer move substantially upon binding substrate, forming a closed dimer similar to the mammalian cyclase heterodimers, in which one interfacial active site is occupied and the quasi-dyad-related active site is occluded. This asymmetry indicates that both active sites cannot simultaneously be catalytically active. Such a mechanism of half-of-sites-reactivity suggests that mammalian heterodimeric adenylyl cyclases may have evolved from gene duplication of a primitive prokaryote-type cyclase, followed by loss of function in one active site. PMID:15678099

  14. Crystallization of the class IV adenylyl cyclase from Yersinia pestis

    SciTech Connect

    Smith, Natasha; Kim, Sook-Kyung; Reddy, Prasad T.; Gallagher, D. Travis

    2006-03-01

    The class IV adenylyl cyclase from Y. pestis has been crystallized in an orthorhombic form suitable for structure determination. The class IV adenylyl cyclase from Yersinia pestis has been cloned and crystallized in both a triclinic and an orthorhombic form. An amino-terminal His-tagged construct, from which the tag was removed by thrombin, crystallized in a triclinic form diffracting to 1.9 Å, with one dimer per asymmetric unit and unit-cell parameters a = 33.5, b = 35.5, c = 71.8 Å, α = 88.7, β = 82.5, γ = 65.5°. Several mutants of this construct crystallized but diffracted poorly. A non-His-tagged native construct (179 amino acids, MW = 20.5 kDa) was purified by conventional chromatography and crystallized in space group P2{sub 1}2{sub 1}2{sub 1}. These crystals have unit-cell parameters a = 56.8, b = 118.6, c = 144.5 Å, diffract to 3 Å and probably have two dimers per asymmetric unit and V{sub M} = 3.0 Å{sup 3} Da{sup −1}. Both crystal forms appear to require pH below 5, complicating attempts to incorporate nucleotide ligands into the structure. The native construct has been produced as a selenomethionine derivative and crystallized for phasing and structure determination.

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

    PubMed

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

    2016-08-19

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

  16. Structural analysis of an oxygen-regulated diguanylate cyclase.

    PubMed

    Tarnawski, Miroslaw; Barends, Thomas R M; Schlichting, Ilme

    2015-11-01

    Cyclic di-GMP is a bacterial second messenger that is involved in switching between motile and sessile lifestyles. Given the medical importance of biofilm formation, there has been increasing interest in understanding the synthesis and degradation of cyclic di-GMPs and their regulation in various bacterial pathogens. Environmental cues are detected by sensing domains coupled to GGDEF and EAL or HD-GYP domains that have diguanylate cyclase and phosphodiesterase activities, respectively, producing and degrading cyclic di-GMP. The Escherichia coli protein DosC (also known as YddV) consists of an oxygen-sensing domain belonging to the class of globin sensors that is coupled to a C-terminal GGDEF domain via a previously uncharacterized middle domain. DosC is one of the most strongly expressed GGDEF proteins in E. coli, but to date structural information on this and related proteins is scarce. Here, the high-resolution structural characterization of the oxygen-sensing globin domain, the middle domain and the catalytic GGDEF domain in apo and substrate-bound forms is described. The structural changes between the iron(III) and iron(II) forms of the sensor globin domain suggest a mechanism for oxygen-dependent regulation. The structural information on the individual domains is combined into a model of the dimeric DosC holoprotein. These findings have direct implications for the oxygen-dependent regulation of the activity of the cyclase domain.

  17. Inhibition of heat shock protein 90 attenuates adenylate cyclase sensitization after chronic morphine treatment.

    PubMed

    Koshimizu, Taka-aki; Tsuchiya, Hiroyoshi; Tsuda, Hidetoshi; Fujiwara, Yoko; Shibata, Katsushi; Hirasawa, Akira; Tsujimoto, Gozoh; Fujimura, Akio

    2010-02-19

    Cellular adaptations to chronic opioid treatment result in enhanced responsiveness of adenylate cyclase and an increase in forskolin- or agonist-stimulated cAMP production. It is, however, not known whether chaperone molecules such as heat shock proteins contribute to this adenylate cyclase sensitization. Here, we report that treatment of cells with geldanamycin, an inhibitor of heat shock protein 90 (Hsp90), led to effective attenuation of morphine-induced adenylate cyclase sensitization. In SK-N-SH human neuroblastoma cells, morphine significantly increased RNA transcript and protein levels of type I adenylate cyclase, leading to sensitization. Whole-genome tiling array analysis revealed that cAMP response element-binding protein, an important mediator for cellular adaptation to morphine, associated with the proximal promoter of Hsp90AB1 not only in SK-N-SH cells but also in rat PC12 and human embryonic kidney cells. Hsp90AB1 transcript and protein levels increased significantly during morphine treatment, and co-application of geldanamycin (0.1-10 nM) effectively suppressed the increase in forskolin-activated adenylate cyclase activation by 56%. Type I adenylate cyclase, but not Hsp90AB1, underwent significant degradation during geldanamycin treatment. These results indicate that Hsp90 is a new pharmacological target for the suppression of adenylate cyclase sensitization induced by chronic morphine treatment.

  18. The Effects of Thrombin on Adenyl Cyclase Activity and a Membrane Protein from Human Platelets

    PubMed Central

    Brodie, G. N.; Baenziger, Nancy Lewis; Chase, Lewis R.; Majerus, Philip W.

    1972-01-01

    Washed human platelets were incubated with 0.1-1.0 U/ml human thrombin and the effects on adenyl cyclase activity and on a platelet membrane protein (designated thrombin-sensitive protein) were studied. Adenyl cyclase activity was decreased 70-90% when intact platelets were incubated with thrombin. The T½ for loss of adenyl cyclase activity was less than 15 sec at 1 U/ml thrombin. There was no decrease of adenyl cyclase activity when sonicated platelets or isolated membranes were incubated with these concentrations of thrombin. Loss of adenyl cyclase activity was relatively specific since the activities of other platelet membrane enzymes were unaffected by thrombin. Prior incubation of platelets with dibutyryl cyclic adenosine monophosphate (AMP), prostaglandin E1, or theophylline protected adenyl cyclase from inhibition by thrombin. Incubation of intact but not disrupted platelets with thrombin resulted in the release of thrombin-sensitive protein from the platelet membrane. The rapid release of this protein (T½ < 15 sec) at low concentrations of thrombin suggested that removal of thrombin-sensitive protein from the platelet membrane is an integral part of the platelet release reaction. This hypothesis is supported by the parallel effects of thrombin on adenyl cyclase activity and thrombin-sensitive protein release in the presence of dibutyryl cyclic AMP, prostaglandin E1, and theophylline at varying concentrations of thrombin. Images PMID:4331802

  19. Product identification and adenylyl cyclase activity in chloroplasts of Nicotiana tabacum.

    PubMed

    Witters, Erwin; Quanten, Lieve; Bloemen, Jo; Valcke, Roland; Van Onckelen, Harry

    2004-01-01

    In view of the ongoing debate on plant cyclic nucleotide metabolism, especially the functional presence of adenylyl cyclase, a novel detection method has been worked out to quantify the reaction product. Using uniformly labelled (15)N-ATP as a substrate for adenylyl cyclase, a qualitative and quantitative liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) method was developed to measure de novo formed (15)N-adenosine 3',5'-cyclic monophosphate. Adenylyl cyclase activity was observed in chloroplasts obtained from Nicotiana tabacum cv. Petit Havana and the kinetic parameters and influence of various metabolic effectors are discussed in their context.

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

    PubMed Central

    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. PMID:27695398

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

    PubMed Central

    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.

  2. Soluble guanylate cyclase as a novel treatment target for osteoporosis.

    PubMed

    Joshua, Jisha; Schwaerzer, Gerburg K; Kalyanaraman, Hema; Cory, Esther; Sah, Robert L; Li, Mofei; Vaida, Florin; Boss, Gerry R; Pilz, Renate B

    2014-12-01

    Osteoporosis is a major health problem leading to fractures that cause substantial morbidity and mortality. Current osteoporosis therapies have significant drawbacks, creating a need for novel bone-anabolic agents. We previously showed that the nitric oxide/cyclic GMP (cGMP)/protein kinase G pathway mediates some of the anabolic effects of estrogens and mechanical stimulation in osteoblasts and osteocytes, leading us to hypothesize that cGMP-elevating agents may have bone-protective effects. We tested cinaciguat, a prototype of a novel class of soluble guanylate cyclase activators, in a mouse model of estrogen deficiency-induced osteoporosis. Compared with sham-operated mice, ovariectomized mice had lower serum cGMP concentrations, which were largely restored to normal by treatment with cinaciguat or low-dose 17β-estradiol. Microcomputed tomography of tibiae showed that cinaciguat significantly improved trabecular bone microarchitecture in ovariectomized animals, with effect sizes similar to those obtained with estrogen replacement therapy. Cinaciguat reversed ovariectomy-induced osteocyte apoptosis as efficiently as estradiol and enhanced bone formation parameters in vivo, consistent with in vitro effects on osteoblast proliferation, differentiation, and survival. Compared with 17β-estradiol, which completely reversed the ovariectomy-induced increase in osteoclast number, cinaciguat had little effect on osteoclasts. Direct guanylate cyclase stimulators have been extremely well tolerated in clinical trials of cardiovascular diseases, and our findings provide proof-of-concept for this new class of drugs as a novel, anabolic treatment strategy for postmenopausal osteoporosis, confirming an important role of nitric oxide/cGMP/protein kinase G signaling in bone. PMID:25188528

  3. Tetrahydrobiopterin protects soluble guanylate cyclase against oxidative inactivation.

    PubMed

    Schmidt, Kurt; Neubauer, Andrea; Kolesnik, Bernd; Stasch, Johannes-Peter; Werner, Ernst R; Gorren, Antonius C F; Mayer, Bernd

    2012-09-01

    Tetrahydrobiopterin (BH4) is a major endogenous vasoprotective agent that improves endothelial function by increasing nitric oxide (NO) synthesis and scavenging of superoxide and peroxynitrite. Therefore, administration of BH4 is considered a promising therapy for cardiovascular diseases associated with endothelial dysfunction and oxidative stress. Here we report on a novel function of BH4 that might contribute to the beneficial vascular effects of the pteridine. Treatment of cultured porcine aortic endothelial cells with nitroglycerin (GTN) or 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) resulted in heme oxidation of soluble guanylate cyclase (sGC), as evident from diminished NO-induced cGMP accumulation that was paralleled by increased cGMP response to a heme- and NO-independent activator of soluble guanylate cyclase [4-([(4-carboxybutyl)[2-(5-fluoro-2-([4'-(trifluoromethyl)biphenyl-4-yl]methoxy)phenyl)ethyl]amino]methyl)benzoic acid (BAY 60-2770)]. Whereas scavenging of superoxide and/or peroxynitrite with superoxide dismutase, tiron, Mn(III)tetrakis(4-benzoic acid)porphyrin, and urate had no protective effects, supplementation of the cells with BH4, either by application of BH4 directly or of its precursors dihydrobiopterin or sepiapterin, completely prevented the inhibition of NO-induced cGMP accumulation by GTN and ODQ. Tetrahydroneopterin had the same effect, and virtually identical results were obtained with RFL-6 fibroblasts, suggesting that our observation reflects a general feature of tetrahydropteridines that is unrelated to NO synthase function and not limited to endothelial cells. Protection of sGC against oxidative inactivation may contribute to the known beneficial effects of BH4 in cardiovascular disorders associated with oxidative stress. PMID:22648973

  4. Dopaminergic modulation of adenylate cyclase stimulation by vasoactive intestinal peptide in anterior pituitary.

    PubMed Central

    Onali, P; Schwartz, J P; Costa, E

    1981-01-01

    The activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by vasoactive intestinal peptide (VIP) was used as a model to investigate the molecular mechanisms triggered by the occupancy of dopamine recognition sites in rat anterior pituitary. Dopamine failed to change the basal enzyme activity, but it inhibited the stimulation of adenylate cyclase elicited by VIP. Apomorphine, 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene, and 2-bromo-alpha-ergocryptine mimicked the effect of dopamine, whereas (-)-sulpiride and and classical neuroleptics antagonized it. Dopamine failed to modulate the activation of pituitary adenylate cyclase by prostaglandin E1, which does not increase prolactin secretion. From these results we infer that stimulation of D-2 (dopamine) receptors may affect pituitary secretion by inhibiting the activation of anterior pituitary adenylate cyclase by VIP or other secretagogues. PMID:6171819

  5. Structural and Biochemical Analysis of the Essential Diadenylate Cyclase CdaA from Listeria monocytogenes*

    PubMed Central

    Rosenberg, Jonathan; Dickmanns, Achim; Neumann, Piotr; Gunka, Katrin; Arens, Johannes; Kaever, Volkhard; Stülke, Jörg; Ficner, Ralf; Commichau, Fabian M.

    2015-01-01

    The recently identified second messenger cyclic di-AMP (c-di-AMP) is involved in several important cellular processes, such as cell wall metabolism, maintenance of DNA integrity, ion transport, transcription regulation, and allosteric regulation of enzyme function. Interestingly, c-di-AMP is essential for growth of the Gram-positive model bacterium Bacillus subtilis. Although the genome of B. subtilis encodes three c-di-AMP-producing diadenlyate cyclases that can functionally replace each other, the phylogenetically related human pathogens like Listeria monocytogenes and Staphylococcus aureus possess only one enzyme, the diadenlyate cyclase CdaA. Because CdaA is also essential for growth of these bacteria, the enzyme is a promising target for the development of novel antibiotics. Here we present the first crystal structure of the L. monocytogenes CdaA diadenylate cyclase domain that is conserved in many human pathogens. Moreover, biochemical characterization of the cyclase revealed an unusual metal cofactor requirement. PMID:25605729

  6. Interactions between lysergic acid diethylamide and dopamine-sensitive adenylate cyclase systems in rat brain.

    PubMed

    Hungen, K V; Roberts, S; Hill, D F

    1975-08-22

    Investigations were carried out on the interactions of the hallucinogenic drug, D-lysergic acid diethylamide (D-LSD), and other serotonin antagonists with catecholamine-sensitive adenylate cyclase systems in cell-free preparations from different regions of rat brain. In equimolar concentration, D-LSD, 2-brono-D-lysergic acid diethylamide (BOL), or methysergide (UML) strongly blocked maximal stimulation of adenylate cyclase activity by either norepinephrine or dopamine in particulate preparations from cerebral cortices of young adult rats. D-LSD also eliminated the stimulation of adenylate cyclase activity of equimolar concentrations of norepinephrine or dopamine in particulate preparations from rat hippocampus. The effects of this hallucinogenic agent on adenylate cyclase activity were most striking in particulate preparations from corpus striatum. Thus, in 10 muM concentration, D-LSD not only completely eradicated the response to 10 muM dopamine in these preparations but also consistently stimulated adenylate cyclase activity. L-LSD (80 muM) was without effect. Significant activation of striatal adenylate cyclase was produced by 0.1 muM D-LSD. Activation of striatal adenylate cyclase of either D-LSD or dopamine was strongly blocked by the dopamine-blocking agents trifluoperazine, thioridazine, chlorpromazine, and haloperidol. The stimulatory effects of D-LSD and dopamine were also inhibited by the serotonin-blocking agents, BOL, 1-methyl-D-lysergic acid diethylamide (MLD), and cyproheptadine, but not by the beta-adrenergic-blocking agent, propranolol. However, these serotonin antagonists by themselves were incapable of stimulating adenylate cyclase activity in the striatal preparations. Several other hallucinogens, which were structurally related to serotonin, were also inactive in this regard, e.g., mescaline, N,N-dimethyltryptamine, psilocin and bufotenine. Serotonin itself produced a small stimulation of adenylate cyclase activity in striatal preparations and

  7. Functional classification of cNMP-binding proteins and nucleotide cyclases with implications for novel regulatory pathways in Mycobacterium tuberculosis.

    PubMed

    McCue, L A; McDonough, K A; Lawrence, C E

    2000-02-01

    We have analyzed the cyclic nucleotide (cNMP)-binding protein and nucleotide cyclase superfamilies using Bayesian computational methods of protein family identification and classification. In addition to the known cNMP-binding proteins (cNMP-dependent kinases, cNMP-gated channels, cAMP-guanine nucleotide exchange factors, and bacterial cAMP-dependent transcription factors), new functional groups of cNMP-binding proteins were identified, including putative ABC-transporter subunits, translocases, and esterases. Classification of the nucleotide cyclases revealed subtle differences in sequence conservation of the active site that distinguish the five classes of cyclases: the multicellular eukaryotic adenylyl cyclases, the eukaryotic receptor-type guanylyl cyclases, the eukaryotic soluble guanylyl cyclases, the unicellular eukaryotic and prokaryotic adenylyl cyclases, and the putative prokaryotic guanylyl cyclases. Phylogenetic distribution of the cNMP-binding proteins and cyclases was analyzed, with particular attention to the 22 complete archaeal and eubacterial genome sequences. Mycobacterium tuberculosis H37Rv and Synechocystis PCC6803 were each found to encode several more putative cNMP-binding proteins than other prokaryotes; many of these proteins are of unknown function. M. tuberculosis also encodes several more putative nucleotide cyclases than other prokaryotic species. PMID:10673278

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

    PubMed Central

    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

  9. Crystal Structures of the Catalytic Domain of Human Soluble Guanylate Cyclase

    PubMed Central

    Allerston, Charles K.; von Delft, Frank; Gileadi, Opher

    2013-01-01

    Soluble guanylate cyclase (sGC) catalyses the synthesis of cyclic GMP in response to nitric oxide. The enzyme is a heterodimer of homologous α and β subunits, each of which is composed of multiple domains. We present here crystal structures of a heterodimer of the catalytic domains of the α and β subunits, as well as an inactive homodimer of β subunits. This first structure of a metazoan, heteromeric cyclase provides several observations. First, the structures resemble known structures of adenylate cyclases and other guanylate cyclases in overall fold and in the arrangement of conserved active-site residues, which are contributed by both subunits at the interface. Second, the subunit interaction surface is promiscuous, allowing both homodimeric and heteromeric association; the preference of the full-length enzyme for heterodimer formation must derive from the combined contribution of other interaction interfaces. Third, the heterodimeric structure is in an inactive conformation, but can be superposed onto an active conformation of adenylate cyclase by a structural transition involving a 26° rigid-body rotation of the α subunit. In the modelled active conformation, most active site residues in the subunit interface are precisely aligned with those of adenylate cyclase. Finally, the modelled active conformation also reveals a cavity related to the active site by pseudo-symmetry. The pseudosymmetric site lacks key active site residues, but may bind allosteric regulators in a manner analogous to the binding of forskolin to adenylate cyclase. This indicates the possibility of developing a new class of small-molecule modulators of guanylate cyclase activity targeting the catalytic domain. PMID:23505436

  10. 3',5'-cyclic adenosine monophosphate and adenylate cyclase in phototransduction by limulus ventral photoreceptors.

    PubMed Central

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

    1984-01-01

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

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

    PubMed

    Piatakova, N V; Severina, I S

    2012-01-01

    The influence of ambroxol--a mucolytic drug--on the activity of human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase and activation of both enzymes by NO-donors (sodium nitroprusside and Sin-1) were investigated. Ambroxol in the concentration range from 0.1 to 10 microM had no effect on the basal activity of both enzymes. Ambroxol inhibited in a concentration-dependent manner the sodium nitroprusside-induced human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase with the IC50 values 3.9 and 2.1 microM, respectively. Ambroxol did not influence the stimulation of both enzymes by protoporphyrin IX. The influence of artemisinin--an antimalarial drug--on human platelet soluble guanylate cyclase activity and the enzyme activation by NO-donors were investigated. Artemisinin (0.1-100 microM) had no effect on the basal activity of the enzyme. Artemisinin inhibited in a concentration-dependent manner the sodium nitroprusside-induced activation of human platelet guanylate cyclase with an IC50 value 5.6 microM. Artemisinin (10 microM) also inhibited (by 71 +/- 4.0%) the activation of the enzyme by thiol-dependent NO-donor the derivative of furoxan, 3,4-dicyano-1,2,5-oxadiazolo-2-oxide (10 microM), but did not influence the stimulation of soluble guanylate cyclase by protoporphyrin IX. It was concluded that the sygnalling system NO-soluble guanylate cyclase-cGMP is involved in the molecular mechanism of the therapeutic action of ambroxol and artemisinin.

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

    PubMed

    Piatakova, N V; Severina, I S

    2012-01-01

    The influence of ambroxol--a mucolytic drug--on the activity of human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase and activation of both enzymes by NO-donors (sodium nitroprusside and Sin-1) were investigated. Ambroxol in the concentration range from 0.1 to 10 microM had no effect on the basal activity of both enzymes. Ambroxol inhibited in a concentration-dependent manner the sodium nitroprusside-induced human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase with the IC50 values 3.9 and 2.1 microM, respectively. Ambroxol did not influence the stimulation of both enzymes by protoporphyrin IX. The influence of artemisinin--an antimalarial drug--on human platelet soluble guanylate cyclase activity and the enzyme activation by NO-donors were investigated. Artemisinin (0.1-100 microM) had no effect on the basal activity of the enzyme. Artemisinin inhibited in a concentration-dependent manner the sodium nitroprusside-induced activation of human platelet guanylate cyclase with an IC50 value 5.6 microM. Artemisinin (10 microM) also inhibited (by 71 +/- 4.0%) the activation of the enzyme by thiol-dependent NO-donor the derivative of furoxan, 3,4-dicyano-1,2,5-oxadiazolo-2-oxide (10 microM), but did not influence the stimulation of soluble guanylate cyclase by protoporphyrin IX. It was concluded that the sygnalling system NO-soluble guanylate cyclase-cGMP is involved in the molecular mechanism of the therapeutic action of ambroxol and artemisinin. PMID:22642150

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

    PubMed

    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).

  14. Cloning, chromosomal mapping, and expression of human fetal brain type I adenylyl cyclase

    SciTech Connect

    Villacres, E.C.; Xia, Z.; Bookbinder, L.H.; Edelhoff, S.; Disteche, C.M.; Storm, D.R.

    1993-05-01

    The neural-specific calmodulin-sensitive adenylyl cyclase (type I), which was first cloned from bovine brain, has been implicated in learning and memory. The objective of this study was to clone and determine the chromosomal localization of human fetal brain type I adenylyl cyclase. A 3.8-kb cDNA clone was isolated that contained sequence coinciding with the 3{prime} end 2553 nucleotides of the bovine open reading frame. This clone shows 87% nucleotide and 92% translated amino acid sequence identity to the bovine clone. The most significant sequence differences were in the carboxy-terminal 100 amino acid residues. This region contains one of several possible calmodulin binding domains and the only putative cAMP-dependent protein kinase A phosphorylation site. A chimera was constructed that contained the 5{prime} half of the bovine type I adenylyl cyclase and the 3{prime} half of the human type I adenylyl cyclase. The activity of the chimeric gene product and its sensitivity to calmodulin and calcium were indistinguishable from those of the bovine type I adenylyl cyclase. In situ hybridization was used to localize the human type I adenylyl cyclase gene to the proximal portion of the short arm of chromosome 7. 36 refs., 4 figs.

