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Sample records for adp receptor p2y12

  1. Identification of Determinants Required for Agonistic and Inverse Agonistic Ligand Properties at the ADP Receptor P2Y12

    PubMed Central

    Schmidt, Philipp; Ritscher, Lars; Dong, Elizabeth N.; Hermsdorf, Thomas; Cöster, Maxi; Wittkopf, Doreen; Meiler, Jens

    2013-01-01

    The ADP receptor P2Y12 belongs to the superfamily of G protein–coupled receptors (GPCRs), and its activation triggers platelet aggregation. Therefore, potent antagonists, such as clopidogrel, are of high clinical relevance in prophylaxis and treatment of thromboembolic events. P2Y12 displays an elevated basal activity in vitro, and as such, inverse agonists may be therapeutically beneficial compared with antagonists. Only a few inverse agonists of P2Y12 have been described. To expand this limited chemical space and improve understanding of structural determinants of inverse agonist-receptor interaction, this study screened a purine compound library for lead structures using wild-type (WT) human P2Y12 and 28 constitutively active mutants. Results showed that ATP and ATP derivatives are agonists at P2Y12. The potency at P2Y12 was 2-(methylthio)-ADP > 2-(methylthio)-ATP > ADP > ATP. Determinants required for agonistic ligand activity were identified. Molecular docking studies revealed a binding pocket for the ATP derivatives that is bordered by transmembrane helices 3, 5, 6, and 7 in human P2Y12, with Y105, E188, R256, Y259, and K280 playing a particularly important role in ligand interaction. N-Methyl-anthraniloyl modification at the 3′-OH of the 2′-deoxyribose leads to ligands (mant-deoxy-ATP [dATP], mant-deoxy-ADP) with inverse agonist activity. Inverse agonist activity of mant-dATP was found at the WT human P2Y12 and half of the constitutive active P2Y12 mutants. This study showed that, in addition to ADP and ATP, other ATP derivatives are not only ligands of P2Y12 but also agonists. Modification of the ribose within ATP can result in inverse activity of ATP-derived ligands. PMID:23093496

  2. P2Y12-ADP receptor antagonists: Days of future and past

    PubMed Central

    Laine, Marc; Paganelli, Franck; Bonello, Laurent

    2016-01-01

    Antiplatelet therapy is the cornerstone of the therapeutic arsenal in coronary artery disease. Thanks to a better understanding in physiology, pharmacology and pharmacogenomics huge progress were made in the field of platelet reactivity inhibition thus allowing the expansion of percutaneous coronary intervention. Stent implantation requires the combination of two antiplatelet agents acting in a synergistic way. Asprin inhibit the cyclo-oxygenase pathway of platelet activation while clopidogrel is a P2Y12 adenosine diphosphate (ADP)-receptor antagonist. This dual antiplatelet therapy has dramatically improved the prognosis of stented patients. However, due to pharmacological limitations of clopidogrel (interindividual variability in its biological efficacy, slow onset of action, mild platelet reactivity inhibition) ischemic recurrences remained high following stent implantation especially in acute coronary syndrome patients. Thus, more potent P2Y12-ADP receptor inhibitors were developped including prasugrel, ticagrelor and more recently cangrelor to overcome these pitfalls. These new agents reduced the rate of thrombotic events in acute coronary syndrome patients at the cost of an increased bleeding risk. The abundance in antiplatelet agents allow us to tailor our strategy based on the thrombotic/bleeding profile of each patient. Recently, the ACCOAST trial cast a doubt on the benefit of pre treatment in non-ST segment elevation acute coronary syndrome. The aim of the present review is to summarize the results of the main studies dealing with antiplatelet therapy in stented/acute coronary syndromes patients. PMID:27231519

  3. P2Y12-ADP receptor antagonists: Days of future and past.

    PubMed

    Laine, Marc; Paganelli, Franck; Bonello, Laurent

    2016-05-26

    Antiplatelet therapy is the cornerstone of the therapeutic arsenal in coronary artery disease. Thanks to a better understanding in physiology, pharmacology and pharmacogenomics huge progress were made in the field of platelet reactivity inhibition thus allowing the expansion of percutaneous coronary intervention. Stent implantation requires the combination of two antiplatelet agents acting in a synergistic way. Asprin inhibit the cyclo-oxygenase pathway of platelet activation while clopidogrel is a P2Y12 adenosine diphosphate (ADP)-receptor antagonist. This dual antiplatelet therapy has dramatically improved the prognosis of stented patients. However, due to pharmacological limitations of clopidogrel (interindividual variability in its biological efficacy, slow onset of action, mild platelet reactivity inhibition) ischemic recurrences remained high following stent implantation especially in acute coronary syndrome patients. Thus, more potent P2Y12-ADP receptor inhibitors were developped including prasugrel, ticagrelor and more recently cangrelor to overcome these pitfalls. These new agents reduced the rate of thrombotic events in acute coronary syndrome patients at the cost of an increased bleeding risk. The abundance in antiplatelet agents allow us to tailor our strategy based on the thrombotic/bleeding profile of each patient. Recently, the ACCOAST trial cast a doubt on the benefit of pre treatment in non-ST segment elevation acute coronary syndrome. The aim of the present review is to summarize the results of the main studies dealing with antiplatelet therapy in stented/acute coronary syndromes patients. PMID:27231519

  4. Agonist-bound structure of the human P2Y12 receptor

    PubMed Central

    Zhang, Jin; Zhang, Kaihua; Gao, Zhan-Guo; Paoletta, Silvia; Zhang, Dandan; Han, Gye Won; Li, Tingting; Ma, Limin; Zhang, Wenru; Müller, Christa E.; Yang, Huaiyu; Jiang, Hualiang; Cherezov, Vadim; Katritch, Vsevolod; Jacobson, Kenneth A.; Stevens, Raymond C.; Wu, Beili; Zhao, Qiang

    2014-01-01

    The P2Y12 receptor (P2Y12R), one of eight members of the P2YR family expressed in humans, has been identified as one of the most prominent clinical drug targets for inhibition of platelet aggregation. Consequently, extensive mutagenesis and modeling studies of the P2Y12R have revealed many aspects of agonist/antagonist binding1-4. However, the details of agonist and antagonist recognition and function at the P2Y12R remain poorly understood at the molecular level. Here, we report the structures of the human P2Y12R in complex with a full agonist 2-methylthio-adenosine-5′-diphosphate (2MeSADP, a close analogue of endogenous agonist ADP) at 2.5 Å resolution, and the corresponding ATP derivative 2-methylthio-adenosine-5′-triphosphate (2MeSATP) at 3.1 Å resolution. Analysis of these structures, together with the structure of the P2Y12R with antagonist ethyl 6-(4-((benzylsulfonyl)carbamoyl)piperidin-1-yl)-5-cyano-2-methylnicotinate (AZD1283)5, reveals dramatic conformational changes between nucleotide and non-nucleotide ligand complexes in the extracellular regions, providing the first insight into a different ligand binding landscape in the δ-group of class A G protein-coupled receptors (GPCRs). Agonist and non-nucleotide antagonist adopt different orientations in the P2Y12R, with only partially overlapped binding pockets. The agonist-bound P2Y12R structure answers long-standing ambiguities surrounding P2Y12R-agonist recognition, and reveals interactions with several residues that had not been reported to be involved in agonist binding. As a first example of a GPCR where agonist access to the binding pocket requires large scale rearrangements in the highly malleable extracellular region, the structural studies therefore will provide invaluable insight into the pharmacology and mechanisms of action of agonists and different classes of antagonists for the P2Y12R and potentially for other closely related P2YRs. PMID:24784220

  5. Contribution of the P2Y12 receptor-mediated pathway to platelet hyperreactivity in hypercholesterolemia

    PubMed Central

    Nagy, Béla; Jin, Jianguo; Ashby, Barrie; Reilly, Michael P.; Kunapuli, Satya P.

    2011-01-01

    Summary Background In hypercholesterolemia, platelets demonstrate increased reactivity and promote the development of cardiovascular disease. Objective This study was carried out to investigate the contribution of the ADP receptor P2Y12-mediated pathway in platelet hyperreactivity due to hypercholesterolemia. Methods Low-density lipoprotein receptor deficient mice and C57Bl/6 wild type mice were fed on normal chow and high-fat (Western or Paigen) diets for 8 weeks to generate differently elevated cholesterol levels. P2Y12 receptor induced functional responses via Gi signaling were studied ex vivo when washed murine platelets were activated by 2MeSADP and PAR4 agonist AYPGKF in the presence and absence of indomethacin. Platelet aggregation, secretion, αIIbβ3 receptor activation and the phosphorylation of extracellular signal-regulated protein kinase (ERK) and Akt were analyzed. Results Plasma cholesterol levels ranged from 69±10 to 1011±185 mg/dl depending on diet in mice with different genotypes. Agonist-dependent aggregation, dense and α-granule secretion and JON/A binding were gradually and significantly (P < 0.05) augmented at low agonist concentration in correlation with the increasing plasma cholesterol levels even if elevated thromboxane generation was blocked. These functional responses were induced via increased level of Gi mediated ERK and Akt phosphorylation in hypercholesterolemic mice versus normocholesterolemic animals. In addition, blocking of the P2Y12 receptor by AR-C69931MX (Cangrelor) resulted in strongly reduced platelet aggregation in mice with elevated cholesterol levels compared to normocholesterolemic controls. Conclusions These data revealed that the P2Y12 receptor pathway was substantially involved in platelet hyperreactivity associated with mild and severe hypercholesterolemia. PMID:21261805

  6. Platelet P2Y12 receptors enhance signalling towards procoagulant activity and thrombin generation. A study with healthy subjects and patients at thrombotic risk.

    PubMed

    van der Meijden, Paola E J; Feijge, Marion A H; Giesen, Peter L A; Huijberts, Maya; van Raak, Lisette P M; Heemskerk, Johan W M

    2005-06-01

    Activated platelets participate in arterial thrombosis by forming aggregates and potentiating the coagulation through exposure of procoagulant phosphatidylserine. The function of the two receptors for ADP, P2Y(1) and P2Y(12), is well-established in aggregation, but is incompletely understood in the platelet procoagulant response. We established that, in PRP from healthy subjects, ADP accelerated and potentiated tissue factor induced thrombin generation exclusively via stimulation of P2Y(12) and not via P2Y(1) receptors. The P2Y(12) receptors also mediated the potentiating effect of PAR-1 stimulation on thrombin generation. Furthermore, ADP enhanced in a P2Y(12)-dependent manner the Ca(2+) response induced by thrombin, which was either added externally or generated in-situ. This ADP effect was in part dependent of phosphoinositide 3-kinase and was paralleled by increased phosphatidylserine exposure. In PRP from (young) patients with either stroke or type-II diabetes, platelet-dependent thrombin generation was similarly enhanced byADP or SFLLRN as in healthy subjects. In PRP from stroke patients of older age, the P2Y(12)-mediated contribution to thrombin generation was variably reduced by two weeks of clopidogrel medication. Remaining P2Y(12) activity after medication correlated with remaining P2Y(12)-dependent P-selectin exposure, i.e. Ca(2+)-dependent secretion, likely due to incomplete antagonism of P2Y(12) receptors. Together, these results indicate that physiological platelet agonists amplify phosphatidylserine exposure and subsequent thrombin generation by release of ADP and P2Y(12)-receptor stimulation. This P2Y(12) response is accomplished by a novel Ca(2+) signalling pathway. It is similarly active in platelets from control subjects and patients at thrombotic risk. Finally, the thrombogram method is useful for measuring incomplete P2Y(12) inhibition with clopidogrel. PMID:15968399

  7. Microglial P2Y12 Receptors Regulate Microglial Activation and Surveillance during Neuropathic Pain

    PubMed Central

    Gu, Nan; Eyo, Ukpong B.; Murugan, Madhuvika; Peng, Jiyun; Matta, Sanjana; Dong, Hailong; Wu, Long-Jun