  15. Bicarbonate Modulates Photoreceptor Guanylate Cyclase (ROS-GC) Catalytic Activity.

    PubMed

    Duda, Teresa; Wen, Xiao-Hong; Isayama, Tomoki; Sharma, Rameshwar K; Makino, Clint L

    2015-04-24

    By generating the second messenger cGMP in retinal rods and cones, ROS-GC plays a central role in visual transduction. Guanylate cyclase-activating proteins (GCAPs) link cGMP synthesis to the light-induced fall in [Ca(2+)]i to help set absolute sensitivity and assure prompt recovery of the response to light. The present report discloses a surprising feature of this system: ROS-GC is a sensor of bicarbonate. Recombinant ROS-GCs synthesized cGMP from GTP at faster rates in the presence of bicarbonate with an ED50 of 27 mM for ROS-GC1 and 39 mM for ROS-GC2. The effect required neither Ca(2+) nor use of the GCAPs domains; however, stimulation of ROS-GC1 was more powerful in the presence of GCAP1 or GCAP2 at low [Ca(2+)]. When applied to retinal photoreceptors, bicarbonate enhanced the circulating current, decreased sensitivity to flashes, and accelerated flash response kinetics. Bicarbonate was effective when applied either to the outer or inner segment of red-sensitive cones. In contrast, bicarbonate exerted an effect when applied to the inner segment of rods but had little efficacy when applied to the outer segment. The findings define a new regulatory mechanism of the ROS-GC system that affects visual transduction and is likely to affect the course of retinal diseases caused by cGMP toxicity. PMID:25767116

  16. Control of outflow resistance by soluble adenylyl cyclase.

    PubMed

    Lee, Yong Suk; Marmorstein, Alan D

    2014-01-01

    Abstract Glaucoma is a leading cause of blindness in the United States affecting as many as 2.2 million Americans. All current glaucoma treatment strategies aim to reduce intraocular pressure, even in patients with normal tension glaucoma. Typically, this is accomplished by reducing the rate of aqueous flow by limiting aqueous production or enhancing drainage using drugs and surgery. Whereas these strategies are effective in diminishing vision loss, some patients continue to lose vision and many discontinue use of their medications because of undesirable side effects. Drugs known to be effective in altering conventional outflow have for the most part been abandoned from modern clinical practice due to undesirable side effects. Identification of new drugs that could enhance conventional outflow, would offer additional options in the treatment of glaucoma and ocular hypertension. To this end, our laboratory has recently uncovered a novel pathway for regulation of conventional outflow by the ciliary body. This pathway is dependent on soluble adenylyl cyclase, an enzyme that catalyzes the generation of cyclic adenosine 3',5' monophosphate (cAMP) in response to bicarbonate.

  17. Guanylyl cyclase C signaling axis and colon cancer prevention

    PubMed Central

    Pattison, Amanda M; Merlino, Dante J; Blomain, Erik S; Waldman, Scott A

    2016-01-01

    Colorectal cancer (CRC) is a major cause of cancer-related mortality and morbidity worldwide. While improved treatments have enhanced overall patient outcome, disease burden encompassing quality of life, cost of care, and patient survival has seen little benefit. Consequently, additional advances in CRC treatments remain important, with an emphasis on preventative measures. Guanylyl cyclase C (GUCY2C), a transmembrane receptor expressed on intestinal epithelial cells, plays an important role in orchestrating intestinal homeostatic mechanisms. These effects are mediated by the endogenous hormones guanylin (GUCA2A) and uroguanylin (GUCA2B), which bind and activate GUCY2C to regulate proliferation, metabolism and barrier function in intestine. Recent studies have demonstrated a link between GUCY2C silencing and intestinal dysfunction, including tumorigenesis. Indeed, GUCY2C silencing by the near universal loss of its paracrine hormone ligands increases colon cancer susceptibility in animals and humans. GUCY2C’s role as a tumor suppressor has opened the door to a new paradigm for CRC prevention by hormone replacement therapy using synthetic hormone analogs, such as the FDA-approved oral GUCY2C ligand linaclotide (Linzess™). Here we review the known contributions of the GUCY2C signaling axis to CRC, and relate them to a novel clinical strategy targeting tumor chemoprevention. PMID:27688649

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

    PubMed

    Desai, Kevin K; Bingman, Craig A; Cheng, Chin L; Phillips, George N; Raines, Ronald T

    2014-10-01

    RNA 3'-phosphate cyclase (RtcA) catalyzes the ATP-dependent cyclization of a 3'-phosphate to form a 2',3'-cyclic phosphate at RNA termini. Cyclization proceeds through RtcA-AMP and RNA(3')pp(5')A covalent intermediates, which are analogous to intermediates formed during catalysis by the tRNA ligase RtcB. Here we present a crystal structure of Pyrococcus horikoshii RtcA in complex with a 3'-phosphate terminated RNA and adenosine in the AMP-binding pocket. Our data reveal that RtcA recognizes substrate RNA by ensuring that the terminal 3'-phosphate makes a large contribution to RNA binding. Furthermore, the RNA 3'-phosphate is poised for in-line attack on the P-N bond that links the phosphorous atom of AMP to N(ε) of His307. Thus, we provide the first insights into RNA 3'-phosphate termini recognition and the mechanism of 3'-phosphate activation by an Rtc enzyme.

  19. Guanylyl cyclase C signaling axis and colon cancer prevention.

    PubMed

    Pattison, Amanda M; Merlino, Dante J; Blomain, Erik S; Waldman, Scott A

    2016-09-28

    Colorectal cancer (CRC) is a major cause of cancer-related mortality and morbidity worldwide. While improved treatments have enhanced overall patient outcome, disease burden encompassing quality of life, cost of care, and patient survival has seen little benefit. Consequently, additional advances in CRC treatments remain important, with an emphasis on preventative measures. Guanylyl cyclase C (GUCY2C), a transmembrane receptor expressed on intestinal epithelial cells, plays an important role in orchestrating intestinal homeostatic mechanisms. These effects are mediated by the endogenous hormones guanylin (GUCA2A) and uroguanylin (GUCA2B), which bind and activate GUCY2C to regulate proliferation, metabolism and barrier function in intestine. Recent studies have demonstrated a link between GUCY2C silencing and intestinal dysfunction, including tumorigenesis. Indeed, GUCY2C silencing by the near universal loss of its paracrine hormone ligands increases colon cancer susceptibility in animals and humans. GUCY2C's role as a tumor suppressor has opened the door to a new paradigm for CRC prevention by hormone replacement therapy using synthetic hormone analogs, such as the FDA-approved oral GUCY2C ligand linaclotide (Linzess™). Here we review the known contributions of the GUCY2C signaling axis to CRC, and relate them to a novel clinical strategy targeting tumor chemoprevention. PMID:27688649

  20. Murine Guanylate Cyclase C Regulates Colonic Injury and Inflammation1

    PubMed Central

    Steinbrecher, Kris A.; Harmel-Laws, Eleana; Garin-Laflam, Monica P.; Mann, Elizabeth A.; Bezerra, Lucas D.; Hogan, Simon P.; Cohen, Mitchell B.

    2011-01-01

    Guanylate cyclase C (GUCY2C or GC-C) and its ligands, guanylin (GUCA2A or Gn) and uroguanylin (GUCA2B or Ugn), are expressed in intestinal epithelial cells (IECs) and regulate ion secretion, intestinal barrier function, and epithelial monolayer homeostasis via cGMP-dependent signaling pathways. The aim of this study was to determine if GC-C and its ligands direct the course of intestinal inflammation. Here, we show that DSS-induced clinical disease and histological damage to the colonic mucosa were significantly less severe in GC-C−/− mice and moderately reduced in Gn−/− animals. Relative to wildtype controls, GC-C−/− and Gn−/− mice had reduced apoptosis and increased proliferation of IECs during DSS colitis. Basal and DSS-induced production of resistin-like molecule β (RELMβ) was substantially diminished in GC-C−/− mice. RELMβ is thought to stimulate cytokine production in macrophages in this disease model and, consistent with this, TNFα and IFNγ production was minimal in GC-C−/− animals. RELMβ and cytokine levels were similar to wildtype in Gn−/− mice, however. Colonic instillation of recombinant RELMβ by enema into GC-C−/− mice restores sensitivity to DSS-mediated mucosal injury. These findings demonstrate a novel role for GC-C signaling in facilitating mucosal wounding and inflammation and further suggest that this may be mediated, in part, through control of RELMβ production. PMID:21555532

  1. Soluble adenylyl cyclase is essential for proper lysosomal acidification.

    PubMed

    Rahman, Nawreen; Ramos-Espiritu, Lavoisier; Milner, Teresa A; Buck, Jochen; Levin, Lonny R

    2016-10-01

    Lysosomes, the degradative organelles of the endocytic and autophagic pathways, function at an acidic pH. Lysosomes are acidified by the proton-pumping vacuolar ATPase (V-ATPase), but the molecular processes that set the organelle's pH are not completely understood. In particular, pH-sensitive signaling enzymes that can regulate lysosomal acidification in steady-state physiological conditions have yet to be identified. Soluble adenylyl cyclase (sAC) is a widely expressed source of cAMP that serves as a physiological pH sensor in cells. For example, in proton-secreting epithelial cells, sAC is responsible for pH-dependent translocation of V-ATPase to the luminal surface. Here we show genetically and pharmacologically that sAC is also essential for lysosomal acidification. In the absence of sAC, V-ATPase does not properly localize to lysosomes, lysosomes fail to fully acidify, lysosomal degradative capacity is diminished, and autophagolysosomes accumulate. PMID:27670898

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

    NASA Technical Reports Server (NTRS)

    Lee, Y. C.; Martin, E.; Murad, F.

    2000-01-01

    The alpha1- and beta1-subunits of human soluble guanylate cyclase (sGC) were coexpressed in the Sf9 cells/baculovirus system. In addition to the native enzyme, constructs with hexahistidine tag at the amino and carboxyl termini of each subunit were coexpressed. This permitted the rapid and efficient purification of active recombinant enzyme on a nickel-affinity column. The enzyme has one heme per heterodimer and was readily activated with the NO donor sodium nitroprusside or 3-(5'-hydroxymethyl-2'furyl)-1-benzyl-indazole (YC-1). Sodium nitroprusside and YC-1 treatment potentiated each other in combination and demonstrated a remarkable 2,200-fold stimulation of the human recombinant sGC. The effects were inhibited with 1H-(1,2, 4)oxadiazole(4,3-a)quinoxalin-1one (ODQ). The kinetics of the recombinant enzyme with respect to GTP was examined. The products of the reaction, cGMP and pyrophosphate, inhibited the enzyme. The extent of inhibition by cGMP depended on the activation state of the enzyme, whereas inhibition by pyrophosphate was not affected by the enzyme state. Both reaction products displayed independent binding and cooperativity with respect to enzyme inhibition. The expression of large quantities of active enzyme will facilitate structural characterization of the protein.

  3. Guanylyl cyclase C signaling axis and colon cancer prevention

    PubMed Central

    Pattison, Amanda M; Merlino, Dante J; Blomain, Erik S; Waldman, Scott A

    2016-01-01

    Colorectal cancer (CRC) is a major cause of cancer-related mortality and morbidity worldwide. While improved treatments have enhanced overall patient outcome, disease burden encompassing quality of life, cost of care, and patient survival has seen little benefit. Consequently, additional advances in CRC treatments remain important, with an emphasis on preventative measures. Guanylyl cyclase C (GUCY2C), a transmembrane receptor expressed on intestinal epithelial cells, plays an important role in orchestrating intestinal homeostatic mechanisms. These effects are mediated by the endogenous hormones guanylin (GUCA2A) and uroguanylin (GUCA2B), which bind and activate GUCY2C to regulate proliferation, metabolism and barrier function in intestine. Recent studies have demonstrated a link between GUCY2C silencing and intestinal dysfunction, including tumorigenesis. Indeed, GUCY2C silencing by the near universal loss of its paracrine hormone ligands increases colon cancer susceptibility in animals and humans. GUCY2C’s role as a tumor suppressor has opened the door to a new paradigm for CRC prevention by hormone replacement therapy using synthetic hormone analogs, such as the FDA-approved oral GUCY2C ligand linaclotide (Linzess™). Here we review the known contributions of the GUCY2C signaling axis to CRC, and relate them to a novel clinical strategy targeting tumor chemoprevention.

  4. Alterations in adipocyte adenylate cyclase activity in morbidly obese and formerly morbidly obese humans.

    PubMed

    Martin, L F; Klim, C M; Vannucci, S J; Dixon, L B; Landis, J R; LaNoue, K F

    1990-08-01

    Studies examining animal models of genetic obesity have identified defects in adipocyte hormone-stimulated lipolysis that involve the adenylate cyclase transmembrane signaling system, specifically those components that decrease adenylate cyclase activity. To determine whether obese people demonstrate alterations in adenylate cyclase activity that could contribute to the maintenance of obesity by inhibiting lipolysis, we examined human adipocytes from patients who were lean, obese, or formerly obese. Fat samples were obtained from the lower abdomen of 14 women who were morbidly obese (obese group), from 10 women who were formerly morbidly obese and had lost weight after gastric stapling (postobese group), and from 10 similarly aged women of normal weight (controls). Adipocyte adenylate cyclase activity was determined under ligand-free (no stimulatory or inhibitory influences present), hormone-stimulated (isoproterenol, 10(-6) mmol/L), and maximal (cells stimulated with 10 mumol/L forskolin) conditions by measuring cyclic adenosine monophosphate (cAMP) levels by radioimmunoassay. The activity of adenylate cyclase was significantly different (p less than 0.01) in the three groups. Adipocytes from obese women had lower levels of cyclase activity under both ligand-free (5% vs 16% of maximal) and hormone-stimulated conditions (76% vs 100% of maximal) than adipocytes from normal women. Postobese women had levels of hormone-stimulated cAMP identical to those of normal women but still had abnormal ligand-free levels (under 5%). These results suggest the presence of an alteration in adipocyte adenylate cyclase regulation in morbidly obese women that is not entirely corrected when weight is lost after food intake is reduced by gastric stapling. This alteration in ligand-free cAMP activity may contribute to the development and maintenance of obesity. PMID:2166354

  5. A lycopene β-cyclase/lycopene ε-cyclase/light-harvesting complex-fusion protein from the green alga Ostreococcus lucimarinus can be modified to produce α-carotene and β-carotene at different ratios.

    PubMed

    Blatt, Andreas; Bauch, Matthias E; Pörschke, Yvonne; Lohr, Martin

    2015-05-01

    Biosynthesis of asymmetric carotenoids such as α-carotene and lutein in plants and green algae involves the two enzymes lycopene β-cyclase (LCYB) and lycopene ε-cyclase (LCYE). The two cyclases are closely related and probably resulted from an ancient gene duplication. While in most plants investigated so far the two cyclases are encoded by separate genes, prasinophyte algae of the order Mamiellales contain a single gene encoding a fusion protein comprised of LCYB, LCYE and a C-terminal light-harvesting complex (LHC) domain. Here we show that the lycopene cyclase fusion protein from Ostreococcus lucimarinus catalyzed the simultaneous formation of α-carotene and β-carotene when heterologously expressed in Escherichia coli. The stoichiometry of the two products in E. coli could be altered by gradual truncation of the C-terminus, suggesting that the LHC domain may be involved in modulating the relative activities of the two cyclase domains in the algae. Partial deletions of the linker region between the cyclase domains or replacement of one or both cyclase domains with the corresponding cyclases from the green alga Chlamydomonas reinhardtii resulted in pronounced shifts of the α-carotene-to-β-carotene ratio, indicating that both the relative activities of the cyclase domains and the overall structure of the fusion protein have a strong impact on the product stoichiometry. The possibility to tune the product ratio of the lycopene cyclase fusion protein from Mamiellales renders it useful for the biotechnological production of the asymmetric carotenoids α-carotene or lutein in bacteria or fungi.

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

    PubMed Central

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

    1980-01-01

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

  7. Role of soluble guanylate cyclase in the molecular mechanism underlying the physiological effects of nitric oxide.

    PubMed

    Severina, I S

    1998-07-01

    In this review the molecular mechanisms underlying the antihypertensive and antiaggregatory actions of nitric oxide (NO) are discussed. It has been shown that these effects are directly connected with the activation of soluble guanylate cyclase and the accumulation of cyclic 3;,5;-guanosine monophosphate (cGMP). The mechanism of guanylate cyclase activation by NO is analyzed, especially the role and biological significance of the nitrosyl--heme complex formed as a result of interaction of guanylate cyclase heme with NO and the role of sulfhydryl groups of the enzyme in this process. Using new approaches for studying the antihypertensive and antiaggregatory actions of nitric oxide in combination with the newly obtained data on the regulatory role of guanylate cyclase in the platelet aggregation process, the most important results were obtained regarding the molecular bases providing for a directed search for and creation of new effective antihypertensive and antiaggregatory preparations. In studying the molecular mechanism for directed activation of soluble guanylate cyclase by new NO donors, a series of hitherto unknown enzyme activators generating NO and involved in the regulation of hemostasis and vascular tone were revealed. PMID:9721331

  8. Overexpression and characterization of lycopene cyclase (CrtY) from marine bacterium Paracoccus haeundaensis.

    PubMed

    Jeong, Tae Hyug; Ji, Keunho; Kim, Young Tae

    2013-02-01

    Lycopene cyclase converts lycopene to beta-carotene by catalyzing the formation of two beta-rings at each end of the linear carotene structure. This reaction takes place as a two-step reaction in which both sides of of the lycopene molecule are cyclized into beta-carotene rings via the monocyclic gamma-carotene as an intermediate. The crtY gene coding for lycopene cyclase from Paracoccus haeundaensis consists of 1,158 base pairs encoding 386 amino acids residues. An expression plasmid containing the crtY gene (pET44a-CrtY) was constructed and expressed in Escherichia coli, and produced a recombinant protein of approximately 43 kDa, corresponding to the molecular mass of lycopene cyclase. The expressed protein was purified to homogeneity by His-tag affinity chromatography and showed enzymatic activity corresponding to lycopene cyclase. We also determined the lycopene substrate specificity and NADPH cofactor requirements of the purified protein. The Km values for lycopene and NADPH were 3.5 microM and 2 mM, respectively. The results obtained from this study will provide a wider base of knowledge on the enzyme characterization of lycopene cyclase at the molecular level.

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

    PubMed Central

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

    2012-01-01

    Δ9-Tetrahydrocannabinol (THC) and other cannabinoids are responsible for the psychoactive and medicinal properties of Cannabis sativa L. (marijuana). The first intermediate in the cannabinoid biosynthetic pathway is proposed to be olivetolic acid (OA), an alkylresorcinolic acid that forms the polyketide nucleus of the cannabinoids. OA has been postulated to be synthesized by a type III polyketide synthase (PKS) enzyme, but so far type III PKSs from cannabis have been shown to produce catalytic byproducts instead of OA. We analyzed the transcriptome of glandular trichomes from female cannabis flowers, which are the primary site of cannabinoid biosynthesis, and searched for polyketide cyclase-like enzymes that could assist in OA cyclization. Here, we show that a type III PKS (tetraketide synthase) from cannabis trichomes requires the presence of a polyketide cyclase enzyme, olivetolic acid cyclase (OAC), which catalyzes a C2–C7 intramolecular aldol condensation with carboxylate retention to form OA. OAC is a dimeric α+β barrel (DABB) protein that is structurally similar to polyketide cyclases from Streptomyces species. OAC transcript is present at high levels in glandular trichomes, an expression profile that parallels other cannabinoid pathway enzymes. Our identification of OAC both clarifies the cannabinoid pathway and demonstrates unexpected evolutionary parallels between polyketide biosynthesis in plants and bacteria. In addition, the widespread occurrence of DABB proteins in plants suggests that polyketide cyclases may play an overlooked role in generating plant chemical diversity. PMID:22802619

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

    PubMed

    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.

  11. Sweet tastants stimulate adenylate cyclase coupled to GTP-binding protein in rat tongue membranes.

    PubMed

    Striem, B J; Pace, U; Zehavi, U; Naim, M; Lancet, D

    1989-05-15

    Sucrose and other saccharides, which produce an appealing taste in rats, were found to significantly stimulate the activity of adenylate cyclase in membranes derived from the anterior-dorsal region of rat tongue. In control membranes derived from either tongue muscle or tongue non-sensory epithelium, the effect of sugars on adenylate cyclase activity was either much smaller or absent. Sucrose enhanced adenylate cyclase activity in a dose-related manner, and this activation was dependent on the presence of guanine nucleotides, suggesting the involvement of a GTP-binding protein ('G-protein'). The activation of adenylate cyclase by various mono- and di-saccharides correlated with their electrophysiological potency. Among non-sugar sweeteners, sodium saccharin activated the enzyme, whereas aspartame and neohesperidin dihydrochalcone did not, in correlation with their sweet-taste effectiveness in the rat. Sucrose activation of the enzyme was partly inhibited by Cu2+ and Zn2+, in agreement with their effect on electrophysiological sweet-taste responses. Our results are consistent with a sweet-taste transduction mechanism involving specific receptors, a guanine-nucleotide-binding protein and the cyclic AMP-generating enzyme adenylate cyclase.

  12. Alternative splicing impairs soluble guanylyl cyclase function in aortic aneurysm.

    PubMed

    Martin, Emil; Golunski, Eva; Laing, Susan T; Estrera, Anthony L; Sharina, Iraida G

    2014-12-01

    Nitric oxide (NO) receptor soluble guanylyl cyclase (sGC) is a key regulator of several important vascular functions and is important for maintaining cardiovascular homeostasis and vascular plasticity. Diminished sGC expression and function contributes to pathogenesis of several cardiovascular diseases. However, the processes that control sGC expression in vascular tissue remain poorly understood. Previous work in animal and cell models revealed the complexity of alternative splicing of sGC genes and demonstrated its importance in modulation of sGC function. The aim of this study was to examine the role of alternative splicing of α1 and β1 sGC in healthy and diseased human vascular tissue. Our study found a variety of α1 and β1 sGC splice forms expressed in human aorta. Their composition and abundance were different between samples of aortic tissue removed during surgical repair of aortic aneurysm and samples of aortas without aneurysm. Aortas with aneurysm demonstrated decreased sGC activity, which correlated with increased expression of dysfunctional sGC splice variants. In addition, the expression of 55-kDa oxidation-resistant α1 isoform B sGC (α1-IsoB) was significantly lower in aortic samples with aneurysm. The α1-IsoB splice variant was demonstrated to support sGC activity in aortic lysates. Together, our results suggest that alternative splicing contributes to diminished sGC function in vascular dysfunction. Precise understanding of sGC splicing regulation could help to design new therapeutic interventions and to personalize sGC-targeting therapies in treatments of vascular disease.

  13. Biochemistry and physiology of the natriuretic peptide receptor guanylyl cyclases.

    PubMed

    Tremblay, Johanne; Desjardins, Richard; Hum, David; Gutkowska, Jolanta; Hamet, Pavel

    2002-01-01

    Guanylyl cyclases (GC) exist as soluble and particulate, membrane-associated enzymes which catalyse the conversion of GTP to cGMP, an intracellular signalling molecule. Several membrane forms of the enzyme have been identified up to now. Some of them serve as receptors for the natriuretic peptides, a family of peptides which includes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP), three peptides known to play important roles in renal and cardiovascular physiology. These are transmembrane proteins composed of a single transmembrane domain, a variable extracellular natriuretic peptide-binding domain, and a more conserved intracellular kinase homology domain (KHD) and catalytic domain. GC-A, the receptor for ANP and BNP, also named natriuretic peptide receptor-A or -1 (NPR-A or NPR- 1), has been studied widely. Its mode of activation by peptide ligands and mechanisms of regulation serve as prototypes for understanding the function of other particulate GC. Activation of this enzyme by its ligand is a complex process requiring oligomerization, ligand binding, KHD phosphorylation and ATP binding. Gene knockout and genetic segregation studies have provided strong evidence for the importance of GC-A in the regulation of blood pressure and heart and renal functions. GC-B is the main receptor for CNP, the latter having a more paracrine role at the vascular and venous levels. The structure and regulation of GC-B is similar to that of GC-A. This chapter reviews the structure and roles of GC-A and GC-B in blood pressure regulation and cardiac and renal pathophysiology. PMID:11952095

  14. Adenylate cyclase regulates elongation of mammalian primary cilia

    SciTech Connect

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

    2009-10-01

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

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

    PubMed

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

    2014-04-01

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

  16. Molecular characterization of tick salivary gland glutaminyl cyclase.

    PubMed

    Adamson, Steven W; Browning, Rebecca E; Chao, Chien-Chung; Bateman, Robert C; Ching, Wei-Mei; Karim, Shahid

    2013-09-01

    Glutaminyl cyclase (QC) catalyzes the cyclization of N-terminal glutamine residues into pyroglutamate. This post-translational modification extends the half-life of peptides and, in some cases, is essential in binding to their cognate receptor. Due to its potential role in the post-translational modification of tick neuropeptides, we report the molecular, biochemical and physiological characterization of salivary gland QC during the prolonged blood feeding of the black-legged tick (Ixodes scapularis) and the gulf-coast tick (Amblyomma maculatum). QC sequences from I. scapularis and A. maculatum showed a high degree of amino acid identity to each other and other arthropods and residues critical for zinc binding/catalysis (D159, E202, and H330) or intermediate stabilization (E201, W207, D248, D305, F325, and W329) are conserved. Analysis of QC transcriptional gene expression kinetics depicts an upregulation during the bloodmeal of adult female ticks prior to fast-feeding phases in both I. scapularis and A. maculatum suggesting a functional link with bloodmeal uptake. QC enzymatic activity was detected in saliva and extracts of tick salivary glands and midguts. Recombinant QC was shown to be catalytically active. Furthermore, knockdown of QC transcript by RNA interference resulted in lower enzymatic activity, and small, unviable egg masses in both studied tick species as well as lower engorged tick weights for I. scapularis. These results suggest that the post-translational modification of neurotransmitters and other bioactive peptides by QC is critical to oviposition and potentially other physiological processes. Moreover, these data suggest that tick-specific QC-modified neurotransmitters/hormones or other relevant parts of this system could potentially be used as novel physiological targets for tick control. PMID:23770496

  17. Renal Phosphate Wasting in the Absence of Adenylyl Cyclase 6

    PubMed Central

    Fenton, Robert A.; Murray, Fiona; Dominguez Rieg, Jessica A.; Tang, Tong; Levi, Moshe

    2014-01-01

    Parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF-23) enhance phosphate excretion by the proximal tubule of the kidney by retrieval of the sodium-dependent phosphate transporters (Npt2a and Npt2c) from the apical plasma membrane. PTH activates adenylyl cyclase (AC) through PTH 1 receptors and stimulates the cAMP/PKA signaling pathway. However, the precise role and isoform(s) of AC in phosphate homeostasis are not known. We report here that mice lacking AC6 (AC6−/−) have increased plasma PTH and FGF-23 levels compared with wild-type (WT) mice but comparable plasma phosphate concentrations. Acute activation of the calcium-sensing receptor or feeding a zero phosphate diet almost completely suppressed plasma PTH levels in both AC6−/− and WT mice, indicating a secondary cause for hyperparathyroidism. Pharmacologic blockade of FGF receptors resulted in a comparable increase in plasma phosphate between genotypes, whereas urinary phosphate remained significantly higher in AC6−/− mice. Compared with WT mice, AC6−/− mice had reduced renal Npt2a and Npt2c protein abundance, with approximately 80% of Npt2a residing in lysosomes. WT mice responded to exogenous PTH with redistribution of Npt2a from proximal tubule microvilli to intracellular compartments and lysosomes alongside a PTH-induced dose–response relationship for fractional phosphate excretion and urinary cAMP excretion. These responses were absent in AC6−/− mice. In conclusion, AC6 in the proximal tubule modulates cAMP formation, Npt2a trafficking, and urinary phosphate excretion, which are highlighted by renal phosphate wasting in AC6−/− mice. PMID:24854272

  18. Role of Adenylate Cyclase 1 in Retinofugal Map Development

    PubMed Central

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

    2013-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2001-01-01

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

  20. Ontogeny of regulatory mechanisms for beta-adrenoceptor control of rat cardiac adenylyl cyclase: targeting of G-proteins and the cyclase catalytic subunit.