    2016-01-01

    Microglial cells are critical in the pathogenesis of neuropathic pain and several microglial receptors have been proposed to mediate this process. Of these receptors, the P2Y12 receptor is a unique purinergic receptor that is exclusively expressed by microglia in the central nervous system (CNS). In this study, we set forth to investigate the role of P2Y12 receptors in microglial electrophysiological and morphological (static and dynamic) activation during spinal nerve transection (SNT)-induced neuropathic pain in mice. First, we found that a genetic deficiency of the P2Y12 receptor (P2Y12−/− mice) ameliorated pain hypersensitivities during the initiation phase of neuropathic pain. Next, we characterized both the electrophysiological and morphological properties of microglia in the superficial spinal cord dorsal horn following SNT injury. We show dramatic alterations including a peak at 3 days post injury in microglial electrophysiology while high resolution two-photon imaging revealed significant changes of both static and dynamic microglial morphological properties by 7 days post injury. Finally, in P2Y12−/− mice, these electrophysiological and morphological changes were ameliorated suggesting roles for P2Y12 receptors in SNT-induced microglial activation. Our results therefore indicate that P2Y12 receptors regulate microglial electrophysiological as well as static and dynamic microglial properties after peripheral nerve injury, suggesting that the microglial P2Y12 receptor could be a potential therapeutic target for the treatment of neuropathic pain. PMID:26576724

  8. Identification of a New Morpholine Scaffold as a P2Y12 Receptor Antagonist.

    PubMed

    Ahn, Young Ha; Lee, Joo-Youn; Park, Hee Dong; Kim, Tae Hun; Park, Min Chul; Choi, Gildon; Kim, Sunghoon

    2016-01-01

    The P2Y12 receptor is critical for platelet activation and is an attractive drug target for the prevention of atherothrombotic events. Despite the proven antithrombotic efficacy of P2Y12 inhibitors, these thienopyridine scaffolds are prodrugs that lack important features of the ideal antithrombotic agent. For this reason, ticagrelor-a new chemical class of P2Y12 receptor antagonist-was developed, but it can cause shortness of breath and various types of bleeding. Moreover, ticagrelor is a cytochrome P450 3A4 substrate/inhibitor and, therefore, caution should be exercised when it is used concomitantly with strong CYP3A4 inducers/inhibitors. There is a need for novel P2Y12 receptor antagonist scaffolds that are reversible and have high efficacy without associated side effects. Here, we describe a novel antagonist containing a morpholine moiety that was identified by screening libraries of commercially available compounds. The molecule, Compound E, acted on P2Y12, but not P2Y1 and P2Y13, and exhibited pharmacological characteristics that were distinct from those of ticagrelor, acting instead on P2Y12 via an allosteric mechanism. These results provide a basis for the development/optimization of a new class of P2Y12 antagonists. PMID:27563870

  9. Racial Differences in Resistance to P2Y12 Receptor Antagonists in Type 2 Diabetic Subjects

    PubMed Central

    Duvernay, Matthew T.; Holinstat, Michael; Colowick, Nancy E.; Hudson, Willie J.; Song, Yanna; Harrell, Frank E.

    2014-01-01

    Although resistance to the P2Y12 antagonist clopidogrel is linked to altered drug metabolism, some studies suggest that these pharmacokinetic abnormalities only partially account for drug resistance. To circumvent pharmacokinetic complications and target P2Y12 receptor function we applied the direct P2Y12 antagonist 2-methylthio-AMP (2-methylthioadenosine 5′-monophosphate triethylammonium salt) to purified platelets ex vivo. Platelets were purified from healthy and type 2 diabetes mellitus (T2DM) patients and stimulated with thrombin or the selective protease-activated receptor agonists, protease-activated receptor 1–activating peptide (PAR1-AP), or PAR4-AP. Platelet activation as measured by αIIbβ3 activation, and P-selectin expression was monitored in 141 subjects. Our results demonstrate that, compared with healthy subjects, platelets from diabetic patients are resistant to inhibition by 2-methylthio-AMP, demonstrating P2Y12 pharmacodynamic defects among diabetic patients. Inhibition of thrombin-mediated αIIbβ3 activation by 2-methylthio-AMP was lower in diabetic platelets versus healthy platelets. Subgroup analysis revealed a racial difference in the resistance to 2-methylthio-AMP. We found no resistance in platelets from diabetic African Americans; they were inhibited by 2-methylthio-AMP equally as well as platelets from healthy African Americans. In contrast, platelets from Caucasian patients with diabetes were resistant to P2Y12 antagonism compared with healthy Caucasians. Multivariable analysis demonstrated that other variables, such as obesity, age, or gender, could not account for the differential resistance to 2-methylthio-AMP among races. These results suggest that in addition to altered drug metabolism, P2Y12 receptor function itself is altered in the Caucasian diabetic population. The racial difference in platelet function in T2DM is a novel finding, which may lead to differences in treatment as well as new targets for antiplatelet therapy

  10. The active metabolite of Clopidogrel disrupts P2Y12 receptor oligomers and partitions them out of lipid rafts

    PubMed Central

    Savi, Pierre; Zachayus, Jean-Luc; Delesque-Touchard, Nathalie; Labouret, Catherine; Hervé, Caroline; Uzabiaga, Marie-Françoise; Pereillo, Jean-Marie; Culouscou, Jean-Michel; Bono, Françoise; Ferrara, Pascual; Herbert, Jean-Marc

    2006-01-01

    P2Y12, a G protein-coupled receptor that plays a central role in platelet activation has been recently identified as the receptor targeted by the antithrombotic drug, clopidogrel. In this study, we further deciphered the mechanism of action of clopidogrel and of its active metabolite (Act-Met) on P2Y12 receptors. Using biochemical approaches, we demonstrated the existence of homooligomeric complexes of P2Y12 receptors at the surface of mammalian cells and in freshly isolated platelets. In vitro treatment with Act-Met or in vivo oral administration to rats with clopidogrel induced the breakdown of these oligomers into dimeric and monomeric entities in P2Y12 expressing HEK293 and platelets respectively. In addition, we showed the predominant association of P2Y12 oligomers to cell membrane lipid rafts and the partitioning of P2Y12 out of rafts in response to clopidogrel and Act-Met. The raft-associated P2Y12 oligomers represented the functional form of the receptor, as demonstrated by binding and signal transduction studies. Finally, using a series of receptors individually mutated at each cysteine residue and a chimeric P2Y12/P2Y13 receptor, we pointed out the involvement of cysteine 97 within the first extracellular loop of P2Y12 in the mechanism of action of Act-Met. PMID:16835302

  11. P2Y12 receptor gene mutation associated with postoperative hemorrhage in a Greater Swiss Mountain dog.

    PubMed

    Boudreaux, Mary K; Martin, Monica

    2011-06-01

    A novel hereditary disorder of platelets was identified across 5 generations of a family of Greater Swiss Mountain dogs. The first dog identified with the mutation bled excessively following routine ovariohysterectomy and required multiple transfusions. Coagulation screening assays, platelet counts, and von Willebrand factor antigen activity were within reference intervals. Flow cytometric studies indicated that platelets from the affected dog expressed normal levels of glycoproteins IIb and IIIa and responded to 2 platelet-activating agents, convulxin and platelet-activating factor, but not to ADP. Based on DNA studies, a 3 base-pair deletion predicted to result in elimination of a serine from the extracellular domain was identified in the gene encoding P2Y12, an ADP receptor protein located on platelet membranes. Flow cytometric analysis of platelets and studies of DNA performed concurrently on 2 unrelated Greater Swiss Mountain dogs were unremarkable. The mutation was subsequently identified in the sire, the maternal grand-dam, a maternal great grandparent, a paternal great grandparent, and a great-great grandparent. The sire was homozygous, but had not yet been identified as having a hemostatic disorder; the other 4 dogs were carriers. This is the first report of a mutation in the gene encoding the ADP receptor P2Y12 in a domestic animal. P2Y12 is the same receptor targeted by ticlopidine and clopidogrel, platelet inhibitors used in lieu of aspirin in people at risk for cardiovascular disease; thus, spontaneous bleeding is not expected unless there are other contributing factors. This disorder is particularly troublesome because spontaneous hemorrhage is absent to mild in affected dogs; however, following routine surgical procedures or trauma, excessive bleeding could occur and have possible fatal consequences.

  12. P2Y12 Receptor Antagonists and Morphine: A Dangerous Liaison?

    PubMed

    Giannopoulos, Georgios; Deftereos, Spyridon; Kolokathis, Fotios; Xanthopoulou, Ioanna; Lekakis, John; Alexopoulos, Dimitrios

    2016-09-01

    P2Y12 receptor antagonists, concurrently administered with aspirin in what has come to be commonly called dual antiplatelet therapy, are a mainstay of treatment for patients with acute coronary syndromes. Morphine, on the contrary, is a commonly used drug in the acute phase of acute coronary syndromes to relieve pain-with the added potential benefit of attenuating acutely raised sympathetic tone. In current guidelines, though, morphine is recommended with decreasing strength of recommendation. One reason is that it raises concern regarding the potentially significant interaction with antiplatelet agents, leading to impaired inhibition of platelet activation. In any case, it is still considered a mandatory part of the inventory of available medications in prehospital acute myocardial infarction management. The goal of the present review is to present published evidence on morphine and its potential interactions with P2Y12 receptor antagonists, as well as on the central issue of whether such interactions may underlie clinically significant effects on patient outcomes.

  13. Modeling ligand recognition at the P2Y12 receptor in light of X-ray structural information.

    PubMed

    Paoletta, Silvia; Sabbadin, Davide; von Kügelgen, Ivar; Hinz, Sonja; Katritch, Vsevolod; Hoffmann, Kristina; Abdelrahman, Aliaa; Straßburger, Jens; Baqi, Younis; Zhao, Qiang; Stevens, Raymond C; Moro, Stefano; Müller, Christa E; Jacobson, Kenneth A

    2015-08-01

    The G protein-coupled P2Y12 receptor (P2Y12R) is an important antithrombotic target and of great interest for pharmaceutical discovery. Its recently solved, highly divergent crystallographic structures in complex either with nucleotides (full or partial agonist) or with a nonnucleotide antagonist raise the question of which structure is more useful to understand ligand recognition. Therefore, we performed extensive molecular modeling studies based on these structures and mutagenesis, to predict the binding modes of major classes of P2Y12R ligands previously reported. Various nucleotide derivatives docked readily to the agonist-bound P2Y12R, but uncharged nucleotide-like antagonist ticagrelor required a hybrid receptor resembling the agonist-bound P2Y12R except for the top portion of TM6. Supervised molecular dynamics (SuMD) of ticagrelor binding indicated interactions with the extracellular regions of P2Y12R, defining possible meta-binding sites. Ureas, sulfonylureas, sulfonamides, anthraquinones and glutamic acid piperazines docked readily to the antagonist-bound P2Y12R. Docking dinucleotides at both agonist- and antagonist-bound structures suggested interactions with two P2Y12R pockets. Thus, our structure-based approach consistently rationalized the main structure-activity relationships within each ligand class, giving useful information for designing improved ligands.

  14. P2Y12 Receptor Antagonists and Morphine: A Dangerous Liaison?

    PubMed

    Giannopoulos, Georgios; Deftereos, Spyridon; Kolokathis, Fotios; Xanthopoulou, Ioanna; Lekakis, John; Alexopoulos, Dimitrios

    2016-09-01

    P2Y12 receptor antagonists, concurrently administered with aspirin in what has come to be commonly called dual antiplatelet therapy, are a mainstay of treatment for patients with acute coronary syndromes. Morphine, on the contrary, is a commonly used drug in the acute phase of acute coronary syndromes to relieve pain-with the added potential benefit of attenuating acutely raised sympathetic tone. In current guidelines, though, morphine is recommended with decreasing strength of recommendation. One reason is that it raises concern regarding the potentially significant interaction with antiplatelet agents, leading to impaired inhibition of platelet activation. In any case, it is still considered a mandatory part of the inventory of available medications in prehospital acute myocardial infarction management. The goal of the present review is to present published evidence on morphine and its potential interactions with P2Y12 receptor antagonists, as well as on the central issue of whether such interactions may underlie clinically significant effects on patient outcomes. PMID:27586412

  15. The physical association of the P2Y12 receptor with PAR4 regulates arrestin-mediated Akt activation.

    PubMed

    Khan, Aasma; Li, Dongjun; Ibrahim, Salam; Smyth, Emer; Woulfe, Donna S

    2014-07-01

    It is now well accepted that protease activated receptor (PAR) 1 and PAR4 have differential roles in platelet activation. PAR4, a low-affinity thrombin receptor in human platelets, participates in sustained platelet activation in a P2Y12-dependent manner; however, the mechanisms are not defined. Our previous studies demonstrated that thrombin induces the association of PAR4 with P2Y12, together with arrestin recruitment to the complex. Here we show that PAR4 and P2Y12 directly interact to coregulate Akt signaling after PAR4 activation. We observed direct and specific interaction of P2Y12 with PAR4 but not PAR1 by bioluminescent resonance energy transfer when the receptors were coexpressed in human embryonic kidney 293T cells. PAR4-P2Y12 dimerization was promoted by PAR4-AP and inhibited by P2Y12 antagonist. By using sequence comparison of the transmembrane domains of PAR1 and PAR4, we designed a mutant form of PAR4, "PAR4SFT," by replacing LGL194-196 at the base of transmembrane domain 4 with the corresponding aligned PAR1 residues SFT 220-222. PAR4SFT supported only 8.74% of PAR4-P2Y12 interaction, abolishing P2Y12-dependent arrestin recruitment to PAR4 and Akt activation. Nonetheless, PAR4SFT still supported homodimerization with PAR4. PAR4SFT failed to induce a calcium flux when expressed independently; however, coexpression of increasing concentrations of PAR4SFT, together with PAR4 potentiated PAR4-mediated calcium flux, suggested that PAR4 act as homodimers to signal to Gq-coupled calcium responses. In conclusion, PAR4 LGL (194-196) governs agonist-dependent association of PAR4 with P2Y12 and contributes to Gq-coupled calcium responses. PAR4-P2Y12 association supports arrestin-mediated sustained signaling to Akt. Hence, PAR4-P2Y12 dimerization is likely to be important for the PAR4-P2Y12 dependent stabilization of platelet thrombi.