    PubMed

    Zeiders, J L; Seidler, F J; Slotkin, T A

    1997-02-01

    Fetal and neonatal tissues are resistant to catecholamine-induced desensitization of essential physiological responses. We examined the mechanisms underlying the ontogeny of desensitization in neonatal rat heart for the beta-adrenergic receptor/adenylyl cyclase signaling cascade. Animals of different ages received isoproterenol daily or 4 days and cardiac membrane preparations were evaluated on the 5th day (6, 15, 25 days old and adults). Measurements were made of basal activity, activity stimulated by two agonists (isoproterenol or glucagon) that operate at different receptors but that share Gs as the transduction intermediate, or by forskolin-Mn' to assess total catalytic capacity of the cyclase subunit; we also assessed inhibition of activity by carbachol which acts via muscarinic cholinergic receptors and G. Adult rats exhibited robust desensitization of the adenylyl cyclase response but the effect was heterologous in that equivalent loss of activity was seen for basal, isoproterenol- and glucagon-stimulated activity forskolin-Mn(2+)-stimulated activity was also decreased. Two factors contributed to desensitization; generalized reduction in membrane protein concentrations caused by cell enlargement (reduced surface-to-volume ratio), and specific interference with the G-protein component that couples receptors to the cyclase. Thus, after adjustment for changes in membrane protein, the desensitization of the forskolin-Mn2, response was no longer evident, but the effects on the other measures were still present. In addition, isoproterenol treatment produced crosstalk with the carbachol/Gi signaling pathway, with significant reductions in the ability of carbachol to inhibit adenylyl cyclase activity. Heterologous desensitization by isoproterenol was also present in 15 and 25 day old rats, but involved only selective components of the effects seen in adults. At 25 days, uncoupling of signals operating through Gs and Gi was obtained without a reduction in forskolin

  1. Crystal structure of the Alpha subunit PAS domain from soluble guanylyl cyclase

    PubMed Central

    Purohit, Rahul; Weichsel, Andrzej; Montfort, William R

    2013-01-01

    Soluble guanylate cyclase (sGC) is a heterodimeric heme protein of ∼150 kDa and the primary nitric oxide receptor. Binding of NO stimulates cyclase activity, leading to regulation of cardiovascular physiology and providing attractive opportunities for drug discovery. How sGC is stimulated and where candidate drugs bind remains unknown. The α and β sGC chains are each composed of Heme-Nitric Oxide Oxygen (H-NOX), Per-ARNT-Sim (PAS), coiled-coil and cyclase domains. Here, we present the crystal structure of the α1 PAS domain to 1.8 Å resolution. The structure reveals the binding surfaces of importance to heterodimer function, particularly with respect to regulating NO binding to heme in the β1 H-NOX domain. It also reveals a small internal cavity that may serve to bind ligands or participate in signal transduction. PMID:23934793

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

    PubMed Central

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

    1979-01-01

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

  3. Pseudomonas aeruginosa Exotoxin Y Is a Promiscuous Cyclase That Increases Endothelial Tau Phosphorylation and Permeability*

    PubMed Central

    Ochoa, Cristhiaan D.; Alexeyev, Mikhail; Pastukh, Viktoriya; Balczon, Ron; Stevens, Troy

    2012-01-01

    Exotoxin Y (ExoY) is a type III secretion system effector found in ∼ 90% of the Pseudomonas aeruginosa isolates. Although it is known that ExoY causes inter-endothelial gaps and vascular leak, the mechanisms by which this occurs are poorly understood. Using both a bacteria-delivered and a codon-optimized conditionally expressed ExoY, we report that this toxin is a dual soluble adenylyl and guanylyl cyclase that results in intracellular cAMP and cGMP accumulation. The enzymatic activity of ExoY caused phosphorylation of endothelial Tau serine 214, accumulation of insoluble Tau, inter-endothelial cell gap formation, and increased macromolecular permeability. To discern whether the cAMP or cGMP signal was responsible for Tau phosphorylation and barrier disruption, pulmonary microvascular endothelial cells were engineered for the conditional expression of either wild-type guanylyl cyclase, which synthesizes cGMP, or a mutated guanylyl cyclase, which synthesizes cAMP. Sodium nitroprusside stimulation of the cGMP-generating cyclase resulted in transient Tau serine 214 phosphorylation and gap formation, whereas stimulation of the cAMP-generating cyclase induced a robust increase in Tau serine 214 phosphorylation, gap formation, and macromolecular permeability. These results indicate that the cAMP signal is the dominant stimulus for Tau phosphorylation. Hence, ExoY is a promiscuous cyclase and edema factor that uses cAMP and, to some extent, cGMP to induce the hyperphosphorylation and insolubility of endothelial Tau. Because hyperphosphorylated and insoluble Tau are hallmarks in neurodegenerative tauopathies such as Alzheimer disease, acute Pseudomonas infections cause a pathophysiological sequela in endothelium previously recognized only in chronic neurodegenerative diseases. PMID:22637478

  4. Inhibition of hormonally regulated adenylate cyclase by the beta gamma subunit of transducin.

    PubMed Central

    Bockaert, J; Deterre, P; Pfister, C; Guillon, G; Chabre, M

    1985-01-01

    Transducin (T), the GTP-binding protein of the retina activates the cGMP phosphodiesterase system, and presents analogies with the proteins GS and Gi which respectively mediate adenylate cyclase activation and inhibition by hormone receptors. These proteins are all comprised of an alpha subunit carrying the GTP-binding site and a beta gamma subunit made of two peptides. The beta peptide (35 kd) appears similar in the three proteins. We demonstrate here that purified T beta gamma inhibits adenylate cyclase from human platelet membranes. This inhibition was observed when adenylate cyclase was stimulated by GTP, prostaglandin E1 (PGE1), NaF and forskolin, but not when stimulated by GTP(gamma)S. In the presence of GTP and forskolin, the T beta gamma-induced maximal inhibition was not additive with the alpha 2-receptor-induced adenylate cyclase inhibition mediated by Gi. Both inhibitions were suppressed at high Mg2+ concentrations, which as also known to dissociate T beta gamma from T alpha-GDP. This suggests that these adenylate cyclase inhibitions are due to the formation of inactive complexes of GS alpha-GDP with T beta gamma or Gi beta gamma. T beta gamma-induced inhibition did not require detergent and could be suppressed by simple washing. T beta gamma effects are dependent on its concentration rather than on its total amount. This suggests that T beta gamma can operate in solution with no integration into the membrane. Similar inhibitory effects of T beta gamma are observed on adenylate cyclase from anterior pituitary and lymphoma S49 cell lines. PMID:3861319

  5. Changed sensitivity of adenylate cyclase signaling system to biogenic amines and peptide hormones in tissues of starving rats.

    PubMed

    Shpakov, A O; Kuznetsova, L A; Plesneva, S A; Pertseva, M N

    2007-07-01

    In the myocardium and skeletal muscles of rats deprived of food for 2 days, basal activity of adenylate cyclase decreased, while the sensitivity of adenylate cyclase signaling system to the stimulating effects of non-hormonal agents (guanine nucleotides and NaF) and beta-agonist isoproterinol modulating adenylate cyclase through stimulating G proteins increased. In starving organism, the regulatory effects of hormones realizing their effects through inhibitory G proteins (somatostatin in the myocardium and bromocryptin in the brain) weakened. Their inhibitory effects on forskolin-stimulated adenylate cyclase activity and stimulating effects on binding of guanosine triphosphate decreased. In the brain of starving rats, the differences in the sensitivity of the adenylate cyclase signaling system to hormones and nonhormonal agents were less pronounced than in the muscle tissues, which attested to tissue-specific changes in the functional state of this system under conditions of 2-day starvation.

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

    SciTech Connect

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

    2005-01-01

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

  7. [Reactivity of the adenylyl cyclase system in rat tissues to biogenic amines and peptide hormones under starvation condition].

    PubMed

    Shpakov, A O; Kuznetsova, L A; Plesneva, S A; Pertseva, M N

    2007-04-01

    Under starvation condition, sensitivity of the adenylyl cyclase system to regulatory action of biogenic amines and peptide hormones in rat tissues are changed. In the myocardium and skeletal muscles, after 2 and 4 days of starvation, the regulatory effects of isoproterenol and relaxin acting via G,-proteins on the adenylyl cyclase activity and the G-protein GTP-binding are significantly increased compared with control. At the same time, regulatory effects ofsomatostatin which are realized via Gi-proteins, on adenylyl cyclase system in the myocardium are decreased. Under prolonged starvation consisting of two consecutive 4-days periods, the effects of hormones acting via Gs-proteins on the adenylyl cyclase activity in muscle tissues are decreased to control value levels. The effects of hormones acting via Gi-proteins are largely reduced. In the brain, intensification of adenylyl cyclase stimulating hormonal effects was late and only observed after a 4-day starvation. Unlike muscle tissues, the increase of adenylyl cyclase stimulating effects in the brain is preserved after two-period starvation. The weakening of adenylyl cyclase inhibiting hormonal signals both in the brain and muscles is observed after a 2-day starvation and then the weakening is intensified. Possible role of glucose level and basal adenylyl cyclase activity in determination of the sensitivity of the adenylyl cyclase system to hormones under study is discussed. It is suggested that one of the key causes of physiological changes in animal organism under starvation involves alteration of hormonal signalling systems sensitivity, in particular that of the adenylyl cyclase system, to hormone regulatory action.

  8. Cytidylyl- and Uridylyl Cyclase Activity of Bacillus anthracis Edema Factor and Bordetella pertussis CyaA

    PubMed Central

    Göttle, Martin; Dove, Stefan; Kees, Frieder; Schlossmann, Jens; Geduhn, Jens; König, Burkhard; Shen, Yuequan; Tang, Wei-Jen; Kaever, Volkhard; Seifert, Roland

    2010-01-01

    Cyclic adenosine 3′:5′-monophosphate (cAMP) and cyclic guanosine 3′:5′-monophosphate (cGMP) are second messengers for a numerous mammalian cell functions. The natural occurrence and synthesis of a third cyclic nucleotide (cNMP), cyclic cytidine 3′:5′-monophosphate (cCMP) is discussed controversially, and almost nothing is known about cyclic uridine 3′:5′-monophosphate (cUMP). Bacillus anthracis and Bordetella pertussis secrete the adenylyl cyclase (AC) toxins edema factor (EF) and CyaA, respectively, weakening immune responses and facilitating bacterial proliferation. A cell-permeable cCMP analog inhibits human neutrophil superoxide production. Here, we report that EF and CyaA also possess cytidylyl cyclase (CC) and uridylyl cyclase (UC) activity. CC- and UC activity was determined by a radiometric assay, using [α-32P]CTP and [α-32P]UTP as substrates, respectively, and by an HPLC method. The identity of cNMPs was confirmed by mass spectrometry. Based on available crystal structures, we developed a model illustrating conversion of CTP to cCMP by bacterial toxins. In conclusion, we have shown both EF and CyaA have a rather broad substrate-specificity and exhibit cytidylyl- and uridylyl cyclase activity. Both cCMP and cUMP may contribute to toxin actions. PMID:20521845

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

    NASA Astrophysics Data System (ADS)

    Kovaleva, V. D.; Uzdensky, A. B.

    2016-04-01

    Photodynamic therapy (PDT) is a potential tool for selective destruction of malignant brain tumors. However, not only malignant but also healthy neurons and glial cells may be damaged during PDT. Nitric oxide is an important modulator of cell viability and intercellular neuroglial communications. NO have been already shown to participate in PDT-induced injury of neurons and glial cells. As soluble guanylyl cyclase is the only known receptor for NO, we have studied the possible role of soluble guanylyl cyclase in the regulation of survival and death of neurons and surrounding glial cells under photo-oxidative stress induced by photodynamic treatment (PDT). The crayfish stretch receptor consisting of a single identified sensory neuron enveloped by glial cells is a simple but informative model object. It was photosensitized with alumophthalocyanine photosens (10 nM) and irradiated with a laser diode (670 nm, 0.4 W/cm2). Using inhibitory analysis we have shown that during PDT soluble guanylyl cyclase, probably, has proapoptotic and antinecrotic effect on the glial cells of the isolated crayfish stretch receptor. Proapoptotic effect of soluble guanylyl cyclase could be mediated by protein kinase G (PKG). Thus, the involvement of NO/sGC/cGMP/PKG signaling pathway in PDT-induced apoptosis of glial cells was indirectly demonstrated.

  10. Defective responsiveness of adenylate cyclase to forskolin in the Drosophila memory mutant rutabaga.

    PubMed

    Dudai, Y; Sher, B; Segal, D; Yovell, Y

    1985-12-01

    The Drosophila memory mutant rutabaga (rut) has been previously shown to have a defective subpopulation (or functional state) of the enzyme adenylate cyclase. We report here that the reduced adenylate cyclase activity is also associated with a defective responsiveness of the enzyme to forskolin. Forskolin activation isotherms of the enzyme in normal membranes reveal low- and high-affinity forskolin-interacting components; the residual enzyme in the mutant shows a smaller proportion of the high-affinity response. In addition, in mutant membrane preparations, forskolin fails to shift the Km of the enzyme for free Mg2+ and for MgATP, in contrast to the situation in the normal tissue. The defect in the responsiveness to forskolin in rut is even more pronounced in a Lubrol-solubilized enzyme preparation, and is due to intrinsic properties of the cyclase system rather than to the absence (or presence) of a soluble, or detergent solubilized, factor in rut. The reduced forskolin responsiveness maps to the X chromosomal segment 12F5-6 to 13A1-5, within the region previously reported to span the locus that controls both the abortive memory and the lack of Ca2+-stimulation of adenylate cyclase in rut17. The possible relevance of the findings to postulated molecular mechanisms of short-term memory formation is discussed. PMID:3935769

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

    SciTech Connect

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

    1987-12-01

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

  12. Mode of coupling between the beta-adrenergic receptor and adenylate cyclase in turkey erythrocytes.

    PubMed

    Tolkovsky, A M; Levitzki, A

    1978-09-01

    The mode of coupling of the beta-adrenergic receptor to the enzyme adenylate cyclase in turkey erythrocyte membranes was analyzed in detail. A number of experimental techniques have been used: (1) measurement of the kinetics of cyclase activation to its permanetly active state in the presence of guanylyl imidodiphosphate, as a function of hormone concentrations; (2) measurement of antagonist and agoinst binding to the beta-adrenergic receptor prior and subsequent to the enzyme activation by hormone and guanylyl imidodiphosphate. On the bases of these two approaches, all the models of receptor to enzyme coupling which involve an equilibrium between the enzyme and the receptor can be rejected. The binding and the kinetic data, however, can be fitted by two diametrically opposed models of receptor to enzyme coupling: (a) the precouped enzyme-receptor model where activation of the enzyme occurs, according to the following scheme: formula (see text) where H is the hormone, RE is the precoupled respetor-enzyme complex, k1 and k2 are the rate constants describing hormone binding, and k is the rate constant characterizing the formation of HRE' from the intermediate HRE. According to this model, the activated complex is composed of all of the interacting species. (b) The other model is the collision coupling mechanism: formula (see test) wheere KH is the horome-receptor dissociation constant, k1 is the bimolecular rate constant governing the formation of HRE, and k3 the rate constant governing the activation of the enzyme. In this case the intermediate never accumulates and constitutes only a small fraction of the total receptor and adenylate cyclase concentrations. In order to establish which of the two mechanisms governs the mode of adenylate cyclase activation by its receptor, a diagnostic experiment was performed: Progressive inactivation of the beta receptor by a specific affinity label was found to cause a decrease in the maximal binding capacity of the receptor and a

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

    PubMed

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

    1987-12-01

    Thermogenesis in brown adipose tissue (BAT) serves as a regulator of body temperature and weight maintenance. Thermogenesis can be stimulated by catecholamine activation of adenylate cyclase through the beta-adrenergic receptor. To investigate the effects of sucrose feeding, food deprivation, and cold exposure on the beta-adrenergic pathway, adenylate cyclase activity and beta-adrenergic receptors were assessed in rat BAT after 2 wk of sucrose feeding, 2 days of food deprivation, or 2 days of cold exposure. beta-Adrenergic receptors were identified in BAT using [125I]iodocyanopindolol. Binding sites had the characteristics of mixed beta 1- and beta 2-type adrenergic receptors at a ratio of 60/40. After sucrose feeding or cold exposure, there was the expected increase in BAT mitochondrial mass as measured by total cytochrome-c oxidase activity but a decrease in beta-adrenergic receptor density due to a loss of the beta 1-adrenergic subtype. This BAT beta-adrenergic receptor downregulation was tissue specific, since myocardial beta-adrenergic receptors were unchanged with either sucrose feeding or cold exposure. In contrast, food deprivation did not alter BAT beta-adrenergic receptor density. Forskolin-stimulated adenylate cyclase activity increased in BAT after sucrose feeding or cold exposure but not after food deprivation. The ratio of isoproterenol-stimulated to forskolin-stimulated adenylate cyclase activity decreased in the sucrose-fed and cold-exposed rats but not in the food-deprived rats. These data suggest that in BAT, sucrose feeding or cold exposure result in downregulation of beta-adrenergic receptors and that isoproterenol-stimulated adenylate cyclase activity was limited by receptor availability. PMID:2827501

  14. Regional distribution of somatostatin receptor binding and modulation of adenylyl cyclase activity in Alzheimer's disease brain.

    PubMed

    Bergström, L; Garlind, A; Nilsson, L; Alafuzoff, I; Fowler, C J; Winblad, B; Cowburn, R F

    1991-10-01

    We have previously reported a reduction in the inhibitory effect of somatostatin on adenylyl cyclase activity in the superior temporal cortex of a group of Alzheimer's disease cases, compared to a group of matched controls. In the present study, the levels of high affinity 125I-Tyr11-somatostatin-14 binding, its modulation by guanine nucleotides and the effects of somatostatin on adenylyl cyclase activity have been measured in preparations of frontal cortex, hippocampus, caudate nucleus and cerebellum from the same patient and control groups. A significant reduction in 125I-Tyr11-somatostatin-14 binding was observed in the frontal cortex, but not other regions, of the Alzheimer's disease group, compared with control values. The profiles of inhibition of specific 125I-Tyr11-somatostatin-14 binding by Gpp(NH)p were similar in all regions in both groups. No significant differences in basal, forskolin-stimulated, or somatostatin and neuropeptide Y inhibitions of adenylyl cyclase activity were found between the two groups. The pattern of change of somatostatin binding in the Alzheimer's disease cases observed in the present study differs from the reported pattern of loss of somatostatin neurons and may be secondary to the degeneration of somatostatin receptor-bearing cholinergic afferents arising from the nucleus basalis. The results of this study indicate that impaired somatostatin modulation of adenylyl cyclase is not a global phenomenon in Alzheimer's disease brain and also that there are no major disruptions of somatostatin receptor-G-protein coupling or of adenylyl cyclase catalytic activity in this disorder. PMID:1684616

  15. Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942.

    PubMed Central

    Cunningham, F X; Sun, Z; Chamovitz, D; Hirschberg, J; Gantt, E

    1994-01-01

    A gene encoding the enzyme lycopene cyclase in the cyanobacterium Synechococcus sp strain PCC7942 was mapped by genetic complementation, cloned, and sequenced. This gene, which we have named crtL, was expressed in strains of Escherichia coli that were genetically engineered to accumulate the carotenoid precursors lycopene, neurosporene, and zeta-carotene. The crtL gene product converts the acyclic hydrocarbon lycopene into the bicyclic beta-carotene, an essential component of the photosynthetic apparatus in oxygen-evolving organisms and a source of vitamin A in human and animal nutrition. The enzyme also converts neurosporene to the monocyclic beta-zeacarotene but does not cyclize zeta-carotene, indicating that desaturation of the 7-8 or 7'-8' carbon-carbon bond is required for cyclization. The bleaching herbicide 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA) effectively inhibits both cyclization reactions. A mutation that confers resistance to MPTA in Synechococcus sp PCC7942 was identified as a point mutation in the promoter region of crtL. The deduced amino acid sequence of lycopene cyclase specifies a polypeptide of 411 amino acids with a molecular weight of 46,125 and a pI of 6.0. An amino acid sequence motif indicative of FAD utilization is located at the N terminus of the polypeptide. DNA gel blot hybridization analysis indicated a single copy of crtL in Synechococcus sp PCC7942. Other than the FAD binding motif, the predicted amino acid sequence of the cyanobacterial lycopene cyclase bears little resemblance to the two known lycopene cyclase enzymes from nonphotosynthetic bacteria. Preliminary results from DNA gel blot hybridization experiments suggest that, like two earlier genes in the pathway, the Synechococcus gene encoding lycopene cyclase is homologous to plant and algal genes encoding this enzyme. PMID:7919981

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

    SciTech Connect

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

    1986-08-12

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

  17. Properties of Adenyl Cyclase from Human Jejunal Mucosa during Naturally Acquired Cholera and Convalescence

    PubMed Central

    Chen, Lincoln C.; Rohde, Jon E.; Sharp, Geoffrey W. G.