  16. The physical association of the P2Y12 receptor with PAR4 regulates arrestin-mediated Akt activation.

    PubMed

    Khan, Aasma; Li, Dongjun; Ibrahim, Salam; Smyth, Emer; Woulfe, Donna S

    2014-07-01

    It is now well accepted that protease activated receptor (PAR) 1 and PAR4 have differential roles in platelet activation. PAR4, a low-affinity thrombin receptor in human platelets, participates in sustained platelet activation in a P2Y12-dependent manner; however, the mechanisms are not defined. Our previous studies demonstrated that thrombin induces the association of PAR4 with P2Y12, together with arrestin recruitment to the complex. Here we show that PAR4 and P2Y12 directly interact to coregulate Akt signaling after PAR4 activation. We observed direct and specific interaction of P2Y12 with PAR4 but not PAR1 by bioluminescent resonance energy transfer when the receptors were coexpressed in human embryonic kidney 293T cells. PAR4-P2Y12 dimerization was promoted by PAR4-AP and inhibited by P2Y12 antagonist. By using sequence comparison of the transmembrane domains of PAR1 and PAR4, we designed a mutant form of PAR4, "PAR4SFT," by replacing LGL194-196 at the base of transmembrane domain 4 with the corresponding aligned PAR1 residues SFT 220-222. PAR4SFT supported only 8.74% of PAR4-P2Y12 interaction, abolishing P2Y12-dependent arrestin recruitment to PAR4 and Akt activation. Nonetheless, PAR4SFT still supported homodimerization with PAR4. PAR4SFT failed to induce a calcium flux when expressed independently; however, coexpression of increasing concentrations of PAR4SFT, together with PAR4 potentiated PAR4-mediated calcium flux, suggested that PAR4 act as homodimers to signal to Gq-coupled calcium responses. In conclusion, PAR4 LGL (194-196) governs agonist-dependent association of PAR4 with P2Y12 and contributes to Gq-coupled calcium responses. PAR4-P2Y12 association supports arrestin-mediated sustained signaling to Akt. Hence, PAR4-P2Y12 dimerization is likely to be important for the PAR4-P2Y12 dependent stabilization of platelet thrombi. PMID:24723492

  17. Central P2Y12 receptor blockade alleviates inflammatory and neuropathic pain and cytokine production in rodents.

    PubMed

    Horváth, Gergely; Gölöncsér, Flóra; Csölle, Cecilia; Király, Kornél; Andó, Rómeó D; Baranyi, Mária; Koványi, Bence; Máté, Zoltán; Hoffmann, Kristina; Algaier, Irina; Baqi, Younis; Müller, Christa E; Von Kügelgen, Ivar; Sperlágh, Beáta

    2014-10-01

    In this study the role of P2Y12 receptors (P2Y12R) was explored in rodent models of inflammatory and neuropathic pain and in acute thermal nociception. In correlation with their activity to block the recombinant human P2Y12R, the majority of P2Y12R antagonists alleviated mechanical hyperalgesia dose-dependently, following intraplantar CFA injection, and after partial ligation of the sciatic nerve in rats. They also caused an increase in thermal nociceptive threshold in the hot plate test. Among the six P2Y12R antagonists evaluated in the pain studies, the selective P2Y12 receptor antagonist PSB-0739 was most potent upon intrathecal application. P2Y12R mRNA and IL-1β protein were time-dependently overexpressed in the rat hind paw and lumbar spinal cord following intraplantar CFA injection. This was accompanied by the upregulation of TNF-α, IL-6 and IL-10 in the hind paw. PSB-0739 (0.3mg/kg i.t.) attenuated CFA-induced expression of cytokines in the hind paw and of IL-1β in the spinal cord. Subdiaphragmatic vagotomy and the α7 nicotinic acetylcholine receptor antagonist MLA occluded the effect of PSB-0739 (i.t.) on pain behavior and peripheral cytokine induction. Denervation of sympathetic nerves by 6-OHDA pretreatment did not affect the action of PSB-0739. PSB-0739, in an analgesic dose, did not influence motor coordination and platelet aggregation. Genetic deletion of the P2Y12R in mice reproduced the effect of P2Y12R antagonists on mechanical hyperalgesia in inflammatory and neuropathic pain models, on acute thermal nociception and on the induction of spinal IL-1β. Here we report the robust involvement of the P2Y12R in inflammatory pain. The anti-hyperalgesic effect of P2Y12R antagonism could be mediated by the inhibition of both central and peripheral cytokine production and involves α7-receptor mediated efferent pathways.

  18. Central P2Y12 receptor blockade alleviates inflammatory and neuropathic pain and cytokine production in rodents

    PubMed Central

    Horváth, Gergely; Gölöncsér, Flóra; Csölle, Cecilia; Király, Kornél; Andó, Rómeó D.; Baranyi, Mária; Koványi, Bence; Máté, Zoltán; Hoffmann, Kristina; Algaier, Irina; Baqi, Younis; Müller, Christa E.; Von Kügelgen, Ivar; Sperlágh, Beáta

    2014-01-01

    In this study the role of P2Y12 receptors (P2Y12R) was explored in rodent models of inflammatory and neuropathic pain and in acute thermal nociception. In correlation with their activity to block the recombinant human P2Y12R, the majority of P2Y12R antagonists alleviated mechanical hyperalgesia dose-dependently, following intraplantar CFA injection, and after partial ligation of the sciatic nerve in rats. They also caused an increase in thermal nociceptive threshold in the hot plate test. Among the six P2Y12R antagonists evaluated in the pain studies, the selective P2Y12 receptor antagonist PSB-0739 was most potent upon intrathecal application. P2Y12R mRNA and IL-1β protein were time-dependently overexpressed in the rat hind paw and lumbar spinal cord following intraplantar CFA injection. This was accompanied by the upregulation of TNF-α, IL-6 and IL-10 in the hind paw. PSB-0739 (0.3 mg/kg i.t.) attenuated CFA-induced expression of cytokines in the hind paw and of IL-1β in the spinal cord. Subdiaphragmatic vagotomy and the α7 nicotinic acetylcholine receptor antagonist MLA occluded the effect of PSB-0739 (i.t.) on pain behavior and peripheral cytokine induction. Denervation of sympathetic nerves by 6-OHDA pretreatment did not affect the action of PSB-0739. PSB-0739, in an analgesic dose, did not influence motor coordination and platelet aggregation. Genetic deletion of the P2Y12R in mice reproduced the effect of P2Y12R antagonists on mechanical hyperalgesia in inflammatory and neuropathic pain models, on acute thermal nociception and on the induction of spinal IL-1β. Here we report the robust involvement of the P2Y12R in inflammatory pain. The anti-hyperalgesic effect of P2Y12R antagonism could be mediated by the inhibition of both central and peripheral cytokine production and involves α7-receptor mediated efferent pathways. PMID:24971933

  19. Inhibition of PAR-4 and P2Y12 receptor-mediated platelet activation produces distinct hepatic pathologies in experimental xenobiotic-induced cholestatic liver disease.

    PubMed

    Joshi, Nikita; Kopec, Anna K; Ray, Jessica L; Luyendyk, James P

    2016-07-15

    Emerging evidence supports a protective effect of platelets in experimental cholestatic liver injury and cholangiofibrosis. Coagulation-mediated platelet activation has been shown to inhibit experimental chronic cholestatic liver necrosis and biliary fibrosis. This occurs through thrombin-mediated activation of protease activated receptor-4 (PAR-4) in mice. However, it is not known whether other pathways of platelet activation, such as adenosine diphosphate (ADP)-mediated receptor P2Y12 activation is also protective. We tested the hypothesis that inhibition of P2Y12-mediated platelet activation exacerbates hepatic injury and cholangiofibrosis, and examined the impact of P2Y12 inhibition in both the presence and absence of PAR-4. Treatment of wild-type mice with the P2Y12 receptor antagonist clopidogrel increased biliary hyperplasia and cholangiofibrosis in wild-type mice exposed to the xenobiotic alpha-naphthylisothiocyanate (ANIT) for 4 weeks compared to vehicle-treated mice exposed to ANIT. Interestingly, this effect of clopidogrel occurred without a corresponding increase in hepatocellular necrosis. Whereas biliary hyperplasia and cholangiofibrosis were increased in PAR-4(-/-) mice, clopidogrel treatment failed to further increase these pathologies in PAR-4(-/-) mice. The results indicate that inhibition of receptor P2Y12-mediated platelet activation exacerbates bile duct fibrosis in ANIT-exposed mice, independent of hepatocellular necrosis. Moreover, the lack of an added effect of clopidogrel administration on the exaggerated pathology in ANIT-exposed PAR-4(-/-) mice reinforces the prevailing importance of coagulation-mediated platelet activation in limiting this unique liver pathology. PMID:27475285

  20. P2Y12 receptor expression is a critical determinant of functional responsiveness to ATX's MORFO domain.

    PubMed

    Dennis, Jameel; Morgan, Magdalena K; Graf, Martin R; Fuss, Babette

    2012-06-01

    In the central nervous system, the formation of the myelin sheath and the differentiation of the myelinating cells, namely oligodendrocytes, are regulated by complex signaling networks that involve purinergic receptors and the extracellular matrix. However, the exact nature of the molecular interactions underlying these networks still needs to be defined. In this respect, the data presented here reveal a signaling mechanism that is characterized by an interaction between the purinergic P2Y(12) receptor and the matricellular extracellular matrix protein autotaxin (ATX), also known as ENPP2, phosphodiesterase-Iα/ATX, or lysoPLD. ATX has been previously described by us to mediate intermediate states of oligodendrocyte adhesion and to enable changes in oligodendrocyte morphology that are thought to be crucial for the formation of a fully functional myelin sheath. This functional property of ATX is mediated by ATX's modulator of oligodendrocyte remodeling and focal adhesion organization (MORFO) domain. Here, we show that the expression of the P2Y(12) receptor is necessary for ATX's MORFO domain to exert its effects on differentiating oligodendrocytes. In addition, our data demonstrate that exogenous expression of the P2Y(12) receptor can render cells responsive to the known effects of ATX's MORFO domain, and they identify Rac1 as an intracellular factor mediating the effect of ATX-MORFO-P2Y(12) signaling on the assembly of focal adhesions. Our data further support the idea that a physical interaction between ATX and the P2Y(12) receptor provides the basis for an ATX-MORFO-P2Y(12) signaling axis that is crucial for mediating cellular states of intermediate adhesion and morphological/structural plasticity.