    1972-01-01

    The enterotoxin of Vibrio cholerae causes copious fluid production throughout the lenght of the small intestine. As this is thought to be mediated by stimulation of adenyl cyclase, a study has been made of the activity and properties of this enzyme in jejunal biopsy tissue taken from patients during the diarrheal phase of cholera and after recovery. Adenyl cyclase activity during cholera was increased more than twofold relative to the enzyme in convalescence. Under both conditions stimulation by prostaglandin E1 (PGE1) and by fluoride was observed. The responsiveness to PGE1 was not altered in cholera; the total activity of the fluoride-stimulated enzyme was similar, a finding that suggests cholera toxin stimulates pre-existing enzyme in the intestinal cell. The enzymes during cholera and convalescence were similar in all other properties examined. Optimal Mg++ concentration was 10 mM; Mn++ at 5 mM stimulated the enzyme but could not replace Mg++ except in the presence of 10 mM fluoride. Calcium was markedly inhibitory at concentrations greater than 10-4 M. The pH optimum was 7.5 and the Michaelis constant (Km) for ATP concentration approximated 10-4 M. Thus the interaction of cholera toxin with human intestinal adenyl cyclase does not alter the basic properties of the enzyme. When biopsy specimens were maintained intact in oxygenated Ringer's solution at 0°C, no loss of activity was observed at 1½ and 3 hr. In contrast, when the cells were homogenized, rapid loss of activity, with a half-life of 90 min was seen even at 0°C. Consequently for comparative assays of human jejunal adenyl cyclase, strict control of the experimental conditions is required. It was under such conditions that a twofold increase in basal adenyl cyclase activity during cholera was observed. Images PMID:4335441

  18. Abscisic acid signaling through cyclic ADP-ribose in hydroid regeneration.

    PubMed

    Puce, Stefania; Basile, Giovanna; Bavestrello, Giorgio; Bruzzone, Santina; Cerrano, Carlo; Giovine, Marco; Arillo, Attilio; Zocchi, Elena

    2004-09-17

    Cyclic ADP-ribose (cADPR) is an intracellular calcium (Ca(2+)(i)) mobilizer involved in fundamental cell functions from protists to higher plants and mammals. Biochemical similarities between the drought-signaling cascade in plants and the temperature-sensing pathway in marine sponges suggest an ancient evolutionary origin of a signaling cascade involving the phytohormone abscisic acid (ABA), cADPR, and Ca(2+)(i). In Eudendrium racemosum (Hydrozoa, Cnidaria), exogenously added ABA stimulated ADP-ribosyl cyclase activity via a protein kinase A (PKA)-mediated phosphorylation and increased regeneration in the dark to levels observed under light conditions. Light stimulated endogenous ABA synthesis, which was conversely inhibited by the inhibitor of plant ABA synthesis Fluridone. The signal cascade of light-induced regeneration uncovered in E. racemosum: light --> increasing ABA --> PKA --> cyclase activation --> increasing [cADPR](i) --> increasing [Ca(2+)](i) --> regeneration is the first report of a complete signaling pathway in Eumetazoa involving a phytohormone.

  19. Stimulation of intestinal mucosal adenyl cyclase by cholera enterotoxin and prostaglandins

    PubMed Central

    Kimberg, Daniel V.; Field, Michael; Johnson, Judith; Henderson, Antonia; Gershon, Elaine

    1971-01-01

    The effects of several prostaglandins (PG) and a highly purified preparation of cholera enterotoxin (CT) on intestinal mucosal adenyl cyclase activity and the effect of CT on intestinal mucosal cyclic 3′,5′-adenosine monophosphate concentration were determined in guinea pig and rabbit small intestine and were correlated with the effects of the same agents on ion transport. Adenyl cyclase activity, measured in a crude membrane fraction of the mucosa, was found at all levels of the small intestine with the highest activity per milligram protein in the duodenum. The prostaglandins, when added directly to the assay, increased adenyl cyclase activity; the greatest effect (2-fold increase) was obtained with PGE1 (maximal effect at 0.03 mM) and PGE2. The prostaglandins also increased short-circuit current (SCC) in isolated guinea pig ileal mucosa, with PGE1 and PGE2 again giving the greatest effects. The prior addition of theophylline (10 mM) reduced the subsequent SCC response to PGE1 and vice versa. It was concluded, therefore, that the SCC response to PGE1, like the response to theophylline, represented active Cl secretion. CT increased adenyl cyclase activity in guinea pig and rabbit ileal mucosa when preincubated with the mucosa from 1 to 2.5 hr in vitro or for 2.5 hr in vivo but not when added directly to the assay. The increments in activity caused by PGE1 and NaF were the same in CT-treated and control mucosa. Cyclic 3′,5′-AMP concentration in rabbit ileal mucosa was increased 3.5-fold after a 2 hr preincubation with CT in vitro. Phosphodiesterase activity in the crude membrane fraction of the mucosa was unaffected by either CT or PGE1. A variety of other agents including insulin, glucagon, parathormone, thyroid-stimulating hormone, L-thyroxine, thyrocalcitonin, vasopressin, and epinephrine all failed to change adenyl cyclase activity. It is concluded that CT and certain prostaglandins produce small intestinal fluid secretion by increasing mucosal adenyl

  20. Opposing effects of ethanol on pig ovarian adenylyl cyclase desensitized by human choriogonadotropin or isoproterenol.

    PubMed

    Ekstrom, R C; Hunzicker-Dunn, M

    1990-11-01

    Pig ovarian follicular membranes contain a gonadotropin-responsive adenylyl cyclase, which becomes partially desensitized (approximately 40%) upon a 40-min incubation with a saturating concentration of human (h) CG. This in vitro desensitization is time and hormone dependent and also requires the presence of micromolar concentrations of GTP. In this report we show that 10% ethanol present during the desensitization phase of the incubation increases the extent of hCG-induced desensitization of adenylyl cyclase by 2-fold. Ethanol shortened the time necessary to reach maximal hCG-induced desensitization from 20 to 10 min, but had no effect on the dose dependency for GTP. In addition, ethanol had no effect on the affinity of the LH/hCG receptor for 125I-hCG but did cause an increase in the ED50 of hCG for inducing desensitization from 0.25 to 0.75 nM. Interestingly, ethanol decreased the apparent number of LH/hCG-receptor sites by 55%, yet the control hCG-sensitive adenylyl cyclase activity was not reduced. The "hyperdesensitized" state achieved in the presence of ethanol could not be reversed by washing the membranes and incubating them in ethanol-free medium. NaF-sensitive adenylyl cyclase was also not impaired in hCG-desensitized membranes from control or ethanol-treated samples. Thus, hCG-induced desensitization was not due to a defect in the functioning of the stimulatory guanine nucleotide-binding regulatory protein (G8) or catalytic subunits, but rather was caused by an impairment of the coupling of the lutropin (LH)/hCG receptor with G8, which was exacerbated further by ethanol. In spite of the effect of ethanol on hCG-induced desensitization, this agent had an inhibitory effect on isoproterenol-induced desensitization of isoproterenol-responsive luteal adenylyl cyclase. These results indicate that membrane fluidity is important in modulating the structure and functional interaction of the LH/hCG receptor with G8 because ethanol is a well known lipid

  1. Identification of the chlE gene encoding oxygen-independent Mg-protoporphyrin IX monomethyl ester cyclase in cyanobacteria.

    PubMed

    Yamanashi, Kaori; Minamizaki, Kei; Fujita, Yuichi

    2015-08-01

    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."

  2. Effects of adenylate cyclase toxin from Bordetella pertussis on human neutrophil interactions with Coccidioides immitis and Staphylococcus aureus.

    PubMed Central

    Galgiani, J N; Hewlett, E L; Friedman, R L

    1988-01-01

    Bordetella pertussis extract that contained adenylate cyclase toxin produced large increases in human neutrophil cyclic AMP levels and inhibited their oxidative burst, as reflected by luminol-enhanced chemiluminescence and superoxide release. The adenylate cyclase toxin-containing extract blocked neutrophil-mediated inhibition of N-acetylglucosamine incorporation by arthroconidia of Coccidioides immitis in a dose-dependent fashion but had no effect on neutrophil phagocytosis of Candida glabrata and only a slight inhibitory effect on arthroconidial attachment. Neither purified pertussis toxin nor extracts from Bordetella mutants lacking the adenylate cyclase toxin affected neutrophil-mediated inhibition of arthroconidial N-acetylglucosamine incorporation. These studies indicate that adenylate cyclase toxin, alone or in concert with other B. pertussis-elaborated toxins, blocks neutrophil inhibition of arthroconidia, primarily by affecting neutrophil responses other than attachment or phagocytosis. PMID:2894360

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

    SciTech Connect

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

    1987-05-01

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

  4. Antagonism of histamine-activated adenylate cyclase in brain by D-lysergic acid diethylamide.

    PubMed

    Green, J P; Johnson, C L; Weinstein, H; Maayani, S

    1977-12-01

    D-Lysergic acid diethylamide and D-2-bromolysergic acid diethylamide are competitive antagonists of the histamine activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); E.C. 4.6.1.1] in broken cell preparations of the hippocampus and cortex of guinea pig brain. The adenylate cyclase is linked to the histamine H2-receptor. Both D-lysergic acid diethylamide and D-2-bromolysergic acid diethylamide show topological congruency with potent H2-antagonists. D-2-Bromolysergic acid diethylamide is 10 times more potent as an H2-antagonist than cimetidine, which has been the most potent H2-antagonist reported, and D-lysergic acid diethylamide is about equipotent to cimetidine. Blockade of H2-receptors could contribute to the behavioral effects of D-2-bromolysergic acid diethylamide and D-lysergic acid diethylamide.

  5. Overexpression of the Type 1 Adenylyl Cyclase in the Forebrain Leads to Deficits of Behavioral Inhibition

    PubMed Central

    Cao, Hong; Saraf, Amit; Zweifel, Larry S.

    2015-01-01

    The type 1 adenylyl cyclase (AC1) is an activity-dependent, calcium-stimulated adenylyl cyclase expressed in the nervous system that is implicated in memory formation. We examined the locomotor activity, and impulsive and social behaviors of AC1+ mice, a transgenic mouse strain overexpressing AC1 in the forebrain. Here we report that AC1+ mice exhibit hyperactive behaviors and demonstrate increased impulsivity and reduced sociability. In contrast, AC1 and AC8 double knock-out mice are hypoactive, and exhibit increased sociability and reduced impulsivity. Interestingly, the hyperactivity of AC1+ mice can be corrected by valproate, a mood-stabilizing drug. These data indicate that increased expression of AC1 in the forebrain leads to deficits in behavioral inhibition. PMID:25568126

  6. Hypothesis: glutaminyl cyclase inhibitors decrease risks of Alzheimer's disease and related dementias.

    PubMed

    Hennekens, Charles H; Bensadon, Benjamin A; Zivin, Robert; Gaziano, J Michael

    2015-01-01

    Alzheimer's disease and related dementias (ADRD) comprise several progressive and incurable neurodegenerative disorders that some have classified as amyloidosis. With increased aging of the world's population, the prevalence of the sporadic form of ADRD, which comprises over 99% of cases, continues to rise at an alarming rate. The enormous societal burdens of ADRD already rival those of the many other major chronic diseases causing premature morbidity and mortality in the USA and worldwide such as cardiovascular disease and cancer. At present, there is an insufficient totality of evidence concerning the efficacy and safety of any pharmacologic agents to delay slow progression or reduce complications of ADRD. In this context, glutaminyl cyclase (QC) inhibitors have shown some early possible evidence of efficacy with a reassuring safety profile. To reliably test the glutaminyl cyclase (QC) and any other promising hypotheses will require cogent data from large-scale randomized trials of sufficient size and duration. PMID:26450764

  7. Mechanistic Characterisation of Two Sesquiterpene Cyclases from the Plant Pathogenic Fungus Fusarium fujikuroi.

    PubMed

    Burkhardt, Immo; Siemon, Thomas; Henrot, Matthias; Studt, Lena; Rösler, Sarah; Tudzynski, Bettina; Christmann, Mathias; Dickschat, Jeroen S

    2016-07-18

    Two sesquiterpene cyclases from Fusarium fujikuroi were expressed in Escherichia coli and purified. The first enzyme was inactive because of a critical mutation, but activity was restored by sequence correction through site-directed mutagenesis. The mutated enzyme and two naturally functional homologues from other fusaria converted farnesyl diphosphate into guaia-6,10(14)-diene. The second enzyme produced eremophilene. The absolute configuration of guaia-6,10(14)-diene was elucidated by enantioselective synthesis, while that of eremophilene was evident from the sign of its optical rotation and is opposite to that in plants but the same as in Sorangium cellulosum. The mechanisms of both terpene cyclases were studied with various (13) C- and (2) H-labelled FPP isotopomers.

  8. Receptor-type guanylate cyclase is required for carbon dioxide sensation by Caenorhabditis elegans.

    PubMed

    Hallem, Elissa A; Spencer, W Clay; McWhirter, Rebecca D; Zeller, Georg; Henz, Stefan R; Rätsch, Gunnar; Miller, David M; Horvitz, H Robert; Sternberg, Paul W; Ringstad, Niels

    2011-01-01

    CO(2) is both a critical regulator of animal physiology and an important sensory cue for many animals for host detection, food location, and mate finding. The free-living soil nematode Caenorhabditis elegans shows CO(2) avoidance behavior, which requires a pair of ciliated sensory neurons, the BAG neurons. Using in vivo calcium imaging, we show that CO(2) specifically activates the BAG neurons and that the CO(2)-sensing function of BAG neurons requires TAX-2/TAX-4 cyclic nucleotide-gated ion channels and the receptor-type guanylate cyclase GCY-9. Our results delineate a molecular pathway for CO(2) sensing and suggest that activation of a receptor-type guanylate cyclase is an evolutionarily conserved mechanism by which animals detect environmental CO(2).

  9. Receptor-type guanylate cyclase is required for carbon dioxide sensation by Caenorhabditis elegans.

    PubMed

    Hallem, Elissa A; Spencer, W Clay; McWhirter, Rebecca D; Zeller, Georg; Henz, Stefan R; Rätsch, Gunnar; Miller, David M; Horvitz, H Robert; Sternberg, Paul W; Ringstad, Niels

    2011-01-01

    CO(2) is both a critical regulator of animal physiology and an important sensory cue for many animals for host detection, food location, and mate finding. The free-living soil nematode Caenorhabditis elegans shows CO(2) avoidance behavior, which requires a pair of ciliated sensory neurons, the BAG neurons. Using in vivo calcium imaging, we show that CO(2) specifically activates the BAG neurons and that the CO(2)-sensing function of BAG neurons requires TAX-2/TAX-4 cyclic nucleotide-gated ion channels and the receptor-type guanylate cyclase GCY-9. Our results delineate a molecular pathway for CO(2) sensing and suggest that activation of a receptor-type guanylate cyclase is an evolutionarily conserved mechanism by which animals detect environmental CO(2). PMID:21173231

  10. Mechanistic Characterisation of Two Sesquiterpene Cyclases from the Plant Pathogenic Fungus Fusarium fujikuroi.

    PubMed

    Burkhardt, Immo; Siemon, Thomas; Henrot, Matthias; Studt, Lena; Rösler, Sarah; Tudzynski, Bettina; Christmann, Mathias; Dickschat, Jeroen S

    2016-07-18

    Two sesquiterpene cyclases from Fusarium fujikuroi were expressed in Escherichia coli and purified. The first enzyme was inactive because of a critical mutation, but activity was restored by sequence correction through site-directed mutagenesis. The mutated enzyme and two naturally functional homologues from other fusaria converted farnesyl diphosphate into guaia-6,10(14)-diene. The second enzyme produced eremophilene. The absolute configuration of guaia-6,10(14)-diene was elucidated by enantioselective synthesis, while that of eremophilene was evident from the sign of its optical rotation and is opposite to that in plants but the same as in Sorangium cellulosum. The mechanisms of both terpene cyclases were studied with various (13) C- and (2) H-labelled FPP isotopomers. PMID:27294564

  11. Overexpression of the type 1 adenylyl cyclase in the forebrain leads to deficits of behavioral inhibition.

    PubMed

    Chen, Xuanmao; Cao, Hong; Saraf, Amit; Zweifel, Larry S; Storm, Daniel R

    2015-01-01

    The type 1 adenylyl cyclase (AC1) is an activity-dependent, calcium-stimulated adenylyl cyclase expressed in the nervous system that is implicated in memory formation. We examined the locomotor activity, and impulsive and social behaviors of AC1+ mice, a transgenic mouse strain overexpressing AC1 in the forebrain. Here we report that AC1+ mice exhibit hyperactive behaviors and demonstrate increased impulsivity and reduced sociability. In contrast, AC1 and AC8 double knock-out mice are hypoactive, and exhibit increased sociability and reduced impulsivity. Interestingly, the hyperactivity of AC1+ mice can be corrected by valproate, a mood-stabilizing drug. These data indicate that increased expression of AC1 in the forebrain leads to deficits in behavioral inhibition.

  12. Molecular cloning of an orphan G-protein-coupled receptor that constitutively activates adenylate cyclase.

    PubMed Central

    Eggerickx, D; Denef, J F; Labbe, O; Hayashi, Y; Refetoff, S; Vassart, G; Parmentier, M; Libert, F

    1995-01-01

    A human gene encoding an orphan G-protein-coupled receptor named ACCA (adenylate cyclase constitutive activator) was isolated from a genomic library using as a probe a DNA fragment obtained by low-stringency PCR. Human ACCA (hACCA) is a protein of 330 amino acids that exhibits all the structural hallmarks of the main family of G-protein-coupled receptors. Expression of hACCA resulted in a dramatic stimulation of adenylate cyclase, similar in amplitude to that obtained with other Gs-coupled receptors fully activated by their respective ligands. This stimulation was obtained in a large variety of stable cell lines derived from various organs, and originating from different mammalian species. hACCA was found to be the human homologue of a recently reported mouse orphan receptor (GPCR21). The mouse ACCA (mACCA) was therefore recloned by PCR, and expression of mACCA in Cos-7 cells demonstrated that the mouse receptor behaved similarly as a constitutive activator of adenylate cyclase. It is not known presently whether the stimulation of adenylate cyclase is the result of a true constitutive activity of the receptor or, alternatively, is the consequence of a permanent stimulation by a ubiquitous ligand. The tissue distribution of mACCA was determined by RNase protection assay. Abundant transcripts were found in the brain, whereas lower amounts were detected in testis, ovary and eye. Various hypotheses concerning the constitutive activity of ACCA and their potential biological significance are discussed. Images Figure 4 Figure 5 PMID:7639700

  13. Interaction of GCAP1 with retinal guanylyl cyclase and calcium: sensitivity to fatty acylation

    PubMed Central

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

    2012-01-01

    Guanylyl cyclase activating proteins (GCAPs) 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 G2A mutant. At the same time, myristoylation had little effect on the high-affinity Ca2+-binding in the EF-hand proximal to the myristoyl residue in 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. PMID:22371697

  14. Vasorelaxant effect of isoliquiritigenin, a novel soluble guanylate cyclase activator, in rat aorta.

    PubMed Central

    Yu, S M; Kuo, S C

    1995-01-01

    1. The vasorelaxant activity of isoliquiritigenin, isolated from Dalbergia odorifera T, was investigated in the phenylephrine-precontracted rat aorta by measuring tension, guanylate and adenylate cyclase activities, guanosine 3':5'-cyclic monophosphate (cyclic GMP) and adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels. 2. Isoliquiritigenin concentration-dependently relaxed rat aorta contracted with phenylephrine, KCl, U-46619, endothelin and 5-hydroxytryptamine, with EC50s of 7.4 +/- 1.6, 10.5 +/- 2.3, 14.3 +/- 3.3, 11.8 +/- 2.0 and 13.6 +/- 3.7 microM, respectively. 3. Isoliquiritigenin caused endothelium-independent relaxation of phenylephrine-precontracted rat aortic rings. Neither NG-monomethyl-L-arginine (L-NMMA) (an inhibitor of the L-arginine-NO pathway) nor oxyhaemoglobin (which binds NO) modified the relaxant effect of isoliquiritigenin. The relaxant action of isoliquiritigenin also persisted in intact aorta in the presence of indomethacin or glibenclamide. However, methylene blue, an inhibitor of soluble guanylate cyclase, abolished relaxation induced by isoliquiritigenin. 4. Incubation of rat aorta with isoliquiritigenin not only increased aortic cyclic GMP content but also caused small increases in aortic cyclic AMP content, and greatly potentiated the increases in cyclic AMP observed in the presence of forskolin. The maximum increase in cyclic GMP by isoliquiritigenin was reached earlier than the increase in cyclic AMP. This result suggests that the increases in cyclic GMP caused by isoliquiritigenin might stimulate the accumulation of cyclic AMP. 5. Concentration-dependent increases in soluble guanylate cyclase activity were observed in isoliquiritigenin (1-100 microM)- or sodium nitroprusside (SNP)-treated rat aortic smooth muscle cells, while adenylate cyclase activity was unchanged in isoliquiritigenin (100 microM)-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7599926

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

    PubMed Central

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

    1982-01-01

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

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

    SciTech Connect

    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.

  17. Elevation of lutein content in tomato: a biochemical tug-of-war between lycopene cyclases.

    PubMed

    Giorio, Giovanni; Yildirim, Arzu; Stigliani, Adriana Lucia; D'Ambrosio, Caterina

    2013-11-01

    Lutein is becoming increasingly important in preventive medicine due to its possible role in maintaining good vision and in preventing age-related maculopathy. Average daily lutein intake in developed countries is often below suggested daily consumption levels, and lutein supplementation could be beneficial. Lutein is also valuable in the food and feed industries and is emerging in nutraceutical and pharmaceutical markets. Currently, lutein is obtained at high cost from marigold petals, and synthesis alternatives are thus desirable. Tomato constitutes a promising starting system for production as it naturally accumulates high levels of lycopene. To develop tomato for lutein synthesis, the tomato Red Setter cultivar was transformed with the tomato lycopene ε-cyclase-encoding gene under the control of a constitutive promoter, and the HighDelta (HD) line, characterised by elevated lutein and δ-carotene content in ripe fruits, was selected. HD was crossed to the transgenic HC line and to RS(B) with the aim of converting all residual fruit δ-carotene to lutein. Fruits of both crosses were enriched in lutein and presented unusual carotenoid profiles. The unique genetic background of the crosses used in this study permitted an unprecedented analysis of the role and regulation of the lycopene cyclase enzymes in tomato. A new defined biochemical index, the relative cyclase activity ratio, was used to discern post-transcriptional regulation of cyclases, and will help in the study of carotenoid biosynthesis in photosynthetic plant species and particularly in those, like tomato, that have been domesticated for the production of food, feed or useful by-products.

  18. Control of the Diadenylate Cyclase CdaS in Bacillus subtilis

    PubMed Central

    Mehne, Felix M. P.; Schröder-Tittmann, Kathrin; Eijlander, Robyn T.; Herzberg, Christina; Hewitt, Lorraine; Kaever, Volkhard; Lewis, Richard J.; Kuipers, Oscar P.; Tittmann, Kai; Stülke, Jörg

    2014-01-01

    The Gram-positive bacterium Bacillus subtilis encodes three diadenylate cyclases that synthesize the essential signaling nucleotide cyclic di-AMP. The activities of the vegetative enzymes DisA and CdaA are controlled by protein-protein interactions with their conserved partner proteins. Here, we have analyzed the regulation of the unique sporulation-specific diadenylate cyclase CdaS. Very low expression of CdaS as the single diadenylate cyclase resulted in the appearance of spontaneous suppressor mutations. Several of these mutations in the cdaS gene affected the N-terminal domain of CdaS. The corresponding CdaS mutant proteins exhibited a significantly increased enzymatic activity. The N-terminal domain of CdaS consists of two α-helices and is attached to the C-terminal catalytically active diadenylate cyclase (DAC) domain. Deletion of the first or both helices resulted also in strongly increased activity indicating that the N-terminal domain serves to limit the enzyme activity of the DAC domain. The structure of YojJ, a protein highly similar to CdaS, indicates that the protein forms hexamers that are incompatible with enzymatic activity of the DAC domains. In contrast, the mutations and the deletions of the N-terminal domain result in conformational changes that lead to highly increased enzymatic activity. Although the full-length CdaS protein was found to form hexamers, a truncated version with a deletion of the first N-terminal helix formed dimers with high enzyme activity. To assess the role of CdaS in sporulation, we assayed the germination of wild type and cdaS mutant spores. The results indicate that cyclic di-AMP formed by CdaS is required for efficient germination. PMID:24939848

  19. The calcium-sensor guanylate cyclase activating protein type 2 specific site in rod outer segment membrane guanylate cyclase type 1.

    PubMed

    Duda, Teresa; Fik-Rymarkiewicz, Ewa; Venkataraman, Venkateswar; Krishnan, Ramalingam; Koch, Karl-Wilhelm; Sharma, Rameshwar K

    2005-05-17

    The rod outer segment membrane guanylate cyclase type 1 (ROS-GC1), originally identified in the photoreceptor outer segments, is a member of the subfamily of Ca(2+)-modulated membrane guanylate cyclases. In phototransduction, its activity is tightly regulated by its two Ca(2+)-sensor protein parts, GCAP1 and GCAP2. This study maps the GCAP2-modulatory site in ROS-GC1 through the use of multiple techniques involving surface plasmon resonance binding studies with soluble ROS-GC1 constructs, coimmunoprecipitation, functional reconstitution experiments with deletion mutants, and peptide competition assays. The findings show that the sequence motif of the core GCAP2-modulatory site is Y965-N981 of ROS-GC1. The site is distinct from the GCAP1-modulatory site. It, however, partially overlaps with the S100B-regulatory site. This indicates that the Y965-N981 motif tightly controls the Ca(2+)-dependent specificity of ROS-GC1. Identification of the site demonstrates an intriguing topographical feature of ROS-GC1. This is that the GCAP2 module transmits the Ca(2+) signals to the catalytic domain from its C-terminal side and the GCAP1 module from the distant N-terminal side.

  20. Identification of photoactivated adenylyl cyclases in Naegleria australiensis and BLUF-containing protein in Naegleria fowleri.

    PubMed

    Yasukawa, Hiro; Sato, Aya; Kita, Ayaka; Kodaira, Ken-Ichi; Iseki, Mineo; Takahashi, Tetsuo; Shibusawa, Mami; Watanabe, Masakatsu; Yagita, Kenji

    2013-01-01

    Complete genome sequencing of Naegleria gruberi has revealed that the organism encodes polypeptides similar to photoactivated adenylyl cyclases (PACs). Screening in the N. australiensis genome showed that the organism also encodes polypeptides similar to PACs. Each of the Naegleria proteins consists of a "sensors of blue-light using FAD" domain (BLUF domain) and an adenylyl cyclase domain (AC domain). PAC activity of the Naegleria proteins was assayed by comparing sensitivities of Escherichia coli cells heterologously expressing the proteins to antibiotics in a dark condition and a blue light-irradiated condition. Antibiotics used in the assays were fosfomycin and fosmidomycin. E. coli cells expressing the Naegleria proteins showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light, indicating that the proteins functioned as PACs in the bacterial cells. Analysis of the N. fowleri genome revealed that the organism encodes a protein bearing an amino acid sequence similar to that of BLUF. A plasmid expressing a chimeric protein consisting of the BLUF-like sequence found in N. fowleri and the adenylyl cyclase domain of N. gruberi PAC was constructed to determine whether the BLUF-like sequence functioned as a sensor of blue light. E. coli cells expressing a chimeric protein showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light. These experimental results indicated that the sequence similar to the BLUF domain found in N. fowleri functioned as a sensor of blue light. PMID:24201148

  1. Evidence for an essential histidine residue in 4S-limonene synthase and other terpene cyclases.

    PubMed

    Rajaonarivony, J I; Gershenzon, J; Miyazaki, J; Croteau, R

    1992-11-15

    (4S)-Limonene synthase, isolated from glandular trichome secretory cell preparations of Mentha x piperita (peppermint) leaves, catalyzes the metal ion-dependent cyclization of geranyl pyrophosphate, via 3S-linalyl pyrophosphate, to (-)-(4S)-limonene as the principal product. Treatment of this terpene cyclase with the histidine-directed reagent diethyl pyrocarbonate at a concentration of 0.25 mM resulted in 50% loss of enzyme activity, and this activity could be completely restored by treatment of the preparation with 5 mM hydroxylamine. Inhibition with diethyl pyrocarbonate was distinguished from inhibition with thiol-directed reagents by protection studies with histidine and cysteine carried out at varying pH. Inactivation of the cyclase by dye-sensitized photooxidation in the presence of rose bengal gave further indication of the presence of a readily modified histidine residue. Protection of the enzyme against inhibition with diethyl pyrocarbonate was afforded by the substrate geranyl pyrophosphate in the presence of Mn2+, and by the sulfonium ion analog of the linalyl carbocation intermediate of the reaction in the presence of inorganic pyrophosphate plus Mn2+, suggesting that an essential histidine residue is located at or near the active site. Similar studies on the inhibition of other monoterpene and sesquiterpene cyclases with diethyl pyrocarbonate suggest that a histidine residue (or residues) may play an important role in catalysis by this class of enzymes. PMID:1444454

  2. Dcsbis (PA2771) from Pseudomonas aeruginosa is a highly active diguanylate cyclase with unique activity regulation

    PubMed Central

    Chen, Ying; Liu, Shiheng; Liu, Cuilan; Huang, Yan; Chi, Kaikai; Su, Tiantian; Zhu, Deyu; Peng, Jin; Xia, Zhijie; He, Jing; Xu, Sujuan; Hu, Wei; Gu, Lichuan

    2016-01-01

    C-di-GMP (3’,5’ -Cyclic diguanylic acid) is an important second messenger in bacteria that influences virulence, motility, biofilm formation, and cell division. The level of c-di-GMP in cells is controlled by diguanyl cyclases (DGCs) and phosphodiesterases (PDEs). Here, we report the biochemical functions and crystal structure of the potential diguanylase Dcsbis (PA2771, a diguanylate cyclase with a self-blocked I-site) from Pseudomonas aeruginosa PAO1. The full-length Dcsbis protein contains an N-terminal GAF domain and a C-terminal GGDEF domain. We showed that Dcsbis tightly coordinates cell motility without markedly affecting biofilm formation and is a diguanylate cyclase with a catalytic activity much higher than those of many other DGCs. Unexpectedly, we found that a peptide loop (protecting loop) extending from the GAF domain occupies the conserved inhibition site, thereby largely relieving the product-inhibition effect. A large hydrophobic pocket was observed in the GAF domain, thus suggesting that an unknown upstream signaling molecule may bind to the GAF domain, moving the protecting loop from the I-site and thereby turning off the enzymatic activity. PMID:27388857

  3. The first structure of a bacterial diterpene cyclase: CotB2.

    PubMed

    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. Evolutionary Divergence of Sedoheptulose 7-phosphate Cyclases Leads to Several Distinct Cyclic Products

    PubMed Central

    Asamizu, Shumpei; Xie, Pengfei; Brumsted, Corey J.; Flatt, Patricia M.; Mahmud, Taifo

    2012-01-01

    Sedoheptulose 7-phosphate cyclases are enzymes that utilize the pentose phosphate pathway intermediate, sedoheptulose 7-phosphate, to generate cyclic precursors of many bioactive natural products, such as the antidiabetic drug acarbose, the crop protectant validamycin, and the natural sunscreens mycosporine-like amino acids. These proteins are phylogenetically related to the dehydroquinate (DHQ) synthases from the shikimate pathway, and are part of the more recently recognized superfamily of sugar phosphate cyclases, which includes DHQ synthases, aminoDHQ synthases and 2-deoxy-scyllo-inosose synthases. Through genome mining and biochemical studies, we identified yet another subset of DHQS-like proteins in the actinomycete Actinosynnema mirum and the myxobacterium Stigmatella aurantiaca DW4/3–1. These enzymes catalyze the conversion of sedoheptulose 7-phosphate to 2-epi-valiolone, which is predicted to be an alternative precursor for aminocyclitol biosynthesis. Comparative bioinformatics and biochemical analyses of these proteins with 2-epi-5-epi-valiolone synthases (EEVS) and desmethyl-4-deoxygadusol synthases (DDGS) provided further insights into their genetic diversity, conserved amino acid sequences, and plausible catalytic mechanisms. The results further highlight the uniquely diverse DHQS-like sugar phosphate cyclases, which may provide new tools for chemoenzymatic, stereospecific synthesis of various cyclic molecules. PMID:22741921

  5. HAMP domain-mediated signal transduction probed with a mycobacterial adenylyl cyclase as a reporter.

    PubMed

    Mondéjar, Laura García; Lupas, Andrei; Schultz, Anita; Schultz, Joachim E

    2012-01-01

    HAMP domains, ∼55 amino acid motifs first identified in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases, operate as signal mediators in two-component signal transduction proteins. A bioinformatics study identified a coevolving signal-accepting network of 10 amino acids in membrane-delimited HAMP proteins. To probe the functionality of this network we used a HAMP containing mycobacterial adenylyl cyclase, Rv3645, as a reporter enzyme in which the membrane anchor was substituted by the Escherichia coli chemotaxis receptor for serine (Tsr receptor) and the HAMP domain alternately with that from the protein Af1503 of the archaeon Archaeoglobus fulgidus or the Tsr receptor. In a construct with the Tsr-HAMP, cyclase activity was inhibited by serine, whereas in a construct with the HAMP domain from A. fulgidus, enzyme activity was not responsive to serine. Amino acids of the signal-accepting network were mutually swapped between both HAMP domains, and serine signaling was examined. The data biochemically tentatively established the functionality of the signal-accepting network. Based on a two-state gearbox model of rotation in HAMP domain-mediated signal propagation, we characterized the interaction between permanent and transient core residues in a coiled coil HAMP structure. The data are compatible with HAMP rotation in signal propagation but do not exclude alternative models for HAMP signaling. Finally, we present data indicating that the connector, which links the α-helices of HAMP domains, plays an important structural role in HAMP function.

  6. Cyanobacteriochrome SesA Is a Diguanylate Cyclase That Induces Cell Aggregation in Thermosynechococcus*♦

    PubMed Central

    Enomoto, Gen; Nomura, Ryouhei; Shimada, Takashi; Ni-Ni-Win; Narikawa, Rei; Ikeuchi, Masahiko

    2014-01-01

    Cyanobacteria have unique photoreceptors, cyanobacteriochromes, that show diverse spectral properties to sense near-UV/visible lights. Certain cyanobacteriochromes have been shown to regulate cellular phototaxis or chromatic acclimation of photosynthetic pigments. Some cyanobacteriochromes have output domains involved in bacterial signaling using a second messenger cyclic dimeric GMP (c-di-GMP), but its role in cyanobacteria remains elusive. Here, we characterize the recombinant Tlr0924 from a thermophilic cyanobacterium Thermosynechococcus elongatus, which was expressed in a cyanobacterial system. The protein reversibly photoconverts between blue- and green-absorbing forms, which is consistent with the protein prepared from Escherichia coli, and has diguanylate cyclase activity, which is enhanced 38-fold by blue light compared with green light. Therefore, Tlr0924 is a blue light-activated diguanylate cyclase. The protein's relatively low affinity (10.5 mm) for Mg2+, which is essential for diguanylate cyclase activity, suggests that Mg2+ might also regulate c-di-GMP signaling. Finally, we show that blue light irradiation under low temperature is responsible for Thermosynechococcus vulcanus cell aggregation, which is abolished when tlr0924 is disrupted, suggesting that Tlr0924 mediates blue light-induced cell aggregation by producing c-di-GMP. Given our results, we propose the name “sesA (sessility-A)” for tlr0924. This is the first report for cyanobacteriochrome-dependent regulation of a sessile/planktonic lifestyle in cyanobacteria via c-di-GMP. PMID:25059661

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

    SciTech Connect

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

    1990-12-01

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

  8. Crystallization and preliminary X-ray diffraction studies of the glutaminyl cyclase from Carica papaya latex

    SciTech Connect

    Azarkan, Mohamed; Clantin, Bernard; Bompard, Coralie; Belrhali, Hassan; Baeyens-Volant, Danielle; Looze, Yvan; Wintjens, René

    2005-01-01

    The glutaminyl cyclase isolated from C. papaya latex has been crystallized using the hanging-drop method. Diffraction data have been collected at ESRF beamline BM14 and processed to 1.7 Å resolution. In living systems, the intramolecular cyclization of N-terminal glutamine residues is accomplished by glutaminyl cyclase enzymes (EC 2.3.2.5). While in mammals these enzymes are involved in the synthesis of hormonal and neurotransmitter peptides, the physiological role played by the corresponding plant enzymes still remains to be unravelled. Papaya glutaminyl cyclase (PQC), a 33 kDa enzyme found in the latex of the tropical tree Carica papaya, displays an exceptional resistance to chemical and thermal denaturation as well as to proteolysis. In order to elucidate its enzymatic mechanism and to gain insights into the structural determinants underlying its remarkable stability, PQC was isolated from papaya latex, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 62.82, b = 81.23, c = 108.17 Å and two molecules per asymmetric unit. Diffraction data have been collected at ESRF beamline BM14 and processed to a resolution of 1.7 Å.

  9. Two members of a widely expressed subfamily of hormone-stimulated adenylyl cyclases.

    PubMed Central

    Premont, R T; Chen, J; Ma, H W; Ponnapalli, M; Iyengar, R

    1992-01-01

    cDNA encoding a hormone- and guanine nucleotide-stimulated adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] (type 6) from rat liver and kidney has been cloned and expressed. This enzyme is stimulated by forskolin, guanosine 5'-[gamma-thio]triphosphate, and isoproterenol plus GTP but is not stimulated by beta gamma subunits of guanine nucleotide-binding proteins. A second form (type 5), which is 75% similar to type 6, has also been cloned. Both types 5 and 6 cDNAs have multiple messages. PCR-based detection of the mRNA for the type 5 and 6 enzymes indicates that both are widely distributed. Homology analyses indicate at least four distinct subfamilies of guanine nucleotide stimulatory protein-regulated adenylyl cyclases. Types 5 and 6 enzymes define one distinct subfamily of mammalian adenylyl cyclases. Diversity of one guanine nucleotide-binding protein-regulated effector may allow different modes of regulation of cell-surface signal transmission. Images PMID:1409703

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

    PubMed

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

    2015-12-15

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

  11. The Presence of Two Cyclase Thioesterases Expands the Conformational Freedom of the Cyclic Peptide Occidiofungin

    PubMed Central

    Ravichandran, Akshaya; Gu, Ganyu; Escano, Jerome; Lu, Shi-En; Smith, Leif

    2014-01-01

    Occidiofungin is a cyclic nonribosomally synthesized antifungal peptide with submicromolar activity produced by Gram-negative bacterium Burkholderia contaminans. The biosynthetic gene cluster was confirmed to contain two cyclase thioesterases. NMR analysis revealed that the presence of both thioesterases is used to increase the conformational repertoire of the cyclic peptide. The loss of the OcfN cyclic thioesterase by mutagenesis results in a reduction of conformational variants and an appreciable decrease in bioactivity against Candida species. Presumably, the presence of both asparagine and β-hydroxyasparagine variants coordinate the enzymatic function of both of the cyclase thioesterases. OcfN has presumably evolved to be part of the biosynthetic gene cluster due to its ability to produce structural variants that enhance antifungal activity against some fungi. The enhancement of the antifungal activity from the incorporation of an additional cyclase thioesterase into the biosynthetic gene cluster of occidiofungin supports the need to explore new conformational variants of other therapeutic or potentially therapeutic cyclic peptides. PMID:23394257

  12. Identification of photoactivated adenylyl cyclases in Naegleria australiensis and BLUF-containing protein in Naegleria fowleri.

    PubMed

    Yasukawa, Hiro; Sato, Aya; Kita, Ayaka; Kodaira, Ken-Ichi; Iseki, Mineo; Takahashi, Tetsuo; Shibusawa, Mami; Watanabe, Masakatsu; Yagita, Kenji

    2013-01-01

    Complete genome sequencing of Naegleria gruberi has revealed that the organism encodes polypeptides similar to photoactivated adenylyl cyclases (PACs). Screening in the N. australiensis genome showed that the organism also encodes polypeptides similar to PACs. Each of the Naegleria proteins consists of a "sensors of blue-light using FAD" domain (BLUF domain) and an adenylyl cyclase domain (AC domain). PAC activity of the Naegleria proteins was assayed by comparing sensitivities of Escherichia coli cells heterologously expressing the proteins to antibiotics in a dark condition and a blue light-irradiated condition. Antibiotics used in the assays were fosfomycin and fosmidomycin. E. coli cells expressing the Naegleria proteins showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light, indicating that the proteins functioned as PACs in the bacterial cells. Analysis of the N. fowleri genome revealed that the organism encodes a protein bearing an amino acid sequence similar to that of BLUF. A plasmid expressing a chimeric protein consisting of the BLUF-like sequence found in N. fowleri and the adenylyl cyclase domain of N. gruberi PAC was constructed to determine whether the BLUF-like sequence functioned as a sensor of blue light. E. coli cells expressing a chimeric protein showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light. These experimental results indicated that the sequence similar to the BLUF domain found in N. fowleri functioned as a sensor of blue light.

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

    SciTech Connect

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

    1985-07-01

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

  14. Central role of soluble adenylyl cyclase and cAMP in sperm physiology

    PubMed Central

    Buffone, Mariano G.; Wertheimer, Eva V.; Visconti, Pablo E.; Krapf, Dario

    2014-01-01

    Cyclic adenosine 3′,5′-monophosphate (cAMP), the first second messenger to be described, plays a central role in cell signaling in a wide variety of cell types. Over the last decades, a wide body of literature addressed the different roles of cAMP in cell physiology, mainly in response to neurotransmitters and hormones. cAMP is synthesized by a wide variety of adenylyl cylases that can generally be grouped in two types: transmembrane adenylyl cyclase and soluble adenylyl cyclases. In particular, several aspects of sperm physiology are regulated by cAMP produced by a single atypical adenylyl cyclase (Adcy10, aka sAC, SACY). The signature that identifies sAC among other ACs, is their direct stimulation by bicarbonate. The essential nature of cAMP in sperm function has been demonstrated using gain of function as well as loss of function approaches. This review unifies state of the art knowledge of the role of cAMP and those enzymes involved in cAMP signaling pathways required for the acquisition of fertilizing capacity of mammalian sperm. PMID:25066614

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

    PubMed Central

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

    2016-01-01

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

  16. Guanine-nucleotide-dependent inhibition of adenylate cyclase of rabbit heart by glucagon.

    PubMed

    Kiss, Z; Tkachuk, V A

    1984-07-16

    The present study demonstrates an inhibitory effect of glucagon on the adenylate cyclase system of rabbit heart. Inhibition was maximal (22-40%) at 0.1-0.01 microM glucagon and required the presence of 0.01-0.1 mM GTP or guanosine 5'-[beta, gamma-imido]triphosphate (GuoPP[NH]P). Reduced or no inhibitor effect of glucagon was observed: (a) after limited proteolysis of plasma membrane proteins by trypsin, (b) in the presence of 1 mM Mn2+, (c) in the absence of Na+, and (d) during the first 10 min of incubation if GuoPP[NH]P was the activating ligand. With GTP as the activating ligand, inhibition of cyclase by glucagon occurred without delay. These data are consistent with a mediation of glucagon inhibition by a guanine-nucleotide-binding protein. In the presence of ethanol (0.2 M) or benzyl alcohol (0.05 M), agents which are known to increase the fluidity of biological membranes, glucagon increased the enzyme activity in a guanine-nucleotide-dependent manner. Activation of cyclase in the presence of alcohols was maximal (30-60%) at 0.1-1.0 microM glucagon and 0.01 mM guanine nucleotides. Data suggest that glucagon receptors can interact with both the activatory and inhibitory guanine-nucleotide-binding proteins and the physical state of membranes may play a role in determining which interaction will be preferential.

  17. Developmental changes in ANP-stimulated guanylyl cyclase activity enhanced by ATP in rat lung membrane fractions.

    PubMed Central

    Charoonroje, P; Tokumitsu, Y; Nomura, Y

    1994-01-01

    1. ANP (atrial natriuretic peptides)- or ANP/ATP-stimulated guanylyl cyclase activities were compared in adult (2 month old) and neonatal (5-7 day old) rat lung membrane fractions. 2. The enzyme activities of both membranes depended on the incubation time and ATP concentration: although the activities of both membranes were similar after a short incubation time (4 min), those in adult membranes were lower than those of neonatal membranes after longer incubation times (10 and 30 min) or at lower concentrations of ATP. 3. ANP/ATP gamma S-stimulated guanylyl cyclase activities, which were much higher than ANP/ATP-stimulated activities, were similar in both membranes. 4. ATPase activity of adult membranes was higher than that of neonatal membranes, suggesting that hydrolysis of ATP leads to a decrease of ANP/ATP-guanylyl cyclase activity in adult membranes. Triton X-100 enhanced and diminished ANP/ATP-stimulated guanylyl cyclase activities of adult and neonatal membranes, respectively, and thereby abolished the adult/neonatal difference in the membrane response to ATP. 5. ANP-stimulated activities of both membranes were much more activated by pre-incubation with ATP gamma S than those induced by simultaneous addition of ATP gamma S. The former activities were decreased to levels of the latter by Triton X-100. The latter activities were not affected by Triton X-100. 6. The present results suggested that conformation of lung plasma membranes is related to activation of the ANP receptor/guanylyl cyclase system. PMID:7834209

  18. Intracellular role of adenylyl cyclase in regulation of lateral pseudopod formation during Dictyostelium chemotaxis.

    PubMed

    Stepanovic, Vesna; Wessels, Deborah; Daniels, Karla; Loomis, William F; Soll, David R

    2005-04-01

    Cyclic AMP (cAMP) functions as the extracellular chemoattractant in the aggregation phase of Dictyostelium development. There is some question, however, concerning what role, if any, it plays intracellularly in motility and chemotaxis. To test for such a role, the behavior of null mutants of acaA, the adenylyl cyclase gene that encodes the enzyme responsible for cAMP synthesis during aggregation, was analyzed in buffer and in response to experimentally generated spatial and temporal gradients of extracellular cAMP. acaA- cells were defective in suppressing lateral pseudopods in response to a spatial gradient of cAMP and to an increasing temporal gradient of cAMP. acaA- cells were incapable of chemotaxis in natural waves of cAMP generated by majority control cells in mixed cultures. These results indicate that intracellular cAMP and, hence, adenylyl cyclase play an intracellular role in the chemotactic response. The behavioral defects of acaA- cells were surprisingly similar to those of cells of null mutants of regA, which encodes the intracellular phosphodiesterase that hydrolyzes cAMP and, hence, functions opposite adenylyl cyclase A (ACA). This result is consistent with the hypothesis that ACA and RegA are components of a receptor-regulated intracellular circuit that controls protein kinase A activity. In this model, the suppression of lateral pseudopods in the front of a natural wave depends on a complete circuit. Hence, deletion of any component of the circuit (i.e., RegA or ACA) would result in the same chemotactic defect.

  19. Multifunctional oxidosqualene cyclases and cytochrome P450 involved in the biosynthesis of apple fruit triterpenic acids.

    PubMed

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

    2016-09-01

    Apple (Malus × domestica) accumulates bioactive ursane-, oleanane-, and lupane-type triterpenes in its fruit cuticle, but their biosynthetic pathway is still poorly understood. We used a homology-based approach to identify and functionally characterize two new oxidosqualene cyclases (MdOSC4 and MdOSC5) and one cytochrome P450 (CYP716A175). The gene expression patterns of these enzymes and of previously described oxidosqualene cyclases were further studied in 20 apple cultivars with contrasting triterpene profiles. MdOSC4 encodes a multifunctional oxidosqualene cyclase producing an oleanane-type triterpene, putatively identified as germanicol, as well as β-amyrin and lupeol, in the proportion 82 : 14 : 4. MdOSC5 cyclizes 2,3-oxidosqualene into lupeol and β-amyrin at a ratio of 95 : 5. CYP716A175 catalyses the C-28 oxidation of α-amyrin, β-amyrin, lupeol and germanicol, producing ursolic acid, oleanolic acid, betulinic acid, and putatively morolic acid. The gene expression of MdOSC1 was linked to the concentrations of ursolic and oleanolic acid, whereas the expression of MdOSC5 was correlated with the concentrations of betulinic acid and its caffeate derivatives. Two new multifuntional triterpene synthases as well as a multifunctional triterpene C-28 oxidase were identified in Malus × domestica. This study also suggests that MdOSC1 and MdOSC5 are key genes in apple fruit triterpene biosynthesis. PMID:27214242

  20. Structure of a Sedoheptulose 7-Phosphate Cyclase: ValA from Streptomyces hygroscopicus

    PubMed Central

    2015-01-01

    Sedoheptulose 7-phosphate cyclases (SH7PCs) encompass three enzymes involved in producing the core cyclitol structures of pseudoglycosides and similar bioactive natural products. One such enzyme is ValA from Streptomyces hygroscopicus subsp. jinggangensis 5008, which makes 2-epi-5-epi-valiolone as part of the biosynthesis of the agricultural antifungal agent validamycin A. We present, as the first SH7PC structure, the 2.1 Å resolution crystal structure of ValA in complex with NAD+ and Zn2+ cofactors. ValA has a fold and active site organization resembling those of the sugar phosphate cyclase dehydroquinate synthase (DHQS) and contains two notable, previously unrecognized interactions between NAD+ and Asp side chains conserved in all sugar phosphate cyclases that may influence catalysis. Because the domains of ValA adopt a nearly closed conformation even though no sugar substrate is present, comparisons with a ligand-bound DHQS provide a model for aspects of substrate binding. One striking active site difference is a loop that adopts a distinct conformation as a result of an Asp → Asn change with respect to DHQS and alters the identity and orientation of a key Arg residue. This and other active site differences in ValA are mostly localized to areas where the ValA substrate differs from that of DHQS. Sequence comparisons with a second SH7PC making a product with distinct stereochemistry lead us to postulate that the product stereochemistry of a given SH7PC is not the result of events taking place during catalysis but is accomplished by selective binding of either the α or β pyranose anomer of the substrate. PMID:24832673

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

    PubMed Central

    Lopez, Miguel A.; Saada, Edwin A.