  1. Bradykinin-induced Ca2+ signaling in human subcutaneous fibroblasts involves ATP release via hemichannels leading to P2Y12 receptors activation

    PubMed Central

    2013-01-01

    Background Chronic musculoskeletal pain involves connective tissue remodeling triggered by inflammatory mediators, such as bradykinin. Fibroblast cells signaling involve changes in intracellular Ca2+ ([Ca2+]i). ATP has been related to connective tissue mechanotransduction, remodeling and chronic inflammatory pain, via P2 purinoceptors activation. Here, we investigated the involvement of ATP in bradykinin-induced Ca2+ signals in human subcutaneous fibroblasts. Results Bradykinin, via B2 receptors, caused an abrupt rise in [Ca2+]i to a peak that declined to a plateau, which concentration remained constant until washout. The plateau phase was absent in Ca2+-free medium; [Ca2+]i signal was substantially reduced after depleting intracellular Ca2+ stores with thapsigargin. Extracellular ATP inactivation with apyrase decreased the [Ca2+]i plateau. Human subcutaneous fibroblasts respond to bradykinin by releasing ATP via connexin and pannexin hemichannels, since blockade of connexins, with 2-octanol or carbenoxolone, and pannexin-1, with 10Panx, attenuated bradykinin-induced [Ca2+]i plateau, whereas inhibitors of vesicular exocytosis, such as brefeldin A and bafilomycin A1, were inactive. The kinetics of extracellular ATP catabolism favors ADP accumulation in human fibroblast cultures. Inhibition of ectonucleotidase activity and, thus, ADP formation from released ATP with POM-1 or by Mg2+ removal from media reduced bradykinin-induced [Ca2+]i plateau. Selective blockade of the ADP-sensitive P2Y12 receptor with AR-C66096 attenuated bradykinin [Ca2+]i plateau, whereas the P2Y1 and P2Y13 receptor antagonists, respectively MRS 2179 and MRS 2211, were inactive. Human fibroblasts exhibited immunoreactivity against connexin-43, pannexin-1 and P2Y12 receptor. Conclusions Bradykinin induces ATP release from human subcutaneous fibroblasts via connexin and pannexin-1-containing hemichannels leading to [Ca2+]i mobilization through the cooperation of B2 and P2Y12 receptors. PMID

  2. Long-Term P2Y12-Receptor Antagonists in Post-Myocardial Infarction Patients: Facing a New Trilemma?

    PubMed

    Alexopoulos, Dimitrios; Xanthopoulou, Ioanna; Moulias, Athanasios; Lekakis, John

    2016-09-13

    Physicians considering prescription of P2Y12-receptor antagonist for long-term (>1 year) protection of patients post-myocardial infarction face the trilemma of selecting between clopidogrel, prasugrel, or ticagrelor. Differential ischemic benefits derived from relevant trials may assist in tailoring treatment, although the different bleeding definitions applied make any meaningful comparison of each agent's bleeding potential very difficult. Considering the available data and recognizing the significant limitation of observations obtained thus far from subgroup analyses, prasugrel appears to provide higher anti-ischemic protection than clopidogrel. Ticagrelor seems to be an attractive option for patients with renal dysfunction, peripheral arterial disease, or following a brief P2Y12-receptor antagonist interruption, whereas clopidogrel may be advised in the presence of cost and availability issues. As head-to-head comparative trials between P2Y12-receptor antagonists are lacking, selection of a specific agent by the clinician should be made on the basis of critical appraisal of available large clinical datasets. PMID:27609686

  3. Comparison of P2Y12 receptor inhibition by clopidogrel and prasugrel in patients undergoing percutaneous coronary intervention.

    PubMed

    Haq, M M; Ahsan, C H; Amin, M N; Karim, M R; Ali, M L; Khan, S R; Chowdhury, M Z; Mansur, M; Millat, M H; Rashid, M A

    2013-12-01

    Dual antiplatelet treatment (DAPT) with aspirin and clopidogrel is vital after percutaneous coronary intervention (PCI). Clopidogrel and prasugrel act on P2Y12 platelet surface receptors. Both these P2Y12 inhibitors are pro-drugs and depend on cytochrome system of the liver for their conversion to active metabolite. There is growing concern regarding suboptimal response in platelet inhibition by clopidogrel. Verify Now system got approval by Federal Drug Administration, USA, for assessing platelet function as its result is almost comparable to gold standard Light Transmission Aggregometry (LTA). There are no data on the prevalence of clopidogrel resistance in Bangladeshi population. Prasugrel, as an antiplatelet drug, is a newer introduction in this country. This study will show light on the efficacy of these drugs on our population especially in patients who undergo PCI where DAPT is mandatory. A total 120 (60 diabetics) patients with Acute Coronary Syndrome (ACS), were alternatively given 600 mg clopidogrel loading dose (LD) followed by 75 mg maintenance dose (MD) daily or 60 mg LD of prasugrel followed by 10 mg MD daily. Five samples of blood were taken at different time intervals over a period of 2 weeks. Measurement of percent inhibition of P2Y12 was done by VerifyNow. Patients who showed less than 20% inhibition (clopidogrel resistant) at any stage were switched to prasugrel. The outcomes of clopidogrel, prasugrel and clopidogrel switched to prasugrel groups were then compared. Nearly half (46.7%) of the patients in the clopidogrel group was found resistant to the drug as opposed to none in the prasugrel group. No difference was found between diabetic and non-diabetic subjects with respect to drug resistance. Intracoronary blood samples showed high degree of platelet inhibition with prasugrel. There was a gradual decline of platelet inhibition over two weeks with prasugrel. Almost fifty percent of the population is clopidogrel resistant in our study

  4. P2Y12 Receptor Localizes in the Renal Collecting Duct and Its Blockade Augments Arginine Vasopressin Action and Alleviates Nephrogenic Diabetes Insipidus.

    PubMed

    Zhang, Yue; Peti-Peterdi, Janos; Müller, Christa E; Carlson, Noel G; Baqi, Younis; Strasburg, David L; Heiney, Kristina M; Villanueva, Karie; Kohan, Donald E; Kishore, Bellamkonda K

    2015-12-01

    P2Y12 receptor (P2Y12-R) signaling is mediated through Gi, ultimately reducing cellular cAMP levels. Because cAMP is a central modulator of arginine vasopressin (AVP)-induced water transport in the renal collecting duct (CD), we hypothesized that if expressed in the CD, P2Y12-R may play a role in renal handling of water in health and in nephrogenic diabetes insipidus. We found P2Y12-R mRNA expression in rat kidney, and immunolocalized its protein and aquaporin-2 (AQP2) in CD principal cells. Administration of clopidogrel bisulfate, an irreversible inhibitor of P2Y12-R, significantly increased urine concentration and AQP2 protein in the kidneys of Sprague-Dawley rats. Notably, clopidogrel did not alter urine concentration in Brattleboro rats that lack AVP. Clopidogrel administration also significantly ameliorated lithium-induced polyuria, improved urine concentrating ability and AQP2 protein abundance, and reversed the lithium-induced increase in free-water excretion, without decreasing blood or kidney tissue lithium levels. Clopidogrel administration also augmented the lithium-induced increase in urinary AVP excretion and suppressed the lithium-induced increase in urinary nitrates/nitrites (nitric oxide production) and 8-isoprostane (oxidative stress). Furthermore, selective blockade of P2Y12-R by the reversible antagonist PSB-0739 in primary cultures of rat inner medullary CD cells potentiated the expression of AQP2 and AQP3 mRNA, and cAMP production induced by dDAVP (desmopressin). In conclusion, pharmacologic blockade of renal P2Y12-R increases urinary concentrating ability by augmenting the effect of AVP on the kidney and ameliorates lithium-induced NDI by potentiating the action of AVP on the CD. This strategy may offer a novel and effective therapy for lithium-induced NDI. PMID:25855780

  5. P2Y12 Receptor Localizes in the Renal Collecting Duct and Its Blockade Augments Arginine Vasopressin Action and Alleviates Nephrogenic Diabetes Insipidus.

    PubMed

    Zhang, Yue; Peti-Peterdi, Janos; Müller, Christa E; Carlson, Noel G; Baqi, Younis; Strasburg, David L; Heiney, Kristina M; Villanueva, Karie; Kohan, Donald E; Kishore, Bellamkonda K

    2015-12-01

    P2Y12 receptor (P2Y12-R) signaling is mediated through Gi, ultimately reducing cellular cAMP levels. Because cAMP is a central modulator of arginine vasopressin (AVP)-induced water transport in the renal collecting duct (CD), we hypothesized that if expressed in the CD, P2Y12-R may play a role in renal handling of water in health and in nephrogenic diabetes insipidus. We found P2Y12-R mRNA expression in rat kidney, and immunolocalized its protein and aquaporin-2 (AQP2) in CD principal cells. Administration of clopidogrel bisulfate, an irreversible inhibitor of P2Y12-R, significantly increased urine concentration and AQP2 protein in the kidneys of Sprague-Dawley rats. Notably, clopidogrel did not alter urine concentration in Brattleboro rats that lack AVP. Clopidogrel administration also significantly ameliorated lithium-induced polyuria, improved urine concentrating ability and AQP2 protein abundance, and reversed the lithium-induced increase in free-water excretion, without decreasing blood or kidney tissue lithium levels. Clopidogrel administration also augmented the lithium-induced increase in urinary AVP excretion and suppressed the lithium-induced increase in urinary nitrates/nitrites (nitric oxide production) and 8-isoprostane (oxidative stress). Furthermore, selective blockade of P2Y12-R by the reversible antagonist PSB-0739 in primary cultures of rat inner medullary CD cells potentiated the expression of AQP2 and AQP3 mRNA, and cAMP production induced by dDAVP (desmopressin). In conclusion, pharmacologic blockade of renal P2Y12-R increases urinary concentrating ability by augmenting the effect of AVP on the kidney and ameliorates lithium-induced NDI by potentiating the action of AVP on the CD. This strategy may offer a novel and effective therapy for lithium-induced NDI.

  6. Gene-by-environment effect of house dust mite on purinergic receptor P2Y12 (P2RY12) and lung function in children with asthma

    PubMed Central

    Bunyavanich, Supinda; Boyce, Joshua A.; Raby, Benjamin A.; Weiss, Scott T.

    2012-01-01

    Background Distinct receptors likely exist for leukotriene(LT)E4, a potent mediator of airway inflammation. Purinergic receptor P2Y12 is needed for LTE4-induced airways inflammation, and P2Y12 antagonism attenuates house dust mite-induced pulmonary eosinophilia in mice. Although experimental data support a role for P2Y12 in airway inflammation, its role in human asthma has never been studied. Objective To test for association between variants in the P2Y12 gene (P2RY12) and lung function in human subjects with asthma, and to examine for gene-by-environment interaction with house dust mite exposure. Methods 19 single nucleotide polymorphisms (SNPs) in P2RY12 were genotyped in 422 children with asthma and their parents (n=1266). Using family-based methods, we tested for associations between these SNPs and five lung function measures. We performed haplotype association analyses and tested for gene-by-environment interactions using house dust mite exposure. We used the false discovery rate to account for multiple comparisons. Results Five SNPs in P2RY12 were associated with multiple lung function measures (P values 0.006–0.025). Haplotypes in P2RY12 were also associated with lung function (P values 0.0055–0.046). House dust mite exposure modulated associations between P2RY12 and lung function, with minor allele homozygotes exposed to house dust mite demonstrating worse lung function than those unexposed (significant interaction P values 0.0028–0.040). Conclusions and clinical relevance P2RY12 variants were associated with lung function in a large family-based asthma cohort. House dust mite exposure caused significant gene-by-environment effects. Our findings add the first human evidence to experimental data supporting a role for P2Y12 in lung function. P2Y12 could represent a novel target for asthma treatment. PMID:22010907

  7. Differential endosomal sorting of a novel P2Y12 purinoreceptor mutant.

    PubMed

    Cunningham, Margaret R; Nisar, Shaista P; Cooke, Alexandra E; Emery, Elizabeth D; Mundell, Stuart J

    2013-05-01

    P2Y12 receptor internalization and recycling play an essential role in ADP-induced platelet activation. Recently, we identified a patient with a mild bleeding disorder carrying a heterozygous mutation of P2Y12 (P341A) whose P2Y12 receptor recycling was significantly compromised. Using human cell line models, we identified key proteins regulating wild-type (WT) P2Y12 recycling and investigated P2Y12 -P341A receptor traffic. Treatment with ADP resulted in delayed Rab5-dependent internalization of P341A when compared with WT P2Y12 . While WT P2Y12 rapidly recycled back to the membrane via Rab4 and Rab11 recycling pathways, limited P341A recycling was observed, which relied upon Rab11 activity. Although minimal receptor degradation was evident, P341A was localized in Rab7-positive endosomes with considerable agonist-dependent accumulation in the trans-Golgi network (TGN). Rab7 activity is known to facilitate recruitment of retromer complex proteins to endosomes to transport cargo to the TGN. Here, we identified that P341A colocalized with Vps26; depletion of which blocked limited recycling and promoted receptor degradation. This study has identified key points of divergence in the endocytic traffic of P341A versus WT-P2Y12 . Given that these pathways are retained in human platelets, this research helps define the molecular mechanisms regulating P2Y12 receptor traffic and explain the compromised receptor function in the platelets of the P2Y12 -P341A-expressing patient.