    2014-01-01

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

  2. Ancient conserved domains shared by animal soluble guanylyl cyclases and bacterial signaling proteins

    PubMed Central

    Iyer, Lakshminarayan M; Anantharaman, Vivek; Aravind, L

    2003-01-01

    Background Soluble guanylyl cyclases (SGCs) are dimeric enzymes that transduce signals downstream of nitric oxide (NO) in animals. They sense NO by means of a heme moiety that is bound to their N-terminal extensions. Results Using sequence profile searches we show that the N-terminal extensions of the SGCs contain two globular domains. The first of these, the HNOB (Heme NO Binding) domain, is a predominantly α-helical domain and binds heme via a covalent linkage to histidine. Versions lacking this conserved histidine and are likely to interact with heme non-covalently. We detected HNOB domains in several bacterial lineages, where they occur fused to methyl accepting domains of chemotaxis receptors or as standalone proteins. The standalone forms are encoded by predicted operons that also contain genes for two component signaling systems and GGDEF-type nucleotide cyclases. The second domain, the HNOB associated (HNOBA) domain occurs between the HNOB and the cyclase domains in the animal SGCs. The HNOBA domain is also detected in bacteria and is always encoded by a gene, which occurs in the neighborhood of a gene for a HNOB domain. Conclusion The HNOB domain is predicted to function as a heme-dependent sensor for gaseous ligands, and transduce diverse downstream signals, in both bacteria and animals. The HNOBA domain functionally interacts with the HNOB domain, and possibly binds a ligand, either in cooperation, or independently of the latter domain. Phyletic profiles and phylogenetic analysis suggest that the HNOB and HNOBA domains were acquired by the animal lineage via lateral transfer from a bacterial source. PMID:12590654

  3. Structure of a sedoheptulose 7-phosphate cyclase: ValA from Streptomyces hygroscopicus.

    PubMed

    Kean, Kelsey M; Codding, Sara J; Asamizu, Shumpei; Mahmud, Taifo; Karplus, P Andrew

    2014-07-01

    Sedoheptulose 7-phosphate cyclases (SH7PCs) encompass three enzymes involved in producing the core cyclitol structures of pseudoglycosides and similar bioactive natural products. One such enzyme is ValA from Streptomyces hygroscopicus subsp. jinggangensis 5008, which makes 2-epi-5-epi-valiolone as part of the biosynthesis of the agricultural antifungal agent validamycin A. We present, as the first SH7PC structure, the 2.1 Å resolution crystal structure of ValA in complex with NAD+ and Zn2+ cofactors. ValA has a fold and active site organization resembling those of the sugar phosphate cyclase dehydroquinate synthase (DHQS) and contains two notable, previously unrecognized interactions between NAD+ and Asp side chains conserved in all sugar phosphate cyclases that may influence catalysis. Because the domains of ValA adopt a nearly closed conformation even though no sugar substrate is present, comparisons with a ligand-bound DHQS provide a model for aspects of substrate binding. One striking active site difference is a loop that adopts a distinct conformation as a result of an Asp→Asn change with respect to DHQS and alters the identity and orientation of a key Arg residue. This and other active site differences in ValA are mostly localized to areas where the ValA substrate differs from that of DHQS. Sequence comparisons with a second SH7PC making a product with distinct stereochemistry lead us to postulate that the product stereochemistry of a given SH7PC is not the result of events taking place during catalysis but is accomplished by selective binding of either the α or β pyranose anomer of the substrate.

  4. Insect stage-specific adenylate cyclases regulate social motility in African trypanosomes.

    PubMed

    Lopez, Miguel A; Saada, Edwin A; Hill, Kent L

    2015-01-01

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

  5. Structure, signaling mechanism and regulation of the natriuretic peptide receptor guanylate cyclase.

    SciTech Connect

    Misono, K. S.; Philo, J. S.; Arakawa, T.; Ogata, C. M.; Qiu, Y.; Ogawa, H.; Young, H. S.

    2011-06-01

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

  6. Effects of Ca++ and Prostaglandin E1 on Vasopressin Activation of Renal Adenyl Cyclase

    PubMed Central

    Marumo, Fumiaki; Edelman, Isidore S.

    1971-01-01

    Adenyl cyclase activity was assayed in crude homogenates of the renal cortex, medulla, and papilla of the golden hamster. The specific activity (moles C-AMP/unit of time per mg protein of tissue) of the enzyme under basal conditions, was greatest in papilla, somewhat lower in medulla, and least in cortex. On an absolute scale, the sensitivity to vasopressin was greater in the medullary and papillary than in the cortical homogenates. In addition, at concentrations of 0.1-1.0 mm, CaCl2 inhibited the enzyme in the order papilla > medulla > cortex. These results imply the existence of distinct differences in the composition of the adenyl cyclase-receptor complex in various parts of the kidney. We proposed that Ca++ inhibits the core enzyme directly since at the minimally inhibitory concentration (0.1 mm), CaCl2 reduced to an equivalent extent (a) basal activity, (b) the response to graded doses of vasopressin (0.5 to 50.0 mU/ml) and (c) the response to maximal stimulatory concentrations of NaF (10 mm). Prostaglandin E1 (PGE1 = 10−7m) had no effect on either basal adenyl-cyclase activity or the response to 10 mm NaF in medullary and papillary homogenates. 7-Oxa-13-prostynoic acid (10−4m) similarly had no effect under basal conditions or on stimulation with NaF in medullary homogenates. Both fatty acids, however, inhibited the enzymic response to vasopressin, particularly at low concentrations of the peptide. The straight-chain fatty acid, 11-eicosanoic acid (10−7m), was inactive on basal activity or on the response to vasopressin. The possibility that PGE1 modifies the coupling mechanism between the core enzyme and the hormone-specific receptor is discussed. PMID:4329002

  7. Regulation of cGMP levels by guanylate cyclase in truncated frog rod outer segments

    PubMed Central

    1989-01-01

    Cyclic GMP is the second messenger in phototransduction and regulates the photoreceptor current. In the present work, we tried to understand the regulation mechanism of cytoplasmic cGMP levels in frog photoreceptors by measuring the photoreceptor current using a truncated rod outer segment (tROS) preparation. Since exogenously applied substance diffuses into tROS from the truncated end, we could examine the biochemical reactions relating to the cGMP metabolism by manipulating the cytoplasmic chemical condition. In tROS, exogenously applied GTP produced a dark current whose amplitude was half-maximal at approximately 0.4 mM GTP. The conductance for this current was suppressed by light in a fashion similar to when it is activated by cGMP. In addition, no current was produced in the absence of Mg2+, which is known to be necessary for the guanylate cyclase activity. These results indicate that guanylate cyclase was present in tROS and synthesized cGMP from exogenously applied GTP. The enzyme activity was distributed throughout the rod outer segment. The amount of synthesized cGMP increased as the cytoplasmic Ca2+ concentration of tROS decreased, which indicated the activation of guanylate cyclase at low Ca2+ concentrations. Half-maximal effect of Ca2+ was observed at approximately 100 nM. tROS contained the proteins involved in the phototransduction mechanism and therefore, we could examine the regulation of the light response waveform by Ca2+. At low Ca2+ concentrations, the time course of the light response was speeded up probably because cGMP recovery was facilitated by activation of the cyclase. Then, if the cytoplasmic Ca2+ concentration of a photoreceptor decreases during light stimulation, the Ca2+ decrease may explain the acceleration of the light response during light adaptation. In tROS, however, we did observe an acceleration during repetitive light flashes when the cytoplasmic Ca2+ concentration increased during the stimulation. This result suggests the

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

    PubMed Central

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

    2016-01-01

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

  9. A kinase-anchoring proteins and adenylyl cyclase in cardiovascular physiology and pathology.

    PubMed

    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.

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

    PubMed Central

    Sternweis, P C; Gilman, A G

    1982-01-01

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

  11. Isolated neuronal growth cones from developing rat forebrain possess adenylate cyclase activity which can be augmented by various receptor agonists.

    PubMed

    Lockerbie, R O; Hervé, D; Blanc, G; Tassin, J P; Glowinski, J

    1988-01-01

    Isolated neuronal growth cones from neonatal rat forebrain were found to contain a high specific activity of adenylate cyclase (61 pmol cyclic AMP/min/mg protein) compared to the pelleted starting homogenate (5 pmol cyclic AMP/min/mg protein). Forskolin at 10(-4) M increased adenylate cyclase activity in both the pelleted homogenate and growth cone fraction by 70 and 217 pmol cyclic AMP/min/mg protein, respectively, over basal levels. The incremental effect of forskolin was 3-fold greater in the growth cone fraction than in the pelleted homogenate. However, relative to basal levels in each of the two fractions, forskolin increased adenylate cyclase activity in the growth cone fraction by only approx. 5-fold compared to 15-fold in the pelleted homogenate. Dopamine (10(-4) M), vasoactive intestinal polypeptide (10(-6) M) and isoproterenol (10(-5) M) also augmented adenylate cyclase activity in the two fractions. In the growth cone fraction, dopamine and vasoactive intestinal polypeptide produced a stimulation over basal levels by approx. 20 pmol cyclic AMP/min/mg protein while isoproterenol produced a stimulation of approx. 10 pmol cAMP/min/mg protein. The incremental effects of these receptor agonists in the growth cone fraction are approx. 5-fold greater than in the pelleted homogenate. The dopamine-sensitive adenylate cyclase activity in the growth cone fraction could be blocked by the compound SCH23390, a selective D1 receptor antagonist. At saturating concentrations, all combinations of dopamine, vasoactive intestinal polypeptide and isoproterenol were found to be completely additive on adenylate cyclase activity in the growth cone fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Enzymatic 13C Labeling and Multidimensional NMR Analysis of Miltiradiene Synthesized by Bifunctional Diterpene Cyclase in Selaginella moellendorffii*

    PubMed Central

    Sugai, Yoshinori; Ueno, Yohei; Hayashi, Ken-ichiro; Oogami, Shingo; Toyomasu, Tomonobu; Matsumoto, Sadamu; Natsume, Masahiro; Nozaki, Hiroshi; Kawaide, Hiroshi

    2011-01-01

    Diterpenes show diverse chemical structures and various physiological roles. The diversity of diterpene is primarily established by diterpene cyclases that catalyze a cyclization reaction to form the carbon skeleton of cyclic diterpene. Diterpene cyclases are divided into two types, monofunctional and bifunctional cyclases. Bifunctional diterpene cyclases (BDTCs) are involved in hormone and defense compound biosyntheses in bryophytes and gymnosperms, respectively. The BDTCs catalyze the successive two-step type-B (protonation-initiated cyclization) and type-A (ionization-initiated cyclization) reactions of geranylgeranyl diphosphate (GGDP). We found that the genome of a lycophyte, Selaginella moellendorffii, contains six BDTC genes with the majority being uncharacterized. The cDNA from S. moellendorffii encoding a BDTC-like enzyme, miltiradiene synthase (SmMDS), was cloned. The recombinant SmMDS converted GGDP to a diterpene hydrocarbon product with a molecular mass of 272 Da. Mutation in the type-B active motif of SmMDS abolished the cyclase activity, whereas (+)-copalyl diphosphate, the reaction intermediate from the conversion of GGDP to the hydrocarbon product, rescued the cyclase activity of the mutant to form a diterpene hydrocarbon. Another mutant lacking type-A activity accumulated copalyl diphosphate as the reaction intermediate. When the diterpene hydrocarbon was enzymatically synthesized from [U-13C6]mevalonate, all carbons were labeled with 13C stable isotope (>99%). The fully 13C-labeled product was subjected to 13C-13C COSY NMR spectroscopic analyses. The direct carbon-carbon connectivities observed in the multidimensional NMR spectra demonstrated that the hydrocarbon product by SmMDS is miltiradiene, a putative biosynthetic precursor of tanshinone identified from the Chinese medicinal herb Salvia miltiorrhiza. Hence, SmMDS functions as a bifunctional miltiradiene synthase in S. moellendorffii. In this study, we demonstrate that one-dimensional and

  13. Identification of a gamma subunit associated with the adenylyl cyclase regulatory proteins Ns and Ni.

    PubMed

    Hildebrandt, J D; Codina, J; Risinger, R; Birnbaumer, L

    1984-02-25

    The subunit composition of the Ns and Ni, the human erythrocyte stimulatory and inhibitory regulatory proteins of adenylyl cyclase, respectively, were analyzed by a sodium dodecyl sulfate-containing discontinuous urea and polyacrylamide gradient gel electrophoresis system designed for the study of low molecular weight polypeptides. This system disclosed that these proteins, in addition to their known alpha and beta subunits, contain an additional small peptide of apparent molecular weight of 5,000 (5K). This "5K peptide" is also present in preparations of another protein which we termed "40K protein" on the basis of its hydrodynamic behavior and whose primary protein constituent is the Mr 35,000 beta subunit of the above regulatory proteins. Analyzing Ni, the 5K peptide was functionally related to the protein by showing that its apparent Stokes radius changes from 5.9 to 5.1 nm after treatment with guanyl-5'-yl imidodiphosphate and magnesium in parallel with the alpha and beta subunits. These data are interpreted as evidence for the existence of a third subunit associated with the regulatory proteins of adenylyl cyclase. We call this subunit gamma and propose a minimum subunit structure for these proteins of the alpha beta gamma type. PMID:6321456

  14. E. coli heat-stable enterotoxin and guanylyl cyclase C: new functions and unsuspected actions.

    PubMed Central

    Giannella, Ralph A.; Mann, Elizabeth A.

    2003-01-01

    Some E. coli cause diarrhea by elaborating heat-labile and heat-stable (ST) enterotoxins which stimulate intestinal secretion. E. coli ST's are small peptides which bind to intestinal luminal epithelial cell receptors. The ST receptor, one of a family of receptor-cyclases called guanylyl cyclase C (GC-C), is a membrane spanning protein containing an extracellular binding domain and intracellular protein kinase and catalytic domains. The intestine synthesizes and secretes homologous peptides, guanylin and uroguanylin. The kidney also synthesizes uroguanylin. ST, guanylin or uroguanylin binding to GC-C results in increased cGMP, phosphorylation of the CFTR Cl- channel and secretion. Proguanylin and prouroguanylin circulate in blood and bind to receptors in intestine, kidney, liver, brain etc. In the kidney, they stimulate the excretion of Na+ and K+. Study of GC-C "knock-out" mice reveal that GC-C is important to intestinal salt and water secretion, duodenal bicarbonate secretion, recovery from CCl4-induced liver injury, and to intestinal polyp formation in Min mice lacking GC-C. PMID:12813912

  15. Identification of an adenylyl cyclase inhibitor for treating neuropathic and inflammatory pain.

    PubMed

    Wang, Hansen; Xu, Hui; Wu, Long-Jun; Kim, Susan S; Chen, Tao; Koga, Kohei; Descalzi, Giannina; Gong, Bo; Vadakkan, Kunjumon I; Zhang, Xuehan; Kaang, Bong-Kiun; Zhuo, Min

    2011-01-12

    Neuropathic pain, often caused by nerve injury, is commonly observed among patients with different diseases. Because its basic mechanisms are poorly understood, effective medications are limited. Previous investigations of basic pain mechanisms and drug discovery efforts have focused mainly on early sensory neurons such as dorsal root ganglion and spinal dorsal horn neurons, and few synaptic-level studies or new drugs are designed to target the injury-related cortical plasticity that accompanies neuropathic pain. Our previous work has demonstrated that calcium-stimulated adenylyl cyclase 1 (AC1) is critical for nerve injury-induced synaptic changes in the anterior cingulate cortex. Through rational drug design and chemical screening, we have identified a lead candidate AC1 inhibitor, NB001, which is relatively selective for AC1 over other adenylate cyclase isoforms. Using a variety of behavioral tests and toxicity studies, we have found that NB001, when administered intraperitoneally or orally, has an analgesic effect in animal models of neuropathic pain, without any apparent side effects. Our study thus shows that AC1 could be a productive therapeutic target for neuropathic pain and describes a new agent for the possible treatment of neuropathic pain.

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

    PubMed Central

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

    2016-01-01

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

  17. Expression, purification and crystallization of a plant polyketide cyclase from Cannabis sativa.

    PubMed

    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.

  18. Adenylate cyclase activity in fish gills in relation to salt adaptation

    SciTech Connect

    Guibbolini, M.E.; Lahlou, B.

    1987-07-06

    The influence of salt adaptation on specific adenylate cyclase activity (measured by conversion of (..cap alpha..-/sup 32/P) - ATP into (..cap alpha..-/sup 32/P) - cAMP) was investigated in gill plasma membranes of rainbow trout (Salmo gairdneri) adapted to various salinities (deionized water, DW; fresh water, FW; 3/4 sea water, 3/4 SW; sea water, SW) and in sea water adapted- mullet (Mugil sp.). Basal activity declined by a factor of 2 in trout with increasing external salinity (pmoles cAMP/mg protein/10 min: 530 in DW, 440 in FW, 340 in 3/4 SW; 250 in SW) and was very low in SW adapted-mullet: 35. The Km for ATP was similar (0.5 mM) in both FW adapted- and SW adapted- trout in either the absence (basal activity) or in the presence of stimulating agents (isoproterenol; NaF) while the Vm varied. Analysis of stimulation ratios with respect to basal levels of the enzyme showed that hormones and pharmacological substances (isoproterenol, NaF) display a greater potency in high salt than in low salt adapted- fish gills. In contrast, salt adaptation did not have any effect on the regulation of adenylate cyclase by PGE/sub 1/. These results are interpreted in relation to the general process of osmoregulation. 27 references, 6 figures.

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

    PubMed Central

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

    2016-01-01

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

  20. Diguanylate cyclase DgcP is involved in plant and human Pseudomonas spp. infections.

    PubMed

    Aragon, Isabel M; Pérez-Mendoza, Daniel; Moscoso, Joana A; Faure, Emmanuel; Guery, Benoit; Gallegos, María-Trinidad; Filloux, Alain; Ramos, Cayo

    2015-11-01

    The second messenger cyclic di-GMP (c-di-GMP) controls the transition between different lifestyles in bacterial pathogens. Here, we report the identification of DgcP (diguanylate cyclase conserved in Pseudomonads), whose activity in the olive tree pathogen Pseudomonas savastanoi pv. savastanoi is dependent on the integrity of its GGDEF domain. Furthermore, deletion of the dgcP gene revealed that DgcP negatively regulates motility and positively controls biofilm formation in both the olive tree pathogen P. savastanoi pv. savastanoi and the human opportunistic pathogen Pseudomonas aeruginosa. Overexpression of the dgcP gene in P. aeruginosa PAK led to increased exopolysaccharide production and upregulation of the type VI secretion system; in turn, it repressed the type III secretion system, which is a hallmark of chronic infections and persistence for P. aeruginosa. Deletion of the dgcP gene in P. savastanoi pv. savastanoi NCPPB 3335 and P. aeruginosa PAK reduced their virulence in olive plants and in a mouse acute lung injury model respectively. Our results show that diguanylate cyclase DgcP is a conserved Pseudomonas protein with a role in virulence, and confirm the existence of common c-di-GMP signalling pathways that are capable of regulating plant and human Pseudomonas spp. infections.

  1. Role of guanylate cyclase-activating proteins (GCAPs) in setting the flash sensitivity of rod photoreceptors

    PubMed Central

    Mendez, Ana; Burns, Marie E.; Sokal, Izabela; Dizhoor, Alexander M.; Baehr, Wolfgang; Palczewski, Krzysztof; Baylor, Denis A.; Chen, Jeannie

    2001-01-01

    The retina's photoreceptor cells adjust their sensitivity to allow photons to be transduced over a wide range of light intensities. One mechanism thought to participate in sensitivity adjustments is Ca2+ regulation of guanylate cyclase (GC) by guanylate cyclase-activating proteins (GCAPs). We evaluated the contribution of GCAPs to sensitivity regulation in rods by disrupting their expression in transgenic mice. The GC activity from GCAPs−/− retinas showed no Ca2+ dependence, indicating that Ca2+ regulation of GCs had indeed been abolished. Flash responses from dark-adapted GCAPs−/− rods were larger and slower than responses from wild-type rods. In addition, the incremental flash sensitivity of GCAPs−/− rods failed to be maintained at wild-type levels in bright steady light. GCAP2 expressed in GCAPs−/− rods restored maximal light-induced GC activity but did not restore normal flash response kinetics. We conclude that GCAPs strongly regulate GC activity in mouse rods, decreasing the flash sensitivity in darkness and increasing the incremental flash sensitivity in bright steady light, thereby extending the rod's operating range. PMID:11493703

  2. Adenylyl cyclase 3 haploinsufficiency confers susceptibility to diet-induced obesity and insulin resistance in mice

    PubMed Central

    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

  3. Expression, purification and crystallization of a plant polyketide cyclase from Cannabis sativa.

    PubMed

    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. PMID:26625288

  4. Bicarbonate-sensitive soluble and transmembrane adenylyl cyclases in peripheral chemoreceptors.

    PubMed

    Nunes, Ana R; Holmes, Andrew P S; Sample, Vedangi; Kumar, Prem; Cann, Martin J; Monteiro, Emília C; Zhang, Jin; Gauda, Estelle B

    2013-08-15

    Stimulation of the carotid body (CB) chemoreceptors by hypercapnia triggers a reflex ventilatory response via a cascade of cellular events, which includes generation of cAMP. However, it is not known if molecular CO2/HCO3(-) and/or H(+) mediate this effect and how these molecules contribute to cAMP production. We previously reported that the CB highly expresses HCO3(-)-sensitive soluble adenylyl cyclase (sAC). In the present study we systematically characterize the role of sAC in the CB, comparing the effect of isohydric hypercapnia (IH) in cAMP generation through activation of sAC or transmembrane-adenylyl cyclase (tmAC). Pharmacological deactivation of sAC and tmAC decreased the CB cAMP content in normocapnia and IH with no differences between these two conditions. Changes from normocapnia to IH did not effect the degree of PKA activation and the carotid sinus nerve discharge frequency. sAC and tmAC are functional in CB but intracellular elevations in CO2/HCO3(-) in IH conditions on their own are insufficient to further activate these enzymes, suggesting that the hypercapnic response is dependent on secondary acidosis.

  5. Inhibitory role of monovalent ions on rat brain cortex adenylyl cyclase activity.

    PubMed

    Nikolic, Ivana; Mitrovic, Marina; Zelen, Ivanka; Zaric, Milan; Kastratovic, Tatjana; Stanojevic, Marijana; Nenadovic, Milutin; Stojanovic, Tomislav

    2013-10-01

    Adenylyl cyclases, comprise of a large family of enzymes that catalyze synthesis of the cyclic AMP from ATP. The aim of our study was to determine the effect of monovalent ions on both basal, stimulated adenylate cyclase EC 4.6.1.1 (AC) activity and C unit of AC and on GTPase active G-protein in the synaptic membranes of rat brain cortex. The effect of ion concentration from 30 to 200 mM (1 mM MgCl2) showed dose-dependent and significant inhibition of the basal AC activity, stimulated and unstimulated C unit activity. Stimulation of AC with 5 μM GTPγS in the presence of 50-200 mM of tested salts showed inhibitory effect on the AC activity. From our results it could be postulated that the investigated monovalent ions exert inhibitory effect on the AC complex activity by affecting the intermolecular interaction of the activated α subunit of G/F protein and the C unit of AC complex an inhibitory influence of tested monovalent ions on these molecular interaction.

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

    PubMed

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

    2015-08-01

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

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

    PubMed

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

    2016-09-01

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

  8. CO2/HCO3(-)- and calcium-regulated soluble adenylyl cyclase as a physiological ATP sensor.

    PubMed

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

    2013-11-15

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

  9. Adenylate Cyclase Toxin (ACT) from Bordetella hinzii: Characterization and Differences from ACT of Bordetella pertussis

    PubMed Central

    Donato, Gina M.; Hsia, Hung-Lun J.; Green, Candace S.; Hewlett, Erik L.