  8. From ATP to AZD6140: the discovery of an orally active reversible P2Y12 receptor antagonist for the prevention of thrombosis.

    PubMed

    Springthorpe, Brian; Bailey, Andrew; Barton, Patrick; Birkinshaw, Timothy N; Bonnert, Roger V; Brown, Roger C; Chapman, David; Dixon, John; Guile, Simon D; Humphries, Robert G; Hunt, Simon F; Ince, Francis; Ingall, Anthony H; Kirk, Ian P; Leeson, Paul D; Leff, Paul; Lewis, Richard J; Martin, Barrie P; McGinnity, Dermot F; Mortimore, Michael P; Paine, Stuart W; Pairaudeau, Garry; Patel, Anil; Rigby, Aaron J; Riley, Robert J; Teobald, Barry J; Tomlinson, Wendy; Webborn, Peter J H; Willis, Paul A

    2007-11-01

    Starting from adenosine triphosphate (ATP), the identification of a novel series of P2Y(12) receptor antagonists and exploitation of their SAR is described. Modifications of the acidic side chain and the purine core and investigation of hydrophobic substituents led to a series of neutral molecules. The leading compound, 17 (AZD6140), is currently in a large phase III clinical trial for the treatment of acute coronary syndromes and prevention of thromboembolic clinical sequelae.

  9. A model-based analysis of the clinical and economic impact of personalising P2Y12-receptor inhibition with platelet function testing in acute coronary syndrome patients.

    PubMed

    Straub, Niels; Beivers, Andreas; Lenk, Ekaterina; Aradi, Daniel; Sibbing, Dirk

    2014-02-01

    Although some observational studies reported that the measured level of P2Y12-inhibition is predictive for thrombotic events, the clinical and economic benefit of incorporating PFT to personalize P2Y12-receptor directed antiplatelet treatment is unknown. Here, we assessed the clinical impact and cost-effectiveness of selecting P2Y12-inhibitors based on platelet function testing (PFT) in acute coronary syndrome (ACS) patients undergoing PCI. A decision model was developed to analyse the health economic effects of different strategies. PFT-guided treatment was compared with the three options of general clopidogrel, prasugrel or ticagrelor treatment. In the PFT arm, low responders to clopidogrel received prasugrel, while normal responders carried on with clopidogrel. The associated endpoints in the model were cardiovascular death, stent thrombosis and major bleeding. With a simulated cohort of 10,000 patients treated for one year, there were 93 less events in the PFT arm compared to general clopidogrel. In prasugrel and ticagrelor arms, 110 and 86 events were prevented compared to clopidogrel treatment, respectively. The total expected costs (including event costs, drug costs and PFT costs) for generic clopidogrel therapy were US$ 1,059/patient. In the PFT arm, total costs were US$ 1,494, while in the prasugrel and ticagrelor branches they were US$ 3,102 and US$ 3,771, respectively. The incremental-cost-effectiveness-ratio (ICER) was US$ 46,770 for PFT-guided therapy, US$ 185,783 for prasugrel and US$ 315,360 for ticagrelor. In this model-based analysis, a PFT-guided therapy may have fewer adverse outcomes than general treatment with clopidogrel and may be more cost-effective than prasugrel or ticagrelor treatment in ACS patients undergoing PCI.

  10. The involvement of P2Y12 receptors, NADPH oxidase, and lipid rafts in the action of extracellular ATP on synaptic transmission at the frog neuromuscular junction.

    PubMed

    Giniatullin, A; Petrov, A; Giniatullin, R

    2015-01-29

    Adenosine 5'-triphosphate (ATP) is the main co-transmitter accompanying the release of acetylcholine from motor nerve terminals. Previously, we revealed the direct inhibitory action of extracellular ATP on transmitter release via redox-dependent mechanism. However, the receptor mechanism of ATP action and ATP-induced sources of reactive oxygen sources (ROS) remained not fully understood. In the current study, using microelectrode recordings of synaptic currents from the frog neuromuscular junction, we analyzed the receptor subtype involved in synaptic action of ATP, receptor coupling to NADPH oxidase and potential location of ATP receptors within the lipid rafts. Using subtype-specific antagonists, we found that the P2Y13 blocker 2-[(2-chloro-5-nitrophenyl)azo]-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-4-pyridinecarboxaldehyde did not prevent the depressant action of ATP. In contrast, the P2Y12 antagonist 2-methylthioadenosine 5'-monophosphate abolished the inhibitory action of ATP, suggesting the key role of P2Y12 receptors in ATP action. As the action of ATP is redox-dependent, we also tested potential involvement of the NADPH oxidase, known as a common inducer of ROS. The depressant action of extracellular ATP was significantly reduced by diphenyleneiodonium chloride and 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, two structurally different inhibitors of NADPH oxidase, indicating that this enzyme indeed mediates the action of ATP. Since the location and activity of various receptors are often associated with lipid rafts, we next tested whether ATP-driven inhibition depends on lipid rafts. We found that the disruption of lipid rafts with methyl-beta-cyclodextrin reduced and largely delayed the action of ATP. Taken together, these data revealed key steps in the purinergic control of synaptic transmission via P2Y12 receptors associated with lipid rafts, and identified NADPH oxidase as the main source of ATP-induced inhibitory ROS at the neuromuscular

  11. Characteristics of new P2Y12 inhibitors: selection of P2Y12 inhibitors in clinical practice.

    PubMed

    Golino, Paolo

    2013-12-01

    The options for antithrombotic therapy have recently been expanded, facilitating optimal tailored treatment. Dual antiplatelet therapy with aspirin and an approved adenosine diphosphate P2Y12 receptor antagonist is recommended for the management of patients with acute coronary syndromes (ACS). However, there are a number of controversies: which P2Y12 inhibitor to choose; how long should antiplatelet therapy be used so as to prevent thrombotic events and minimize bleeding risks; whether to use drug-eluting (DES) or bare-metal stents (BMS) and how to manage the individual variability in response to clopidogrel. Clopidogrel in combination with aspirin has been the standard dual antiplatelet regimen for ACS. The new, more potent P2Y12 inhibitors, prasugrel and ticagrelor, have shown improved antithrombotic effects compared with clopidogrel in patients with ACS (with or without ST-segment elevation myocardial infarction) in landmark trials, even if they were associated with an increased risk of major bleeding. Different pharmacogenetic and pharmacodynamic characteristics may explain, in part, the different pharmacologic and clinical responses to these antiplatelet agents. Importantly, both clopidogrel and prasugrel are prodrugs, i.e., they need to be converted in vivo into active metabolites that selectively and irreversibly bind the P2Y12 receptor. Unlike clopidogrel, however, common functional cytochrome P450 genetic variants do not affect prasugrel active metabolite levels or inhibition of platelet aggregation. In contrast, ticagrelor is not a prodrug (i.e., does not require hepatic metabolism to exert its antiplatelet effect) and represents the first oral P2Y12 receptor antagonist that is reversibly bound. Similar to prasugrel, ticagrelor achieves greater and more rapid inhibition of platelet function than clopidogrel. Evidence suggests that the new P2Y12 antagonists may offer improved antithrombotic effects compared with clopidogrel in selected patients for the

  12. BF0801, a novel adenine derivative, inhibits platelet activation via phosphodiesterase inhibition and P2Y12 antagonism.

    PubMed

    Zhang, Si; Hu, Liang; Du, Hongguang; Guo, Yan; Zhang, Yan; Niu, Haixia; Jin, Jianguo; Zhang, Jian; Liu, Junling; Zhang, Xiaohui; Kunapuli, Satya P; Ding, Zhongren

    2010-10-01

    Though antiplatelet drugs are proven beneficial to patients with coronary heart disease and stroke, more effective and safer antiplatelet drugs are still needed. In this study we report the antiplatelet effects and mechanism of BF0801, a novel adenine derivative. BF0801 dramatically inhibited platelet aggregation and ATP release induced by ADP, 2MeSADP, AYPGKF, SFLLRN or convulxin without affecting shape change in vitro . It also potentiated the inhibitory effects of adenosine-based P2Y12 antagonist AR-C69931MX or phosphodiesterase (PDE) inhibitor IBMX on platelet aggregation. The cAMP levels in both resting and forskolin-stimulated platelets were increased by BF0801 suggesting its PDE inhibitor activity, which is further confirmed by the concentration-dependent suppression of BF0801 on the native and recombinant PDE. Similar to AR-C69931MX, BF0801 drastically inhibited 2MeSADP- induced adenylyl cyclase inhibition in platelets indicating its P2Y12 antagonism activity, which is substantiated by the inhibition of BF0801 on the interaction between ADP and P2Y12 receptor expressed in CHO-K1 cells measured by atomic force microscopy. Moreover, we confirmed the antiplatelet effects of BF0801 using platelets from rats intravenously given BF0801. In summary, for the first time we developed a novel adenine derivative bearing dual activities of PDE inhibition and P2Y12 antagonism, which may have therapeutic advantage as a potential antithrombotic drug. PMID:20806121

  13. Optimal timing of initiation of oral P2Y12-receptor antagonist therapy in patients with non-ST elevation acute coronary syndromes. Lessons learnt from the ACCOAST-trial.

    PubMed

    Zeymer, Uwe; Montalescot, Gilles; Ardissino, Diego; Bolognese, Leonardo; Clemmensen, Peter; Collet, Jean-Philippe; Lopez-Sendon, Jose; Widimsky, Petr

    2016-06-01

    The optimal time-point of the initiation of P2Y12 antagonist therapy in patients with non-ST elevation acute coronary syndromes (NTSE-ACS) is still a matter of debate. European guidelines recommend P2Y12 as soon as possible after first medical contact. However, the only trial which compared the two strategies did not demonstrate any benefit of pre-treatment with prasugrel before angiography compared to starting therapy after angiography and just prior to percutaneous coronary intervention (PCI). This paper summarizes the results of pharmacodynamic and previous studies, and gives recommendations for the initiation of P2Y12 antagonist therapy in NSTE-ACS in different clinical situations. PMID:25921481

  14. Membrane coordination of receptors and channels mediating the inhibition of neuronal ion currents by ADP.

    PubMed

    Gafar, Hend; Dominguez Rodriguez, Manuel; Chandaka, Giri K; Salzer, Isabella; Boehm, Stefan; Schicker, Klaus

    2016-09-01

    ADP and other nucleotides control ion currents in the nervous system via various P2Y receptors. In this respect, Cav2 and Kv7 channels have been investigated most frequently. The fine tuning of neuronal ion channel gating via G protein coupled receptors frequently relies on the formation of higher order protein complexes that are organized by scaffolding proteins and harbor receptors and channels together with interposed signaling components. However, ion channel complexes containing P2Y receptors have not been described. Therefore, the regulation of Cav2.2 and Kv7.2/7.3 channels via P2Y1 and P2Y12 receptors and the coordination of these ion channels and receptors in the plasma membranes of tsA 201 cells have been investigated here. ADP inhibited currents through Cav2.2 channels via both P2Y1 and P2Y12 receptors with phospholipase C and pertussis toxin-sensitive G proteins being involved, respectively. The nucleotide controlled the gating of Kv7 channels only via P2Y1 and phospholipase C. In fluorescence energy transfer assays using conventional as well as total internal reflection (TIRF) microscopy, both P2Y1 and P2Y12 receptors were found juxtaposed to Cav2.2 channels, but only P2Y1, and not P2Y12, was in close proximity to Kv7 channels. Using fluorescence recovery after photobleaching in TIRF microscopy, evidence for a physical interaction was obtained for the pair P2Y12/Cav2.2, but not for any other receptor/channel combination. These results reveal a membrane juxtaposition of P2Y receptors and ion channels in parallel with the control of neuronal ion currents by ADP. This juxtaposition may even result in apparent physical interactions between receptors and channels.

  15. An intact PDZ motif is essential for correct P2Y12 purinoceptor traffic in human platelets.

    PubMed

    Nisar, Shaista; Daly, Martina E; Federici, Augusto B; Artoni, Andrea; Mumford, Andrew D; Watson, Stephen P; Mundell, Stuart J

    2011-11-17

    The platelet P2Y(12) purinoceptor (P2Y(12)R), which plays a crucial role in hemostasis, undergoes internalization and subsequent recycling to maintain receptor responsiveness, processes that are essential for normal platelet function. Here, we observe that P2Y(12)R function is compromised after deletion or mutation of the 4 amino acids at the extreme C-terminus of this receptor (ETPM), a putative postsynaptic density 95/disc large/zonula occludens-1 (PDZ)-binding motif. In cell line models, removal of this sequence or mutation of one of its core residues (P341A), attenuates receptor internalization and receptor recycling back to the membrane, thereby blocking receptor resensitization. The physiologic significance of these findings in the regulation of platelet function is shown by identification of a patient with a heterozygous mutation in the PDZ binding sequence of their P2Y(12)R (P341A) that is associated with reduced expression of the P2Y(12)R on the cell surface. Importantly, platelets from this subject showed significantly compromised P2Y(12)R recycling, emphasizing the importance of the extreme C-terminus of this receptor to ensure correct receptor traffic. PMID:21937696

  16. High platelet reactivity after P2Y12-inhibition in patients with atrial fibrillation and coronary stenting.

    PubMed

    Rilinger, Jonathan; Meyer, Melanie; Schnabel, Katharina; Weik, Patrick; Charlet, Anne; Esser, Jennifer S; Zhou, Qian; Bode, Christoph; Moser, Martin; Diehl, Philipp; Olivier, Christoph B

    2016-11-01

    High platelet reactivity (HPR) after P2Y12-inhibition in patients undergoing coronary stenting is associated with an increased risk for thromboembolic events and coronary death. So far it is not known how HPR affects the clinical outcome of different treatment strategies in patients with atrial fibrillation (AF) undergoing coronary stenting. In this single centre, observational study the antiplatelet effect of P2Y12-inhibitors in AF patients undergoing coronary stenting was investigated using impedance aggregometry. Patients received either dual antiplatelet therapy (DAPT) or triple therapy (TT). HPR was defined as the ratio of ADP-to TRAP-induced aggregation (r-ADP-agg) ≥50 %. Thromboembolic and bleeding events were assessed within the first 30 days after stenting. Out of 910 screened patients 167 patients were available for the present analysis. HPR was found in 5 of 43 (12 %) patients treated with DAPT and in 18 of 124 (15 %) patients treated with TT. In patients receiving TT, HPR was not a risk factor for thromboembolic events compared to patients with adequate response to P2Y12-inhibitors (6 vs. 8 %, p = 0.712). There was a trend for less bleeding events in patients with HPR compared to r-ADP-agg <50 % in the TT group (0 vs. 16 %, p = 0.077). Our data suggest that HPR after P2Y12-antagonism in patients receiving TT due to AF and coronary stenting might protect from bleeding without increasing thromboembolic risk. Future studies will need to investigate if patients with AF receiving coronary stenting benefit from a reduction of antithrombotic therapy.

  17. Microglial P2Y12 is necessary for synaptic plasticity in mouse visual cortex.

    PubMed

    Sipe, G O; Lowery, R L; Tremblay, M-È; Kelly, E A; Lamantia, C E; Majewska, A K

    2016-01-01

    Microglia are the resident immune cells of the brain. Increasingly, they are recognized as important mediators of normal neurophysiology, particularly during early development. Here we demonstrate that microglia are critical for ocular dominance plasticity. During the visual critical period, closure of one eye elicits changes in the structure and function of connections underlying binocular responses of neurons in the visual cortex. We find that microglia respond to monocular deprivation during the critical period, altering their morphology, motility and phagocytic behaviour as well as interactions with synapses. To explore the underlying mechanism, we focused on the P2Y12 purinergic receptor, which is selectively expressed in non-activated microglia and mediates process motility during early injury responses. We find that disrupting this receptor alters the microglial response to monocular deprivation and abrogates ocular dominance plasticity. These results suggest that microglia actively contribute to experience-dependent plasticity in the adolescent brain. PMID:26948129

  18. Microglial P2Y12 is necessary for synaptic plasticity in mouse visual cortex

    PubMed Central

    Sipe, G. O.; Lowery,, R. L.; Tremblay, M-È; Kelly, E. A.; Lamantia, C. E.; Majewska, A. K.

    2016-01-01

    Microglia are the resident immune cells of the brain. Increasingly, they are recognized as important mediators of normal neurophysiology, particularly during early development. Here we demonstrate that microglia are critical for ocular dominance plasticity. During the visual critical period, closure of one eye elicits changes in the structure and function of connections underlying binocular responses of neurons in the visual cortex. We find that microglia respond to monocular deprivation during the critical period, altering their morphology, motility and phagocytic behaviour as well as interactions with synapses. To explore the underlying mechanism, we focused on the P2Y12 purinergic receptor, which is selectively expressed in non-activated microglia and mediates process motility during early injury responses. We find that disrupting this receptor alters the microglial response to monocular deprivation and abrogates ocular dominance plasticity. These results suggest that microglia actively contribute to experience-dependent plasticity in the adolescent brain. PMID:26948129

  19. The impact of P2Y12 promoter DNA methylation on the recurrence of ischemic events in Chinese patients with ischemic cerebrovascular disease

    PubMed Central

    Li, Xin-Gang; Ma, Ning; Wang, Bo; Li, Xiao-Qing; Mei, Sheng-Hui; Zhao, Kun; Wang, Yong-Jun; Li, Wei; Zhao, Zhi-Gang; Sun, Shu-Sen; Miao, Zhong-Rong

    2016-01-01

    The primary mechanism of clopidogrel resistance is still unclear. We aimed to investigate whether the methylation status of the P2Y12 promoter has effects on platelet function and clinical ischemic events. Patients with ischemic cerebrovascular disease were enrolled into our study. Venous blood samples were drawn for thrombelastograpy (TEG) and active metabolite assay. Patients were divided into a case- or control-group based on the occurrence of ischemic events during a one year follow-up. Two TEG parameters between the case and control groups were statistically significant [ADP inhibition rate (ADP%): P = 0.018; ADP-induced platelet-fibrin clot strength (MAADP): P = 0.030]. The concentrations of clopidogrel active metabolite had no significant difference (P = 0.281). Sixteen CpG dinucleotides on P2Y12 promoter were tested. Three CpG sites (CpG11 and CpG12 + 13) showed lower methylation status, which correlated with a strong association with increased risk of clinical events. Changes of MAADP and ADP% were also associated with methylation levels of CpG 11 and CpG 12 + 13. Hypomethylation of the P2Y12 promoter is associated with a higher platelet reactivity and increased risk of ischemic events in our patients. Methylation analysis of peripheral blood samples might be a novel molecular marker to help early identification of patients at high risk for clinical ischemic events. PMID:27686864

  20. 4-((R)-2-{[6-((S)-3-Methoxypyrrolidin-1-yl)-2-phenylpyrimidine-4-carbonyl]amino}-3-phosphonopropionyl)piperazine-1-carboxylic Acid Butyl Ester (ACT-246475) and Its Prodrug (ACT-281959), a Novel P2Y12 Receptor Antagonist with a Wider Therapeutic Window in the Rat Than Clopidogrel.

    PubMed

    Caroff, Eva; Hubler, Francis; Meyer, Emmanuel; Renneberg, Dorte; Gnerre, Carmela; Treiber, Alexander; Rey, Markus; Hess, Patrick; Steiner, Beat; Hilpert, Kurt; Riederer, Markus A

    2015-12-10

    Recent post hoc analyses of several clinical trials with P2Y12 antagonists showed the need for new molecules being fully efficacious as antiplatelet agents and having a reduced propensity to cause major bleeding. We have previously reported the discovery of the 2-phenylpyrimidine-4-carboxamide analogs as P2Y12 antagonists with nanomolar potency in the disease-relevant platelet aggregation assay in human plasma. Herein we present the optimization steps that led to the discovery of clinical candidate ACT-246475 (30d). The key step was the replacement of the carboxylic acid functionality by a phosphonic acid group which delivered the most potent molecules of the program. In addition, low in vivo clearance in rat and dog was achieved for the first time. Since the bioavailability of 30d was low in rat and dog, we developed the bis((isopropoxycarbonyl)oxy)methyl ester prodrug (ACT-281959, 45). Compound 30d showed efficacy in the rat ferric chloride thrombosis model when administered intravenously as parent or orally as its prodrug 45. Moreover, 30d displays a wider therapeutic window as compared to clopidogrel in the rat surgical blood loss model.

  1. Protein kinase C and P2Y12 take center stage in thrombin-mediated activation of mammalian target of rapamycin complex 1 in human platelets

    PubMed Central

    Moore, S F; Hunter, R W; Hers, I

    2014-01-01

    Background Rapamycin, an inhibitor of mammalian target of rapamycin complex-1 (mTORC1), reduces platelet spreading, thrombus stability, and clot retraction. Despite an important role of mTORC1 in platelet function, little is known about how it is regulated. The objective of this study was to determine the signaling pathways that regulate mTORC1 in human platelets. Methods Mammalian target of rapamycin complex-1 activation was assessed by measuring the phosphorylation of its downstream substrate ribosomal S6 kinase 1 (p70S6K). Results Thrombin or the protein kinase C (PKC) activator phorbal 12-myristate 13-acetate stimulated activation of mTORC1 in a PKC-dependent, Akt-independent manner that correlated with phosphorylation of tuberin/tuberous sclerosis 2 (TSC2) (Ser939 and Thr1462). In contrast, insulin-like growth factor 1 (IGF-1)–stimulated TSC2 phosphorylation was completely dependent on phosphoinositide 3 kinase (PI3 kinase)/Akt but did not result in any detectable mTORC1 activation. Early (Ser939 and Thr1462) and late (Thr1462) TSC2 phosphorylation in response to thrombin were directly PKC dependent, whereas later TSC2 (Ser939) and p70S6K phosphorylation were largely dependent on paracrine signaling through P2Y12. PKC-mediated adenosine diphosphate (ADP) secretion was essential for thrombin-stimulated mTORC1 activation, as (i) ADP rescued p70S6K phosphorylation in the presence of a PKC inhibitor and (ii) P2Y12 antagonism prevented thrombin-mediated mTORC1 activation. Rescue of mTORC1 activation with exogenous ADP was completely dependent on the Src family kinases but independent of PI3 kinase/Akt. Interestingly, although inhibition of Src blocked the ADP rescue, it had little effect on thrombin-stimulated p70S6K phosphorylation under conditions where PKC was not inhibited. Conclusion These results demonstrate that thrombin activates the mTORC1 pathway in human platelets through PKC-mediated ADP secretion and subsequent activation of P2Y12, in a manner

  2. Reviewing the controversy surrounding pre-treatment with P2Y12 inhibitors in acute coronary syndrome patients.

    PubMed

    Capodanno, Davide; Angiolillo, Dominick J

    2016-07-01

    Pretreatment with oral P2Y12 inhibitors occurs each time clopidogrel, prasugrel, ticagrelor are given to patients with suspected coronary artery disease before definition of the coronary anatomy. In acute coronary syndromes, the practice of administering oral P2Y12 inhibitors upstream has been the object of significant controversy in recent years, following the publication of two trials of pretreatment in non-ST-segment elevation acute coronary syndromes and ST-segment elevation myocardial infarction, respectively. The introduction in the market of cangrelor - the first intravenous P2Y12 inhibitor - represents a new opportunity but also a new challenge for clinicians. This article reviews current recommendations and supporting evidence surrounding pretreatment with oral and intravenous P2Y12 inhibitors in patients with acute coronary syndromes. PMID:26953527

  3. Contemporary registries on P2Y12 inhibitors in patients with acute coronary syndromes in Europe: overview and methodological considerations.

    PubMed

    Jukema, J Wouter; Lettino, Maddalena; Widimský, Petr; Danchin, Nicolas; Bardaji, Alfredo; Barrabes, Jose A; Cequier, Angel; Claeys, Marc J; De Luca, Leonardo; Dörler, Jakob; Erlinge, David; Erne, Paul; Goldstein, Patrick; Koul, Sasha M; Lemesle, Gilles; Lüscher, Thomas F; Matter, Christian M; Montalescot, Gilles; Radovanovic, Dragana; Lopez-Sendón, Jose; Tousek, Petr; Weidinger, Franz; Weston, Clive F M; Zaman, Azfar; Zeymer, Uwe

    2015-10-01

    Patient registries that document real-world clinical experience play an important role in cardiology as they complement the data from randomized controlled trials, provide valuable information on drug use and clinical outcomes, and evaluate to what extent guidelines are followed in practice. The Platelet Inhibition Registry in ACS EvalUation Study (PIRAEUS) project is an initiative of registry holders who are managing national or international registries observing patients with acute coronary syndromes (ACS). The aim of PIRAEUS is to systematically compare and combine available information/insights from various European ACS registries with a focus on P2Y12 inhibitors. The present publication introduces the 17 participating registries in a narrative and tabular form, and describes which ACS groups and which dual antiplatelet therapies were investigated. It sets the basis for upcoming publications that will focus on effectiveness and safety of the antiplatelets used. PMID:27532447

  4. Trends in P2Y12 Inhibitor Use in Patients Referred for Invasive Evaluation of Coronary Artery Disease in Contemporary US Practice.