    2005-01-01

    Bordetella hinzii is a commensal respiratory microorganism in poultry but is increasingly being recognized as an opportunistic pathogen in immunocompromised humans. Although associated with a variety of disease states, practically nothing is known about the mechanisms employed by this bacterium. In this study, we show by DNA sequencing and reverse transcription-PCR that both commensal and clinical strains of B. hinzii possess and transcriptionally express cyaA, the gene encoding adenylate cyclase toxin (ACT) in other pathogenic Bordetella species. By Western blotting, we also found that B. hinzii produces full-length ACT protein in quantities that are comparable to those made by B. pertussis. In contrast to B. pertussis ACT, however, ACT from B. hinzii is less extractable from whole bacteria, nonhemolytic, has a 50-fold reduction in adenylate cyclase activity, and is unable to elevate cyclic AMP levels in host macrophages (nontoxic). The decrease in enzymatic activity is attributable, at least in part, to a decreased binding affinity of B. hinzii ACT for calmodulin, the eukaryotic activator of B. pertussis ACT. In addition, we demonstrate that the lack of intoxication by B. hinzii ACT may be due to the absence of expression of cyaC, the gene encoding the accessory protein required for the acylation of B. pertussis ACT. These results demonstrate the expression of ACT by B. hinzii and represent the first characterization of a potential virulence factor of this organism. PMID:16267282

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

    PubMed

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

    2016-01-01

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

  11. Diguanylate cyclase DgcP is involved in plant and human Pseudomonas spp. infections.

    PubMed

    Aragon, Isabel M; Pérez-Mendoza, Daniel; Moscoso, Joana A; Faure, Emmanuel; Guery, Benoit; Gallegos, María-Trinidad; Filloux, Alain; Ramos, Cayo

    2015-11-01

    The second messenger cyclic di-GMP (c-di-GMP) controls the transition between different lifestyles in bacterial pathogens. Here, we report the identification of DgcP (diguanylate cyclase conserved in Pseudomonads), whose activity in the olive tree pathogen Pseudomonas savastanoi pv. savastanoi is dependent on the integrity of its GGDEF domain. Furthermore, deletion of the dgcP gene revealed that DgcP negatively regulates motility and positively controls biofilm formation in both the olive tree pathogen P. savastanoi pv. savastanoi and the human opportunistic pathogen Pseudomonas aeruginosa. Overexpression of the dgcP gene in P. aeruginosa PAK led to increased exopolysaccharide production and upregulation of the type VI secretion system; in turn, it repressed the type III secretion system, which is a hallmark of chronic infections and persistence for P. aeruginosa. Deletion of the dgcP gene in P. savastanoi pv. savastanoi NCPPB 3335 and P. aeruginosa PAK reduced their virulence in olive plants and in a mouse acute lung injury model respectively. Our results show that diguanylate cyclase DgcP is a conserved Pseudomonas protein with a role in virulence, and confirm the existence of common c-di-GMP signalling pathways that are capable of regulating plant and human Pseudomonas spp. infections. PMID:25809128

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

    PubMed

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

    2016-01-01

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

  13. Enhanced production and action of cyclic ADP-ribose during oxidative stress in small bovine coronary arterial smooth muscle.

    PubMed

    Zhang, Andrew Y; Yi, Fan; Teggatz, Eric G; Zou, Ai-Ping; Li, Pin-Lan

    2004-03-01

    Recent studies in our lab and by others have indicated that cyclic ADP-ribose (cADPR) as a novel second messenger is importantly involved in vasomotor response in various vascular beds. However, the mechanism regulating cADPR production and actions remains poorly understood. The present study determined whether changes in redox status influence the production and action of cADPR in coronary arterial smooth muscle cells (CASMCs) and thereby alters vascular tone in these arteries. HPLC analyses demonstrated that xanthine (X, 40 microM)/xanthine oxidase (XO, 0.1 U/ml), a superoxide-generating system, increased the ADP-ribosyl cyclase activity by 59% in freshly isolated bovine CASMCs. However, hydrogen peroxide (H2O2, 1-100 microM) had no significant effect on ADP-ribosyl cyclase activity. In these CASMCs, X/XO produced a rapid increase in [Ca2+]i (Delta[Ca2+]i=201 nM), which was significantly attenuated by a cADPR antagonist, 8-Br-cADPR. Both inhibition of cADPR production by nicotinamide (Nicot) and blockade of Ca2+-induced Ca2+ release (CICR) by tetracaine (TC) and ryanodine (Rya) significantly reduced X/XO-induced rapid Ca2+ responses. In isolated, perfused, and pressurized small bovine coronary arteries, X at 2.5-80 microM with a fixed XO level produced a concentration-dependent vasoconstriction with a maximal decrease in arterial diameter of 45%. This X/XO-induced vasoconstriction was significantly attenuated by 8-Br-cADPR, Nicot, TC, or Rya. We conclude that superoxide activates cADPR production, and thereby mobilizes intracellular Ca2+ from the SR and produces vasoconstriction in coronary arteries.

  14. The magnesium-protoporphyrin IX (oxidative) cyclase system. Studies on the mechanism and specificity of the reaction sequence.

    PubMed

    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.

  15. Reduced basal and stimulated (isoprenaline, Gpp(NH)p, forskolin) adenylate cyclase activity in Alzheimer's disease correlated with histopathological changes.

    PubMed

    Ohm, T G; Bohl, J; Lemmer, B

    1991-02-01

    Cyclic adenosine monophosphate (cAMP) is an adenylate cyclase borne second messenger involved in basic metabolic events. The beta-adrenoceptor sensitive adenylate cyclase was studied in post-mortem hippocampi of controls and Alzheimer patients. Virtually identical subsets of each hippocampus homogenate were stimulated by 100 mumol isoprenaline, Gpp(NH)p and forskolin, respectively, in presence of an ATP-regenerating system. The determination of cAMP formed was carried out by means of a radioassay. The observed significant 50% reduction in basal as well as in stimulated adenylate cyclase activity in Alzheimer's disease is negatively correlated with semiquantitative evaluations of amyloid plaques (P less than 0.05) but not with neuritic plaques, neurofibrillary tangles or neuropil threads. This reduction in enzyme activity is obviously not due to simple cell loss alone. It is likely that the crucial point of the observed functional disturbance is at the level of the catalytic unit of the adenylate cyclase, since the same degree of reduction is maintained at all steps of the signal cascade. PMID:2054615

  16. Purification, characterization, and N-terminal amino acid sequence of the adenylyl cyclase-activating protease from bovine sperm.

    PubMed

    Adeniran, A J; Shoshani, I; Minuth, M; Awad, J A; Elce, J S; Johnson, R A

    1995-03-01

    We previously reported the extraction of a factor from bovine sperm that activated adenylyl cyclases of rat brain and human platelets, and identified it as a trypsin-like protease that was referred to as "ninhibin." This proteolytic activity was purified to near homogeneity from an alkaline extract of washed sperm particles by sequential chromatography on p-aminobenzamidine agarose and CM-Sephadex. Purification was greater than 100-fold with nearly 30% recovery of protease activity exhibiting a major band of approximately 40 kDa. An approximately 45-kDa form of the protease was also evident in crude extracts and was preferentially isolated when the enzyme was prepared in the presence of a mixture of protease inhibitors. The larger form of the protease was substantially less effective in stimulating adenylyl cyclase than was the smaller form; it is likely to be a zymogen form from which the smaller, more active form is derived. Purified forms of acrosin and ninhibin exhibited similar mobilities on PAGE, similar capacities for activating adenylyl cyclase, similar patterns of proteolytic fragmentation, and similar immunoblot patterns obtained with an antibody against purified bovine acrosin. More importantly, the N-terminal amino acid sequence of bovine ninhibin was found to be identical with that of bovine acrosin and caprine acrosin and more than 75% identical with porcine acrosin. The data support the conclusion that the adenylyl cyclase-activating protease previously referred to as ninhibin is, in fact, acrosin. PMID:7756444

  17. Multiplex PCR Assay Targeting a Diguanylate Cyclase-Encoding Gene, cgcA, To Differentiate Species within the Genus Cronobacter

    PubMed Central

    Carter, L.; Lindsey, L. A.; Grim, C. J.; Sathyamoorthy, V.; Jarvis, K. G.; Gopinath, G.; Lee, C.; Sadowski, J. A.; Trach, L.; Pava-Ripoll, M.; McCardell, B. A.; Tall, B. D.

    2013-01-01

    In a comparison to the widely used Cronobacter rpoB PCR assay, a highly specific multiplexed PCR assay based on cgcA, a diguanylate cyclase gene, that identified all of the targeted six species among 305 Cronobacter isolates was designed. This assay will be a valuable tool for identifying suspected Cronobacter isolates from food-borne investigations. PMID:23144142

  18. Stimulation of guanylate cyclase by sodium nitroprusside, nitroglycerin and nitric oxide in various tissue preparations and comparison to the effects of sodium azide and hydroxylamine.

    PubMed

    Katsuki, S; Arnold, W; Mittal, C; Murad, F

    1977-02-01

    Sodium nitroprusside, nitroglycerin, sodium azide and hydroxylamine increased guanylate cyclase activity in particulate and/or soluble preparations from various tissues. While sodium nitroprusside increased guanylate cyclase activity in most of the preparations examined, the effects of sodium azide, hydroxylamine and nitroglycerin were tissue specific. Nitroglycerin and hydroxylamine were also less potent. Neither the protein activator factor nor catalase which is required for sodium azide effects altered the stimulatory effect of sodium nitroprusside. In the presence of sodium azide, sodium nitroprusside or hydroxylamine, magnesium ion was as effective as manganese ion as a sole cation cofactor for guanylate cyclase. With soluble guanylate cyclase from rat liver and bovine tracheal smooth muscle the concentrations of sodium nitroprusside that gave half-maximal stimulation with Mn2+ were 0.1 mM and 0.01 mM, respectively. Effective concentrations were slightly less with Mg2+ as a sole cation cofactor. The ability of these agents to increase cyclic GMP levels in intact tissues is probably due to their effects on guanylate cyclase activity. While the precise mechanism of guanylate cyclase activation by these agents is not known, activation may be due to the formation of nitric oxide or another reactive material since nitric oxide also increased guanylate cyclase activity. PMID:14978

  19. Magnesium regulation of the beta-receptor-adenylate cyclase complex. II. Sc3+ as a Mg2 antagonist.

    PubMed

    Maguire, M E

    1982-09-01

    Sc3+ bears the same relationship to Mg2+ as La3+ to Ca2+, a similar ionic radius but increased charge. Therefore, the possibility was investigated that Sc3+ would be a Mg2+ antagonist at Mg2+ sites on the beta-adrenergic receptor-adenylate cyclase complex of the murine S49 lymphoma cell. Sc3+ is consistently much more potent than La3+ in inhibiting adenylate cyclase regardless of the mode of activation. IC50 values for Sc3+ of 10-30 microM were observed, whereas those for La3+ were about 300 microM. However, Sc3+ does not block the ability of Mg2+ to increase beta-receptor affinity for agonist nor alter agonist affinity by itself. Furthermore, Sc3+ is a weak inhibitor of the beta-receptor-mediated inhibition of Mg2+ influx. In cyc- S49 membranes, in which the catalytic subunit of cyclase cannot interact with the nucleotide-coupling protein(s), Sc3+ is as potent as in wild-type S49 membranes and again more potent than La3+. Substrate kinetics show that Sc3+, like Mg2+, modulates adenylate cyclase activity by affecting the Vmax without altering the Km for substrate. The data suggest that Sc3+ is a specific antagonist of Mg2+ at the Mg2+ site on the catalytic subunit and support the suggestion that there are two distinct sites for Mg2+ with different functions, one site on the coupling protein(s) and one on the catalytic subunit. It was also found that an apparent complex of Sc3+ and F-, ScF4-, is a potent inhibitor of adenylate cyclase, with an IC50 of 3 microM. PMID:6292689

  20. Calcium-Myristoyl Tug Is a New Mechanism for Intramolecular Tuning of Calcium Sensitivity and Target Enzyme Interaction for Guanylyl Cyclase-activating Protein 1

    PubMed Central

    Peshenko, Igor V.; Olshevskaya, Elena V.; Lim, Sunghyuk; Ames, James B.; Dizhoor, Alexander M.

    2012-01-01

    Guanylyl cyclase-activating protein 1 (GCAP1), a myristoylated Ca2+ sensor in vision, regulates retinal guanylyl cyclase (RetGC). We show that protein-myristoyl group interactions control Ca2+ sensitivity, apparent affinity for RetGC, and maximal level of cyclase activation. Mutating residues near the myristoyl moiety affected the affinity of Ca2+ binding to EF-hand 4. Inserting Phe residues in the cavity around the myristoyl group increased both the affinity of GCAP1 for RetGC and maximal activation of the cyclase. NMR spectra show that the myristoyl group in the L80F/L176F/V180F mutant remained sequestered inside GCAP1 in both Ca2+-bound and Mg2+-bound states. This mutant displayed much higher affinity for the cyclase but reduced Ca2+ sensitivity of the cyclase regulation. The L176F substitution improved affinity of myristoylated and non-acylated GCAP1 for the cyclase but simultaneously reduced the affinity of Ca2+ binding to EF-hand 4 and Ca2+ sensitivity of the cyclase regulation by acylated GCAP1. The replacement of amino acids near both ends of the myristoyl moiety (Leu80 and Val180) minimally affected regulatory properties of GCAP1. N-Lauryl- and N-myristoyl-GCAP1 activated RetGC in a similar fashion. Thus, protein interactions with the central region of the fatty acyl chain optimize GCAP1 binding to RetGC and maximize activation of the cyclase. We propose a dynamic connection (or “tug”) between the fatty acyl group and EF-hand 4 via the C-terminal helix that attenuates the efficiency of RetGC activation in exchange for optimal Ca2+ sensitivity. PMID:22383530

  1. pH sensing via bicarbonate-regulated “soluble” adenylyl cyclase (sAC)

    PubMed Central

    Rahman, Nawreen; Buck, Jochen; Levin, Lonny R.

    2013-01-01

    Soluble adenylyl cyclase (sAC) is a source of the second messenger cyclic adenosine 3′, 5′ monophosphate (cAMP). sAC is directly regulated by bicarbonate (HCO−3) ions. In living cells, HCO−3 ions are in nearly instantaneous equilibrium with carbon dioxide (CO2) and pH due to the ubiquitous presence of carbonic anhydrases. Numerous biological processes are regulated by CO2, HCO−3, and/or pH, and in a number of these, sAC has been shown to function as a physiological CO2/HCO3/pH sensor. In this review, we detail the known pH sensing functions of sAC, and we discuss two highly-studied, pH-dependent pathways in which sAC might play a role. PMID:24324443

  2. An Approach to Mimicking the Sesquiterpene Cyclase Phase by Nickel-Promoted Diene/Alkyne Cooligomerization

    PubMed Central

    Holte, Dane; Götz, Daniel C. G.; Baran, Phil S.

    2012-01-01

    Artificially mimicking the cyclase phase of terpene biosynthesis inspires the invention of new methodologies, since working with carbogenic frameworks containing minimal functionality limits the chemist’s toolbox of synthetic strategies. For example, the construction of terpene skeletons from five-carbon building blocks would be an exciting pathway to mimic in the laboratory. Nature oligomerizes, cyclizes, and then oxidizes γ,γ-dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) to all of the known terpenes. Starting from isoprene, the goal of this work was to mimic Nature’s approach for rapidly building molecular complexity. In principle, the controlled oligomerization of isoprene would drastically simplify the synthesis of terpenes used in the medicine, perfumery, flavor, and materials industries. This article delineates our extensive efforts to cooligomerize isoprene or butadiene with alkynes in a controlled fashion by zero-valent nickel catalysis building off the classic studies by Günther Wilke and coworkers. PMID:22229741

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

    NASA Astrophysics Data System (ADS)

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

    2010-10-01

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

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

    SciTech Connect

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

    1990-01-01

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

  5. Endotoxic lipopolysaccharides stimulate steroidogenesis and adenylate cyclase in adrenal tumor cells.

    PubMed

    Wolff, J; Cook, G H

    1975-12-01

    Lipopolysaccharides (endotoxins) from Escherichia coli, Serratia marcesens and Salmonella typhosa stimulated steroid production in Y-1 adrenal tumor cells in culture with a latent period of 3-4 h. Lipid A, derived from Escherichia coli lipopolysaccharide, also stimulated steroidogenesis. Lipopolysaccharides and lipid A also stimulate adenylate cyclase activity and cause rounding of the cells. In contrast, lipopolysaccharides do not stimulate steroidogenesis in receptor-deficient adrenal tumor cells (OS-3) or Leydig tumor cells (I-10). This tends to rule out contamination by enterotoxin to which these lines respond. Although both hormone and lipopolysaccharide responses are lost in these lines, there was no interaction between these sites as judged by the failure of lipopolysaccharides to block, during their latency, the response to corticotropin in Y-1 cells. The possibility that the lipopolysaccharide effect is one on membrane conformation is discussed.

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

    PubMed

    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.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2010-10-01

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

  9. Squalene hopene cyclases are protonases for stereoselective Brønsted acid catalysis.

    PubMed

    Hammer, Stephan C; Marjanovic, Antonija; Dominicus, Jörg M; Nestl, Bettina M; Hauer, Bernhard

    2015-02-01

    For many important reactions catalyzed in chemical laboratories, the corresponding enzymes are missing, representing a restriction in biocatalysis. Although nature provides highly developed machineries appropriate to catalyze such reactions, their potential is often ignored. This also applies to Brønsted acid catalysis, a powerful method to promote a myriad of chemical transformations. Here, we report on the unique protonation machinery of a squalene hopene cyclase (SHC). Active site engineering of this highly evolvable enzyme yielded a platform for enzymatic Brønsted acid catalysis in water. This is illustrated by activation of different functional groups (alkenes, epoxides and carbonyls), enabling the highly stereoselective syntheses of various cyclohexanoids while uncoupling SHC from polycyclization chemistry. This work highlights the potential of systematic investigation on nature's catalytic machineries to generate unique catalysts.

  10. Regulation of soluble guanylate cyclase by matricellular thrombospondins: implications for blood flow.

    PubMed

    Rogers, Natasha M; Seeger, Franziska; Garcin, Elsa D; Roberts, David D; Isenberg, Jeffrey S

    2014-01-01

    Nitric oxide (NO) maintains cardiovascular health by activating soluble guanylate cyclase (sGC) to increase cellular cGMP levels. Cardiovascular disease is characterized by decreased NO-sGC-cGMP signaling. Pharmacological activators and stimulators of sGC are being actively pursued as therapies for acute heart failure and pulmonary hypertension. Here we review molecular mechanisms that modulate sGC activity while emphasizing a novel biochemical pathway in which binding of the matricellular protein thrombospondin-1 (TSP1) to the cell surface receptor CD47 causes inhibition of sGC. We discuss the therapeutic implications of this pathway for blood flow, tissue perfusion, and cell survival under physiologic and disease conditions.

  11. Characterization of the dopamine stimulated adenylate cyclase in the pedal ganglia of Mytilus edulis: interactions with etorphine, beta-endorphin, DALA, and methionine enkephalin.

    PubMed

    Stefano, G B; Catapane, E J; Kream, R M

    1981-03-01

    The dopamine-stimulated adenylate cyclase activity was studied both in vivo and in vitro in the central nervous system of the bivalve mollusc Mytilus edulis. Dopamine, epinine, and apomorphine stimulated the enzyme system. Fluphenazine, haloperidol, chlorpromaxine, and to a lesser extent BOL inhibited the dopamine-stimulated adenylate cyclase. Etorphine, beta-endorphine, DALA, and methionine enkephalin depressed cyclic AMP levels. This phenomena was naloxone reversible. In addition, the opioids inhibited the stimulation of adenylate cyclase by dopamine. This phenomena was also naloxone reversible. The study demonstrates an interaction among dopamine, the opioids, and cyclic AMP. PMID:6286125

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

    PubMed Central

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

    1998-01-01

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

  13. Activation of Soluble Adenylyl Cyclase Protects against Secretagogue Stimulated Zymogen Activation in Rat Pancreaic Acinar Cells

    PubMed Central

    Kolodecik, Thomas R.; Shugrue, Christine A.; Thrower, Edwin C.; Levin, Lonny R.; Buck, Jochen; Gorelick, Fred S.

    2012-01-01

    An early feature of acute pancreatitis is activation of zymogens, such as trypsinogen, within the pancreatic acinar cell. Supraphysiologic concentrations of the hormone cholecystokinin (CCK; 100 nM), or its orthologue cerulein (CER), induce zymogen activation and elevate levels of cAMP in pancreatic acinar cells. The two classes of adenylyl cyclase, trans-membrane (tmAC) and soluble (sAC), are activated by distinct mechanisms, localize to specific subcellular domains, and can produce locally high concentrations of cAMP. We hypothesized that sAC activity might selectively modulate acinar cell zymogen activation. sAC was identified in acinar cells by PCR and immunoblot. It localized to the apical region of the cell under resting conditions and redistributed intracellularly after treatment with supraphysiologic concentrations of cerulein. In cerulein-treated cells, pre-incubation with a trans-membrane adenylyl cyclase inhibitor did not affect zymogen activation or amylase secretion. However, treatment with a sAC inhibitor (KH7), or inhibition of a downstream target of cAMP, protein kinase A (PKA), significantly enhanced secretagogue-stimulated zymogen activation and amylase secretion. Activation of sAC with bicarbonate significantly inhibited secretagogue-stimulated zymogen activation; this response was decreased by inhibition of sAC or PKA. Bicarbonate also enhanced secretagogue-stimulated cAMP accumulation; this effect was inhibited by KH7. Bicarbonate treatment reduced secretagogue-stimulated acinar cell vacuolization, an early marker of pancreatitis. These data suggest that activation of sAC in the pancreatic acinar cell has a protective effect and reduces the pathologic activation of proteases during pancreatitis. PMID:22844459

  14. Restoring Soluble Guanylyl Cyclase Expression and Function Blocks the Aggressive Course of GliomaS⃞

    PubMed Central

    Zhu, Haifeng; Li, Jessica Tao; Zheng, Fang; Martin, Emil; Kots, Alexander Y.; Krumenacker, Joshua S.; Choi, Byung-Kwon; McCutcheon, Ian E.; Weisbrodt, Norman; Bögler, Oliver; Murad, Ferid

    2011-01-01

    The NO and cGMP signaling pathways are of broad physiological and pathological significance. We compared the NO/soluble guanylyl cyclase (sGC)/cGMP pathway in human glioma tissues and cell lines with that of healthy control samples and demonstrated that sGC expression is significantly lower in glioma preparations. Our analysis of GEO databases (National Cancer Institute) further revealed a statistically significant reduction of sGC transcript levels in human glioma specimens. On the other hand, the expression levels of particulate (membrane) guanylyl cyclases (pGC) and cGMP-specific phosphodiesterase (PDE) were intact in the glioma cells that we have tested. Pharmacologically manipulating endogenous cGMP generation in glioma cells through either stimulating pGC by ANP/BNP, or blocking PDE by 3-isobutyl-1-methylxanthine/zaprinast caused significant inhibition of proliferation and colony formation of glioma cells. Genetically restoring sGC expression also correlated inversely with glioma cells growth. Orthotopic implantation of glioma cells transfected with an active mutant form of sGC (sGCα1β1Cys105) in athymic mice increased the survival time by 4-fold over the control. Histological analysis of xenografts overexpressing α1β1Cys105 sGC revealed changes in cellular architecture that resemble the morphology of normal cells. In addition, a decrease in angiogenesis contributed to glioma inhibition by sGC/cGMP therapy. Our study proposes the new concept that suppressed expression of sGC, a key enzyme in the NO/cGMP pathway, may be associated with an aggressive course of glioma. The sGC/cGMP signaling-targeted therapy may be a favorable alternative to chemotherapy and radiotherapy for glioma and perhaps other tumors. PMID:21908708

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

    PubMed

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

    2007-09-25

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

  16. Engineering of Bacillus subtilis Strains To Allow Rapid Characterization of Heterologous Diguanylate Cyclases and Phosphodiesterases

    PubMed Central

    Gao, Xiaohui; Dong, Xiao; Subramanian, Sundharraman; Matthews, Paige M.; Cooper, Caleb A.; Kearns, Daniel B.