    PubMed

    Fan, Weihong; Plent, Stephanie; Prats, Jayne; Deliargyris, Efthymios N

    2016-05-01

    Practice in patients undergoing invasive evaluation for coronary artery disease is variable regarding choice of P2Y12 inhibitor and timing of treatment initiation and is usually dictated by institutional or even individual operator preference. Limited data are available on the actual patterns of P2Y12 inhibitor use in contemporary practice in the United States. We used electronic medical records from the Cerner "Health Facts" database of adults who underwent coronary angiography with or without percutaneous coronary intervention (PCI) from January 2008 to June 2013 and who received a loading dose of clopidogrel, prasugrel, or ticagrelor at any time from 48 hours before the start of procedure up to 6 hours after. Timing of P2Y12 inhibitor administration was categorized as >2 hours before, 0 to 2 hours before (pretreatment groups), or after the start of procedure. Results were also evaluated according to type of P2Y12 inhibitor and patient clinical presentation. A total of 37,964 patients underwent coronary angiography, and 28,306 proceeded to PCI. Pretreatment with a P2Y12 inhibitor was observed in 28% and 23% in the overall and PCI populations, respectively. Moderate variability of pretreatment rates was noted relative to clinical presentation and P2Y12 inhibitor type. Pretreatment rates remained fairly constant over time with the exception of a decreasing trend with prasugrel. In conclusion, among patients referred for invasive evaluation of coronary artery disease, P2Y12 inhibitor pretreatment was low in contemporary US practice, an observation consistent over time and for all available agents and clinical presentations.

  5. G-Protein–Coupled Receptors Signaling Pathways in New Antiplatelet Drug Development

    PubMed Central

    Gurbel, Paul A.; Kuliopulos, Athan; Tantry, Udaya S.

    2016-01-01

    Platelet G-protein–coupled receptors influence platelet function by mediating the response to various agonists, including ADP, thromboxane A2, and thrombin. Blockade of the ADP receptor, P2Y12, in combination with cyclooxygenase-1 inhibition by aspirin has been among the most widely used pharmacological strategies to reduce cardiovascular event occurrence in high-risk patients. The latter dual pathway blockade strategy is one of the greatest advances in the field of cardiovascular medicine. In addition to P2Y12, the platelet thrombin receptor, protease activated receptor-1, has also been recently targeted for inhibition. Blockade of protease activated receptor-1 has been associated with reduced thrombotic event occurrence when added to a strategy using P2Y12 and cyclooxygenase-1 inhibition. At this time, the relative contributions of these G-protein–coupled receptor signaling pathways to in vivo thrombosis remain incompletely defined. The observation of treatment failure in ≈10% of high-risk patients treated with aspirin and potent P2Y12 inhibitors provides the rationale for targeting novel pathways mediating platelet function. Targeting intracellular signaling downstream from G-protein–coupled receptor receptors with phosphotidylionisitol 3-kinase and Gq inhibitors are among the novel strategies under investigation to prevent arterial ischemic event occurrence. Greater understanding of the mechanisms of G-protein–coupled receptor–mediated signaling may allow the tailoring of antiplatelet therapy. PMID:25633316

  6. The active metabolite of prasugrel inhibits ADP-stimulated thrombo-inflammatory markers of platelet activation: Influence of other blood cells, calcium, and aspirin.

    PubMed

    Frelinger, Andrew L; Jakubowski, Joseph A; Li, Youfu; Barnard, Marc R; Fox, Marsha L; Linden, Matthew D; Sugidachi, Atsuhiro; Winters, Kenneth J; Furman, Mark I; Michelson, Alan D

    2007-07-01

    The novel thienopyridine prodrug prasugrel, a platelet P2Y(12) ADP receptor antagonist, requires in vivo metabolism for activity. Although pharmacological data have been collected on the effects of prasugrel on platelet aggregation, there are few data on the direct effects of the prasugrel's active metabolite, R-138727, on other aspects of platelet function. Here we examined the effects of R-138727 on thrombo-inflammatory markers of platelet activation, and the possible modulatory effects of other blood cells, calcium, and aspirin. Blood (PPACK or citrate anticoagulated) from healthy donors pre- and post-aspirin was incubated with R-138727 and the response to ADP assessed in whole blood or platelet-rich plasma (PRP) by aggregometry and flow cytometric analysis of leukocyte-platelet aggregates, platelet surface P-selectin, and GPIIb-IIIa activation. Low-micromolar concentrations of R-138727 resulted in a rapid and consistent inhibition of these ADP-stimulated thrombo-inflammatory markers. These rapid kinetics required physiological calcium levels, but were largely unaffected by aspirin. Lower IC(50) values in whole blood relative to PRP suggested that other blood cells affect ADP-induced platelet activation and hence the net inhibition by R-138727. R-138727 did not inhibit P2Y(12)-mediated ADP-induced shape change, even at concentrations that completely inhibited platelet aggregation, confirming the specificity of R-138727 for P2Y(12). In conclusion, R-138727, the active metabolite of prasugrel, results in rapid, potent, consistent, and selective inhibition of P2Y(12)-mediated up-regulation of thrombo-inflammatory markers of platelet activation. This inhibition is enhanced in the presence other blood cells and calcium, but not aspirin. PMID:17598013

  7. P2Y12 Inhibitor Pre-Treatment in Non-ST-Elevation Acute Coronary Syndrome: A Decision-Analytic Model.

    PubMed

    Gunton, James; Hartshorne, Trent; Langrish, Jeremy; Chuang, Anthony; Chew, Derek

    2016-01-01

    Current guidelines recommend initiation of a P2Y12 inhibitor for all patients with non-ST-elevation acute coronary syndrome (NSTE-ACS) at the time of diagnosis (pre-treatment); however, there are no randomized trials directly comparing pre-treatment with initiation at the time of angiography to support this practice. We explore clinical and institutional parameters potentially associated with benefit with this strategy in a decision-analytic model based on available evidence from randomised trials. A decision analysis model was constructed comparing three P2Y12 inhibitors in addition to aspirin in patients with NSTE-ACS. Based on clinical trial data, the cumulative probability of 30 day mortality, myocardial infarction (MI) and major bleeding were determined, and used to calculate the net clinical benefit (NCB) with and without pre-treatment. Sensitivity analysis was performed to assess the relationship between NCB and baseline ischemic risk, bleeding risk, time to angiography and local surgical revascularization rates. Pre-treatment with ticagrelor and clopidogrel was associated with a greater than 50% likelihood of providing a >1% increase in 30 day NCB when baseline estimated ischemic risk exceeds 11% and 14%, respectively. Prasugrel pre-treatment did not achieve a greater than 50% probability of an increase in NCB regardless of baseline ischemic risk. Institutional surgical revascularization rates and time to coronary angiography did not correlate with the likelihood of benefit from P2Y12 pre-treatment. In conclusion, pre-treatment with P2Y12 inhibition is unlikely to be beneficial to the majority of patients presenting with NSTE-ACS. A tailored assessment of each patient's individual ischemic and bleeding risk may identify those likely to benefit. PMID:27548237

  8. P2Y12 Inhibitor Pre-Treatment in Non-ST-Elevation Acute Coronary Syndrome: A Decision-Analytic Model

    PubMed Central

    Gunton, James; Hartshorne, Trent; Langrish, Jeremy; Chuang, Anthony; Chew, Derek

    2016-01-01

    Current guidelines recommend initiation of a P2Y12 inhibitor for all patients with non-ST-elevation acute coronary syndrome (NSTE-ACS) at the time of diagnosis (pre-treatment); however, there are no randomized trials directly comparing pre-treatment with initiation at the time of angiography to support this practice. We explore clinical and institutional parameters potentially associated with benefit with this strategy in a decision-analytic model based on available evidence from randomised trials. A decision analysis model was constructed comparing three P2Y12 inhibitors in addition to aspirin in patients with NSTE-ACS. Based on clinical trial data, the cumulative probability of 30 day mortality, myocardial infarction (MI) and major bleeding were determined, and used to calculate the net clinical benefit (NCB) with and without pre-treatment. Sensitivity analysis was performed to assess the relationship between NCB and baseline ischemic risk, bleeding risk, time to angiography and local surgical revascularization rates. Pre-treatment with ticagrelor and clopidogrel was associated with a greater than 50% likelihood of providing a >1% increase in 30 day NCB when baseline estimated ischemic risk exceeds 11% and 14%, respectively. Prasugrel pre-treatment did not achieve a greater than 50% probability of an increase in NCB regardless of baseline ischemic risk. Institutional surgical revascularization rates and time to coronary angiography did not correlate with the likelihood of benefit from P2Y12 pre-treatment. In conclusion, pre-treatment with P2Y12 inhibition is unlikely to be beneficial to the majority of patients presenting with NSTE-ACS. A tailored assessment of each patient’s individual ischemic and bleeding risk may identify those likely to benefit. PMID:27548237

  9. Evaluation of CYP2C19, P2Y12, and ABCB1 polymorphisms and phenotypic response to clopidogrel in healthy Indian adults

    PubMed Central

    Sridharan, Kannan; Kataria, Rachna; Tolani, Drishti; Bendkhale, Shital; Gogtay, Nithya J.; Thatte, Urmila M.

    2016-01-01

    Introduction: CYP2C19 and P2Y12 polymorphisms have been claimed to alter the pharmacodynamic response to clopidogrel. ABCB1 polymorphism has been associated with the efflux of clopidogrel resulting in decreased bioavailability. Due to paucity of data from Indian population, the present study was undertaken to evaluate the association of genetic polymorphisms of CYP2C19, P2Y12, and ABCB1 with inhibition of platelet aggregation (IPA) by clopidogrel. Methods: Healthy adults (n = 90) of either gender were administered single dose of 300 mg clopidogrel. Baseline, 4 h postdose, and day 7 assessment of platelet aggregation and genotype of CYP2C19, P2Y12, and ABCB1 were carried out using standardized laboratory methods. The difference in the maximum platelet aggregation (MPA) between baseline and 4 h postdose was considered as delta-MPA (DMPA), and percentage change of MPA at 4 h from baseline was considered as IPA. Those with an IPA of <30% were considered as poor responders. Inferential statistics was applied to find out significant difference of these parameters between various groups of genetic polymorphisms. Results: Mean (standard deviation [SD]) of MPA (%) at baseline, 4 h postdose, and day 7 were 78 (5), 56 (16), and 71 (8), respectively. Similarly, mean (SD) of DMPA (%) and IPA (%) were 23 (17) and 29 (21), respectively. A total of 54/90 (60%) cases were found to be poor responders to clopidogrel. A wild genotype (*1/*1) of CYP2C19 was observed in 35 (40.2%), 42 (48.3%) had *1/*2, 2 (2.3%) individuals had *1/*3, and 8 (9.2%) had *2/*2 mutant genotypes. Although statistically not significant (P = 0.09), a trend was observed in having decreased inhibition values (both MPA and IPA) as we proceed from wild genotype (*1/*1) to mutant genotypes in the order of *1/*2, *1/*3, and *2/*2. Similarly, in P2Y12, a wild haplotype (H1/H1) was present in 77 (89.5%) and 9 (10.5%) individuals had H1/H2 type. A statistically significant difference in DMPA and IPA was observed with

  10. Association of PON1, P2Y12 and COX1 with Recurrent Ischemic Events in Patients with Extracranial or Intracranial Stenting

    PubMed Central

    Wang, Bo; Sun, Shu-Sen; Gao, Feng; Mo, Da-Peng; Song, Li-Gang; Sun, Xuan; Liu, Lian; Zhao, Xing-Quan; Wang, Yi-Long; Wang, Yong-Jun; Zhao, Zhi-Gang; Miao, Zhong-Rong

    2016-01-01

    Background and Purpose Short-term combined use of clopidogrel and aspirin improves cerebrovascular outcomes in patients with symptomatic extracranial or intracranial stenosis. Antiplatelet non-responsiveness is related to recurrent ischemic events, but the culprit genetic variants responsible for the non-responsiveness have not been well studied. We aimed to identify the genetic variants associated with poor clinical outcomes. Methods Patients with symptomatic extracranial or intracranial stenosis scheduled for stenting and receiving dual antiplatelets (clopidogrel 75 mg and aspirin 100 mg daily) for at least 5 days before intervention were enrolled. Ischemic events including recurrent transient ischemic attack, stroke, myocardial infarction, and vascular-related mortality within 12 months follow-up were recorded. We examined the influence of genetic polymorphisms on treatment outcome in our patients. Results A total of 268 patients were enrolled into our study and ischemic events were observed in 39 patients. For rs662 of paraoxonase 1 (PON1), allele C was associated with an increased risk of ischemic events (OR = 1.64, 95%CI = 1.03–2.62, P = 0.029). The A-allele carriers of rs2046934 of P2Y12 had a significant association with adverse events (OR = 2.01, 95%CI = 1.10–3.67, P = 0.041). The variant T-allele of cyclooxygenase-1 (COX1) rs1330344 significantly increased the risk of recurrent clinical events (OR = 1.85, 95%CI = 1.12–3.03, P = 0.017). The other single nucleotide polymorphism (SNP) had no association with ischemic events. Conclusions PON1, P2Y12 and COX1 polymorphisms were associated with poorer vascular outcomes. Testing for these polymorphisms may be valuable in the identification of patients at risk for recurrent ischemic events. PMID:26870959

  11. Potassium 2-(1-hydroxypentyl)-benzoate inhibits ADP-induced rat platelet aggregation through P2Y1-PLC signaling pathways.