    2014-01-01

    Microbial processes, including biofilm formation, motility, and virulence, are often regulated by changes in the available concentration of cyclic dimeric guanosine monophosphate (c-di-GMP). Generally, high c-di-GMP concentrations are correlated with decreased motility and increased biofilm formation and low c-di-GMP concentrations are correlated with an increase in motility and activation of virulence pathways. The study of c-di-GMP is complicated, however, by the fact that organisms often encode dozens of redundant enzymes that synthesize and hydrolyze c-di-GMP, diguanylate cyclases (DGCs), and c-di-GMP phosphodiesterases (PDEs); thus, determining the contribution of any one particular enzyme is challenging. In an effort to develop a facile system to study c-di-GMP metabolic enzymes, we have engineered a suite of Bacillus subtilis strains to assess the effect of individual heterologously expressed proteins on c-di-GMP levels. As a proof of principle, we characterized all 37 known genes encoding predicted DGCs and PDEs in Clostridium difficile using parallel readouts of swarming motility and fluorescence from green fluorescent protein (GFP) expressed under the control of a c-di-GMP-controlled riboswitch. We found that 27 of the 37 putative C. difficile 630 c-di-GMP metabolic enzymes had either active cyclase or phosphodiesterase activity, with agreement between our motility phenotypes and fluorescence-based c-di-GMP reporter. Finally, we show that there appears to be a threshold level of c-di-GMP needed to inhibit motility in Bacillus subtilis. PMID:25085482

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

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  18. Interaction of retinal guanylate cyclase with the alpha subunit of transducin: potential role in transducin localization.

    PubMed

    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.

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

    PubMed

    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.

  20. Regulation of uterine adenylate cyclase by magnesium, manganese and calcium ions

    SciTech Connect

    Rayford, W.; Sanders, R.B.

    1987-05-01

    The regulation of rat uterine adenylate cyclase (AC) by Mg/sup 2 +/, Mn/sup 2 +/ and Ca/sup 2 +/ was examined during metestrus and proestrus of the estrous cycle and Days 1 and 4 of pseudopregnancy, before and after a mild trauma to the uterus. Mg/sup 2 +/ increased cyclase activity on all days measured. Maximal enzymic activity occurred with 5-10 mM Mg/sup 2 +/ during Day 4 following a mild traumatic stimulus to the uterus. The apparent Ka for Mg/sup 2 +/ was not significantly changed during these days. AC activity as a function of Mn/sup 2 +/ concentration was biphasic. It increased with increasing concentrations of Mn/sup 2 +/ and was maximal at 1.0-2.5 mM during Day 4 following uterine trauma. Higher concentrations of Mn/sup 2 +/ were inhibitory. The apparent Ka for Mn/sup 2 +/ was 0.36 +/- 0.05 mM and was not significantly altered during the days studied. Even though the Ka for Mn/sup 2 +/ was ten-fold lower than that for Mg/sup 2 +/, the Vmaxes shown with both ions were similar. Ca/sup 2 +/ is a potent inhibitor of uterine AC activity. When measured at its I.C./sub 50/, it lowered AC activity as Mg/sup 2 +/ concentrations were increased. Ca/sup 2 +/ did not have a significant effect on AC activated by Mn/sup 2 +/. The data showed that Mg/sup 2 +/, Mn/sup 2 +/ and Ca/sup 2 +/ might have important regulatory roles in the activation and inhibition of uterine AC in the rodent.

  1. Characterization of a novel sesquiterpene cyclase involved in (+)-caryolan-1-ol biosynthesis in Streptomyces griseus.

    PubMed

    Nakano, Chiaki; Horinouchi, Sueharu; Ohnishi, Yasuo

    2011-08-12

    Most terpenoids have been isolated from plants and fungi and only a few from bacteria. However, an increasing number of genome sequences indicate that bacteria possess a variety of terpenoid cyclase genes. We characterized a sesquiterpene cyclase gene (SGR2079, named gcoA) found in Streptomyces griseus. When expressed in Streptomyces lividans, gcoA directed production of a sesquiterpene, isolated and determined to be (+)-caryolan-1-ol using spectroscopic analyses. (+)-Caryolan-1-ol was also detected in the crude cell lysate of wild-type S. griseus but not in a gcoA knockout mutant, indicating that GcoA is a genuine (+)-caryolan-1-ol synthase. Enzymatic properties were characterized using N-terminally histidine-tagged GcoA, produced in Escherichia coli. As expected, incubation of the recombinant GcoA protein with farnesyl diphosphate yielded (+)-caryolan-1-ol. However, a small amount of another sesquiterpene was also detected. This was identified as the bicyclic sesquiterpene hydrocarbon (+)-β-caryophyllene by comparison with an authentic sample using GC-MS. Incorporation of a deuterium atom into the C-9 methylene of (+)-caryolan-1-ol in an in vitro GcoA reaction in deuterium oxide indicated that (+)-caryolan-1-ol was synthesized by a proton attack on the C-8/C-9 double bond of (+)-β-caryophyllene. Several β-caryophyllene synthases have been identified from plants, but these cannot synthesize caryolan-1-ol. Although caryolan-1-ol has been isolated previously from several plants, the enzyme responsible for its biosynthesis has not been identified previously. GcoA is thus the first known caryolan-1-ol synthase. Isolation of caryolan-1-ol from microorganisms is unprecedented. PMID:21693706

  2. Novel metabotropic glutamate receptor negatively coupled to adenylyl cyclase in cultured rat cerebellar astrocytes.

    PubMed

    Kanumilli, Srinivasan; Toms, Nick J; Roberts, Peter J

    2004-04-01

    Several excitatory amino acid ligands were found potently to inhibit forskolin-stimulated cAMP accumulation in rat cultured cerebellar astrocytes: L-cysteine sulfinic acid (L-CSA) = L-aspartate > L-glutamate >/= the glutamate uptake inhibitor, L-PDC. This property did not reflect activation of conventional glutamate receptors, since the selective ionotropic glutamate receptor agonists NMDA, AMPA, and kainate, as well as several mGlu receptor agonists [(1S,3R)-ACPD, (S)-DHPG, DCG-IV, L-AP4, L-quisqualate, and L-CCG-I], were without activity. In addition, the mGlu receptor antagonists, L-AP3, (S)-4CPG, Eglu, LY341495, (RS)-CPPG, and (S)-MCPG failed to reverse 30 microM glutamate-mediated inhibitory responses. L-PDC-mediated inhibition was abolished by the addition of the enzyme glutamate-pyruvate transaminase. This finding suggests that the effect of L-PDC is indirect and that it is mediated through endogenously released L-glutamate. Interestingly, L-glutamate-mediated inhibitory responses were resistant to pertussis toxin, suggesting that G(i)/G(o) type G proteins were not involved. However, inhibition of protein kinase C (PKC, either via the selective PKC inhibitor GF109203X or chronic PMA treatment) augmented glutamate-mediated inhibitory responses. Although mGlu3 receptors (which are negatively coupled to adenylyl cyclase) are expressed in astrocyte populations, in our study Western blot analysis indicated that this receptor type was not expressed in cerebellar astrocytes. We therefore suggest that cerebellar astrocytes express a novel mGlu receptor, which is negatively coupled to adenylyl cyclase, and possesses an atypical pharmacological profile. PMID:14999808

  3. A role for guanylate cyclase C in acid-stimulated duodenal mucosal bicarbonate secretion.

    PubMed

    Rao, S P; Sellers, Z; Crombie, D L; Hogan, D L; Mann, E A; Childs, D; Keely, S; Sheil-Puopolo, M; Giannella, R A; Barrett, K E; Isenberg, J I; Pratha, V S

    2004-01-01

    Luminal acidification provides the strongest physiological stimulus for duodenal HCO3- secretion. Various neurohumoral mechanisms are believed to play a role in acid-stimulated HCO3- secretion. Previous studies in the rat and human duodenum have shown that guanylin and Escherichia coli heat-stable toxin, both ligands of the transmembrane guanylyl cyclase receptor [guanylate cyclase C (GC-C)], are potent stimulators for duodenal HCO3- secretion. We postulated that the GC-C receptor plays an important role in acid-stimulated HCO3- secretion. In vivo perfusion studies performed in wild-type (WT) and GC-C knockout (KO) mice indicated that acid-stimulated duodenal HCO3- secretion was significantly decreased in the GC-C KO animals compared with the WT counterparts. Pretreatment with PD-98059, an MEK inhibitor, resulted in attenuation of duodenal HCO3- secretion in response to acid stimulation in the WT mice with no further effect in the KO mice. In vitro cGMP generation studies demonstrated a significant and comparable increase in cGMP levels on acid exposure in the duodenum of both WT and KO mice. In addition, a rapid, time-dependent phosphorylation of ERK was observed with acid exposure in the duodenum of WT mice, whereas a marked attenuation in ERK phosphorylation was observed in the KO animals despite equivalent levels of ERK in both groups of animals. On the basis of these studies, we conclude that transmembrane GC-C is a key mediator of acid-stimulated duodenal HCO3- secretion. Furthermore, ERK phosphorylation may be an important intracellular mediator of duodenal HCO3- secretion. PMID:12881226

  4. Cloning, sequencing, and expression of a 24-kDa Ca(2+)-binding protein activating photoreceptor guanylyl cyclase.

    PubMed

    Dizhoor, A M; Olshevskaya, E V; Henzel, W J; Wong, S C; Stults, J T; Ankoudinova, I; Hurley, J B

    1995-10-20

    Two vertebrate photoreceptor-specific membrane guanylyl cyclases, RetGC-1 and RetGC-2, are activated by a soluble 24-kDa retinal protein, p24, in a Ca(2+)-sensitive manner (Dizhoor, A.M., Lowe, D.G., Olshevskaya, E.V., Laura, R.P., and Hurley, J.B. (1994) Neuron 12, 1345-1352; Lowe, D.G., Dizhoor, A.M., Liu, K., Gu, O., Laura, R., Lu, L., and Hurley, J.B. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 5535-5539). The primary structure of bovine p24 has been derived from peptide sequencing and from its cDNA. p24 is a new EF-hand-type Ca(2+)-binding protein, related but not identical to another guanylyl cyclase-activating protein, GCAP (Palczewski, K., Subbaraya, I., Gorczyca, W.A., Helekar, B.S., Ruiz, C.C., Ohguro, H. Huang, J., Zhao, X., Crabb, J.W., Johnson, R.S., Walsh, K.A., Gray-Keller, M.P., Detwiler, P.B., and Baehr, W. (1994) Neuron 13, 395-404) and other members of the recovering family of Ca(2+)-binding proteins. Antibodies against a truncated fusion protein and against a p24-specific synthetic peptide specifically recognize retinal p24 on immunoblot. Both antibodies inhibit activation of photoreceptor membrane guanylyl cyclase by purified p24. p24 is found only in retina, and it copurifies with outer segment membranes. Immunocytochemical analysis shows that it is present in rod photoreceptor cells. An immobilized antibody column was used to purify p24 from a heat-treated retinal extract. Purified p24 appears on SDS-polyacrylamide gel electrophoresis as a homogeneous protein not contaminated with GCAP, and it activates photoreceptor guanylyl cyclase in vitro at submicromolar concentrations. Ca2+ inhibits this activation with an EC50 near 200 nM and a Hill coefficient of 1.7. Recombinant p24 expressed in 293 cells effectively stimulates photoreceptor guanylyl cyclase. These findings demonstrate that p24, like GCAP, imparts Ca2+ sensitivity to photoreceptor membrane guanylyl cyclase. We propose that p24 be referred to as GCAP-2 and that GCAP be referred to as

  5. Heavy isotope labeling study of the turnover of forskolin-stimulated adenylate cyclase in BC/sup 3/H1 cell line

    SciTech Connect

    Bouhelal, R.; Bockaert, J.; Mermet-Bouvier, R.; Guillon, G.; Homburger, V.

    1987-06-25

    We have used the method of heavy isotope labeling to study the metabolic turnover of adenylate cyclase in a nonfusing muscle cell line, the BC/sup 3/H1 cells. These cells contains an adenylate cyclase coupled to beta-adrenergic receptors and highly stimulated by forskolin, a potent activator of the enzyme. After transfer of the cells from normal medium to heavy medium (a medium containing heavy labeled amino acids, /sup 3/H, /sup 13/C, /sup 15/N), heavy isotope-labeled adenylate cyclase molecules progressively replace pre-existing light molecules. In sucrose gradient differential sedimentation, after a 5-day switch in heavy medium, the enzyme exhibited a higher mass (s = 8.40 +/- 0.03 S, n = 13) compared to the control enzyme. Indeed, the increase in the sedimentation coefficient of the heavy molecules was due to the synthesis of new molecules of adenylate cyclase labeled with heavy isotope amino acids since in the presence of cycloheximide, an inhibitor of protein synthesis, no change in the sedimentation pattern of the forskolin-stimulated adenylate cyclase occurred. After incorporation of heavy isotope amino acids in the adenylate cyclase molecules, the kinetics parameters of the enzyme did not change. However, adenylate cyclase from cells incubated with heavy medium exhibits an activity about 2-fold lower than control. After switching the cells to the heavy medium, the decrease of the activity of the enzyme occurred during the first 24 h and thereafter remained at a steady state for at least 4 days. In contrast, 24 h after the switch, the sedimentation coefficient of forskolin-stimulated adenylate cyclase was progressively shifted to a higher value.

  6. Cloning and bacterial expression of sesquiterpene cyclase, a key branch point enzyme for the synthesis of sesquiterpenoid phytoalexin capsidiol in UV-challenged leaves of Capsicum annuum.

    PubMed

    Back, K; He, S; Kim, K U; Shin, D H

    1998-09-01

    Sesquiterpene cyclase, a branch point enzyme in the general isoprenoid pathway for the synthesis of phytoalexin capsidiol, was induced in detached leaves of Capsicum annuum (pepper) by UV treatment. The inducibility of cyclase enzyme activities paralleled the absolute amount of cyclase protein(s) of pepper immunodetected by monoclonal antibodies raised against tobacco sesquiterpene cyclase. A cDNA library was constructed with poly(A)+ RNA isolated from 24 h UV-challenged leaves of pepper. A cDNA clone for sesquiterpene cyclase in pepper was isolated by using a tobacco 5-epi aristolochene synthase gene as a heterologous probe. The predicted protein encoded by this cDNA was comprised of 559 amino acids and had a relative molecular mass of 65,095. The primary structural information from the cDNA clone revealed that it shared 77%, 72% and 49% identity with 5-epi aristolochene, vetispiradiene, and cadinene synthase, respectively. The enzymatic product catalyzed by the cDNA clone in bacteria was identified as 5-epi aristolochene, as judged by argentation TLC. RNA blot hybridization demonstrated the induction of an mRNA consistent with the induction of cyclase enzyme activity in UV-treated pepper. PMID:9816674

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

    PubMed

    Al-Younis, Inas; Wong, Aloysius; Gehring, Chris

    2015-12-21

    Adenylate cyclases (ACs) catalyse the formation of the second messenger cyclic adenosine 3',5'-monophosphate (cAMP) from adenosine 5'-triphosphate (ATP). Although cAMP is increasingly recognised as an important signalling 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 AtKUP7(1-100) 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 AtKUP7(1-100) generates cAMP in vitro. PMID:26638082

  8. Molecular characterization of an oxidosqualene cyclase that yields shionone, a unique tetracyclic triterpene ketone of Aster tataricus.

    PubMed

    Sawai, Satoru; Uchiyama, Hiroshi; Mizuno, Syuhei; Aoki, Toshio; Akashi, Tomoyoshi; Ayabe, Shin-ichi; Takahashi, Takeyoshi

    2011-04-01

    Shionone is the major triterpenoid component of Aster tataricus possessing a unique all six-membered tetracyclic skeleton and 3-oxo-4-monomethyl structure. To clarify its biosynthetic process, an oxidosqualene cyclase cDNA was isolated from A. tataricus, and the function of the enzyme was determined in lanosterol synthase-deficient yeast. The cyclase yielded ca. 90% shionone and small amounts of β-amyrin, friedelin, dammara-20,24-dienol, and 4-epishionone and was designated as a shionone synthase (SHS). Transcripts of SHS were detected in A. tataricus organs, confirming its involvement in shionone biosynthesis. SHS was shown to have evolved in the Asteraceae from β-amyrin synthase lineages and acquired characteristic species- and product-specificities.

  9. Crystal structure of a squalene cyclase in complex with the potential anticholesteremic drug Ro48-8071.

    PubMed

    Lenhart, Alexander; Weihofen, Wilhelm A; Pleschke, Axel E W; Schulz, Georg E

    2002-05-01

    Squalene-hopene cyclase (SHC) catalyzes the conversion of squalene into pentacyclic compounds. It is the prokaryotic counterpart of the eukaryotic oxidosqualene cyclase (OSC) that catalyzes the steroid scaffold formation. Because of clear sequence homology, SHC can serve as a model for OSC, which is an attractive target for anticholesteremic drugs. We have established the crystal structure of SHC complexed with Ro48-8071, a potent inhibitor of OSC and therefore of cholesterol biosynthesis. Ro48-8071 is bound in the active-center cavity of SHC and extends into the channel that connects the cavity with the membrane. The binding site of Ro48-8071 is largely identical with the expected site of squalene; it differs from a previous model based on photoaffinity labeling. The knowledge of the inhibitor binding mode in SHC is likely to help develop more potent inhibitors for OSC.

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

    PubMed Central

    Kim, Hyo Kyung

    2016-01-01

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

  11. Isolation and functional characterization of Lycopene β-cyclase (CYC-B) promoter from Solanum habrochaites

    PubMed Central

    2010-01-01

    Background Carotenoids are a group of C40 isoprenoid molecules that play diverse biological and ecological roles in plants. Tomato is an important vegetable in human diet and provides the vitamin A precursor β-carotene. Genes encoding enzymes involved in carotenoid biosynthetic pathway have been cloned. However, regulation of genes involved in carotenoid biosynthetic pathway and accumulation of specific carotenoid in chromoplasts are not well understood. One of the approaches to understand regulation of carotenoid metabolism is to characterize the promoters of genes encoding proteins involved in carotenoid metabolism. Lycopene β-cyclase is one of the crucial enzymes in carotenoid biosynthesis pathway in plants. Its activity is required for synthesis of both α-and β-carotenes that are further converted into other carotenoids such as lutein, zeaxanthin, etc. This study describes the isolation and characterization of chromoplast-specific Lycopene β-cyclase (CYC-B) promoter from a green fruited S. habrochaites genotype EC520061. Results A 908 bp region upstream to the initiation codon of the Lycopene β-cyclase gene was cloned and identified as full-length promoter. To identify promoter region necessary for regulating developmental expression of the ShCYC-B gene, the full-length promoter and its three different 5' truncated fragments were cloned upstream to the initiation codon of GUS reporter cDNA in binary vectors. These four plant transformation vectors were separately transformed in to Agrobacterium. Agrobacterium-mediated transient and stable expression systems were used to study the GUS expression driven by the full-length promoter and its 5' deletion fragments in tomato. The full-length promoter showed a basal level activity in leaves, and its expression was upregulated > 5-fold in flowers and fruits in transgenic tomato plants. Deletion of -908 to -577 bp 5' to ATG decreases the ShCYC-B promoter strength, while deletion of -908 to -437 bp 5' to ATG led to

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2008-01-01

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

  14. Protein-Protein Docking and Analysis Reveal That Two Homologous Bacterial Adenylyl Cyclase Toxins Interact with Calmodulin Differently*S⃞

    PubMed Central

    Guo, Qing; Jureller, Justin E.; Warren, Julia T.; Solomaha, Elena; Florián, Jan; Tang, Wei-Jen

    2008-01-01

    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 (∼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 β-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. PMID:18583346

  15. Receptor number and caveolar co-localization determine receptor coupling efficiency to adenylyl cyclase.

    PubMed

    Ostrom, R S; Gregorian, C; Drenan, R M; Xiang, Y; Regan, J W; Insel, P A

    2001-11-01

    Recent evidence suggests that many signaling molecules localize in microdomains of the plasma membrane, particularly caveolae. In this study, overexpression of adenylyl cyclase was used as a functional probe of G protein-coupled receptor (GPCR) compartmentation. We found that three endogenous receptors in neonatal rat cardiomyocytes couple with different levels of efficiency to the activation of adenylyl cyclase type 6 (AC6), which localizes to caveolin-rich membrane fractions. Overexpression of AC6 enhanced the maximal cAMP response to beta(1)-adrenergic receptor (beta(1)AR)-selective activation 3.7-fold, to beta(2)AR-selective activation only 1.6-fold and to prostaglandin E(2) (PGE(2)) not at all. Therefore, the rank order of efficacy in coupling to AC6 is beta(1)AR > beta(2)AR > prostaglandin E(2) receptor (EP(2)R). beta(2)AR coupling efficiency was greater when we overexpressed the receptor or blocked its desensitization by expressing betaARKct, an inhibitor of G protein-coupled receptor kinase activation, but was not significantly greater when cells were treated with pertussis toxin. Assessment of receptor and AC expression indicated co-localization of AC5/6, beta(1)AR, and beta(2)AR, but not EP(2)R, in caveolin-rich membranes and caveolin-3 immunoprecipitates, likely explaining the observed activation of AC6 by betaAR subtypes but lack thereof by PGE(2). When cardiomyocytes were stimulated with a betaAR agonist, beta(2)AR were no longer found in caveolin-3 immunoprecipitates; an effect that was blocked by expression of betaARKct. Thus, agonist-induced translocation of beta(2)AR out of caveolae causes a sequestration of receptor from effector and likely contributes to the lower efficacy of beta(2)AR coupling to AC6 as compared with beta(1)AR, which do not similarly translocate. Therefore, spatial co-localization is a key determinant of efficiency of coupling by particular extracellular signals to activation of GPCR-linked effectors. PMID:11533056

  16. Extracellular Regulation of Sperm Transmembrane Adenylyl Cyclase by a Forward Motility Stimulating Protein

    PubMed Central

    Dey, Souvik; Roy, Debarun; Majumder, Gopal C.; Bhattacharyya, Debdas

    2014-01-01

    Forward motility stimulating factor (FMSF), a glycoprotein isolated from buffalo serum, binds to the surface of the mature sperm cells to promote their progressive motility. This article reports the mode of signal transduction of this extracellular factor in goat sperm. The mechanism was investigated by assaying intracellular second messenger level and forward motility in presence of different pharmacological modulators. Mg++-dependent Forskolin responsive form of transmembrane adenylyl cyclase (tmAC) of goat spermatozoa was probed for its involvement in FMSF action. Dideoxyadenosine, a selective inhibitor of tmACs, was used to identify the role of this enzyme in the scheme of FMSF-signaling. Involvement of the α-subunit of G-protein in this regard has been inspected using GTPγS. Participation of protein kinase A (PKA) and tyrosine kinase was checked using IP20 and genistein, respectively. FMSF promotes tmAC activity in a dose-dependent manner through receptor/G-protein activation to enhance intracellular cAMP and forward motility. Motility boosting effects of this glycoprotein are almost lost in presence of dideoxyadenosine. But, FMSF displayed substantial motility promoting activity when movement of spermatozoa was inhibited with KH7, the specific inhibitor of soluble adenylyl cyclase indicating tmAC to be the primary target of FMSF action. Involvement of cAMP in mediating FMSF action was confirmed by the application of dibutyryl cAMP. Observed motility regulatory effects with IP20 and genistein indicate contribution of PKA and tyrosine kinase in FMSF activity; enhanced phosphorylation of a tyrosine containing ≈50 kDa protein was detected in this regard. FMSF initiates a novel signaling cascade to stimulate tmAC activity that augments intracellular cAMP, which through downstream crosstalk of phosphokinases leads to enhanced forward motility in mature spermatozoa. Thus, this article for the first time describes conventional tmAC-dependent profound activation

  17. Receptor number and caveolar co-localization determine receptor coupling efficiency to adenylyl cyclase.

    PubMed

    Ostrom, R S; Gregorian, C; Drenan, R M; Xiang, Y; Regan, J W; Insel, P A

    2001-11-01

    Recent evidence suggests that many signaling molecules localize in microdomains of the plasma membrane, particularly caveolae. In this study, overexpression of adenylyl cyclase was used as a functional probe of G protein-coupled receptor (GPCR) compartmentation. We found that three endogenous receptors in neonatal rat cardiomyocytes couple with different levels of efficiency to the activation of adenylyl