    PubMed

    Yang, Hongyan; Xu, Shaofeng; Li, Jiang; Wang, Ling; Wang, Xiaoliang

    2015-09-01

    Potassium 2-(1-hydroxypenty1)-benzoate (dl-PHPB) is a new drug candidate for treatment of ischemic stroke with antiplatelet effect. In this study, we investigated the mechanisms of dl-PHPB in inhibiting platelet aggregation. The ADP-activated P2Y1-Gq-PLC and P2Y12-Gi-AC pathways were observed, respectively. Intravenous injection of dl-PHPB (1.3, 3.9, 12.9 mg/kg) significantly inhibited ADP-, collagen-, and arachidonic acid-induced rat platelet aggregation in a dose-dependent manner, and dl-PHPB had a relatively more potent inhibitory effect on ADP-induced rat platelet aggregation than other agonists. Dl-PHPB also showed a decreased expression of CD62P (a marker for platelet activation) mediated by ADP. Both dl-PHPB and ticlopidine (P2Y12 receptor antagonist) decreased cytoplasmic Ca(2+) concentration. But, dl-PHPB did not reverse the inhibition of PGE1-induced platelet cAMP formation by ADP, which was different from ticlopidine. Further, dl-PHPB instead of ticlopidine showed increasing phospholipase C-β phosphorylation (ser(1105)). The m-3M3FBS, a phospholipase C activator, attenuated the inhibitory effect of dl-PHPB on ADP-induced platelet aggregation and enhanced IP1 accumulation in rat platelets. Dl-PHPB decreased IP1 accumulation induced by ADP but had no effect on IP1 level enhanced by m-3M3FBS. Our results suggest that dl-PHPB has a potent antiplatelet effect, which is mainly through blockade of P2Y1 receptor-PLC-IP3 pathway and decreasing cytoplasmic calcium.

  12. Nucleolin down-regulation is involved in ADP-induced cell cycle arrest in S phase and cell apoptosis in vascular endothelial cells.

    PubMed

    Wang, Wenmeng; Luo, Junqing; Xiang, Fang; Liu, Xueting; Jiang, Manli; Liao, Lingjuan; Hu, Jinyue

    2014-01-01

    High concentration of extracellular ADP has been reported to induce cell apoptosis, but the molecular mechanisms remain not fully elucidated. In this study, we found by serendipity that ADP treatment of human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC) down-regulated the protein level of nucleolin in a dose- and time-dependent manner. ADP treatment did not decrease the transcript level of nucloelin, suggesting that ADP might induce nucleolin protein degradation. HUVEC and HAEC expressed ADP receptor P2Y13 receptor, but did not express P2Y1 or P2Y12 receptors. However, P2Y1, 12, 13 receptor antagonists MRS2179, PSB0739, MRS2211 did not inhibit ADP-induced down-regulation of nucleolin. Moreover, MRS2211 itself down-regulated nucleolin protein level. In addition, 2-MeSADP, an agonist for P2Y1, 12 and 13 receptors, did not down-regulate nucleolin protein. These results suggested that ADP-induced nucleolin down-regulation was not due to the activation of P2Y1, 12, or 13 receptors. We also found that ADP treatment induced cell cycle arrest in S phase, cell apoptosis and cell proliferation inhibition via nucleolin down-regulation. The over-expression of nucleolin by gene transfer partly reversed ADP-induced cell cycle arrest, cell apoptosis and cell proliferation inhibition. Furthermore, ADP sensitized HUVEC to cisplatin-induced cell death by the down-regulation of Bcl-2 expression. Taken together, we found, for the first time to our knowledge, a novel mechanism by which ADP regulates cell proliferation by induction of cell cycle arrest and cell apoptosis via targeting nucelolin. PMID:25290311

  13. The Mitochondrial Fission Receptor MiD51 Requires ADP as a Cofactor

    PubMed Central

    Losón, Oliver C.; Liu, Raymond; Rome, Michael E.; Meng, Shuxia; Kaiser, Jens T.; Shan, Shu-ou; Chan, David C.

    2014-01-01

    SUMMARY Mitochondrial fission requires recruitment of dynamin-related protein 1 (Drp1) to the mitochondrial surface and activation of its GTP-dependent scission function. The Drp1 receptors MiD49 and MiD51 recruit Drp1 to facilitate mitochondrial fission, but their mechanism of action is poorly understood. Using X-ray crystallography, we demonstrate that MiD51 contains a nucleotidyl transferase domain that binds ADP with high affinity. MiD51 recruits Drp1 via a surface loop that functions independently of ADP binding. However, in the absence of nucleotide binding, the recruited Drp1 cannot be activated for fission. Purified MiD51 strongly inhibits Drp1 assembly and GTP hydrolysis in the absence of ADP. Addition of ADP relieves this inhibition and promotes Drp1 assembly into spirals with enhanced GTP hydrolysis. Our results reveal ADP as an essential cofactor for MiD51 during mitochondrial fission. PMID:24508339

  14. The mitochondrial fission receptor MiD51 requires ADP as a cofactor.

    PubMed

    Losón, Oliver C; Liu, Raymond; Rome, Michael E; Meng, Shuxia; Kaiser, Jens T; Shan, Shu-ou; Chan, David C

    2014-03-01

    Mitochondrial fission requires recruitment of dynamin-related protein 1 (Drp1) to the mitochondrial surface and activation of its GTP-dependent scission function. The Drp1 receptors MiD49 and MiD51 recruit Drp1 to facilitate mitochondrial fission, but their mechanism of action is poorly understood. Using X-ray crystallography, we demonstrate that MiD51 contains a nucleotidyl transferase domain that binds ADP with high affinity. MiD51 recruits Drp1 via a surface loop that functions independently of ADP binding. However, in the absence of nucleotide binding, the recruited Drp1 cannot be activated for fission. Purified MiD51 strongly inhibits Drp1 assembly and GTP hydrolysis in the absence of ADP. Addition of ADP relieves this inhibition and promotes Drp1 assembly into spirals with enhanced GTP hydrolysis. Our results reveal ADP as an essential cofactor for MiD51 during mitochondrial fission.

  15. P2Y13 receptors mediate presynaptic inhibition of acetylcholine release induced by adenine nucleotides at the mouse neuromuscular junction.

    PubMed

    Guarracino, Juan F; Cinalli, Alejandro R; Fernández, Verónica; Roquel, Liliana I; Losavio, Adriana S

    2016-06-21

    It is known that adenosine 5'-triphosphate (ATP) is released along with the neurotransmitter acetylcholine (ACh) from motor nerve terminals. At mammalian neuromuscular junctions (NMJs), we have previously demonstrated that ATP is able to decrease ACh secretion by activation of P2Y receptors coupled to pertussis toxin-sensitive Gi/o protein. In this group, the receptor subtypes activated by adenine nucleotides are P2Y12 and P2Y13. Here, we investigated, by means of pharmacological and immunohistochemical assays, the P2Y receptor subtype that mediates the modulation of spontaneous and evoked ACh release in mouse phrenic nerve-diaphragm preparations. First, we confirmed that the preferential agonist for P2Y12-13 receptors, 2-methylthioadenosine 5'-diphosphate trisodium salt hydrate (2-MeSADP), reduced MEPP frequency without affecting MEPP amplitude as well as the amplitude and quantal content of end-plate potentials (EPPs). The effect on spontaneous secretion disappeared after the application of the selective P2Y12-13 antagonists AR-C69931MX or 2-methylthioadenosine 5'-monophosphate triethylammonium salt hydrate (2-MeSAMP). 2-MeSADP was more potent than ADP and ATP in reducing MEPP frequency. Then we demonstrated that the selective P2Y13 antagonist MRS-2211 completely prevented the inhibitory effect of 2-MeSADP on MEPP frequency and EPP amplitude, whereas the P2Y12 antagonist MRS-2395 failed to do this. The preferential agonist for P2Y13 receptors inosine 5'-diphosphate sodium salt (IDP) reduced spontaneous and evoked ACh secretion and MRS-2211 abolished IDP-mediated modulation. Immunohistochemical studies confirmed the presence of P2Y13 but not P2Y12 receptors at the end-plate region. Disappearance of P2Y13 receptors after denervation suggests the presynaptic localization of the receptors. We conclude that, at motor nerve terminals, the Gi/o protein-coupled P2Y receptors implicated in presynaptic inhibition of spontaneous and evoked ACh release are of the subtype P2Y

  16. P2Y13 receptors mediate presynaptic inhibition of acetylcholine release induced by adenine nucleotides at the mouse neuromuscular junction.

    PubMed

    Guarracino, Juan F; Cinalli, Alejandro R; Fernández, Verónica; Roquel, Liliana I; Losavio, Adriana S

    2016-06-21

    It is known that adenosine 5'-triphosphate (ATP) is released along with the neurotransmitter acetylcholine (ACh) from motor nerve terminals. At mammalian neuromuscular junctions (NMJs), we have previously demonstrated that ATP is able to decrease ACh secretion by activation of P2Y receptors coupled to pertussis toxin-sensitive Gi/o protein. In this group, the receptor subtypes activated by adenine nucleotides are P2Y12 and P2Y13. Here, we investigated, by means of pharmacological and immunohistochemical assays, the P2Y receptor subtype that mediates the modulation of spontaneous and evoked ACh release in mouse phrenic nerve-diaphragm preparations. First, we confirmed that the preferential agonist for P2Y12-13 receptors, 2-methylthioadenosine 5'-diphosphate trisodium salt hydrate (2-MeSADP), reduced MEPP frequency without affecting MEPP amplitude as well as the amplitude and quantal content of end-plate potentials (EPPs). The effect on spontaneous secretion disappeared after the application of the selective P2Y12-13 antagonists AR-C69931MX or 2-methylthioadenosine 5'-monophosphate triethylammonium salt hydrate (2-MeSAMP). 2-MeSADP was more potent than ADP and ATP in reducing MEPP frequency. Then we demonstrated that the selective P2Y13 antagonist MRS-2211 completely prevented the inhibitory effect of 2-MeSADP on MEPP frequency and EPP amplitude, whereas the P2Y12 antagonist MRS-2395 failed to do this. The preferential agonist for P2Y13 receptors inosine 5'-diphosphate sodium salt (IDP) reduced spontaneous and evoked ACh secretion and MRS-2211 abolished IDP-mediated modulation. Immunohistochemical studies confirmed the presence of P2Y13 but not P2Y12 receptors at the end-plate region. Disappearance of P2Y13 receptors after denervation suggests the presynaptic localization of the receptors. We conclude that, at motor nerve terminals, the Gi/o protein-coupled P2Y receptors implicated in presynaptic inhibition of spontaneous and evoked ACh release are of the subtype P2Y

  17. Platelet morphologic changes and fibrinogen receptor localization. Initial responses in ADP-activated human platelets.

    PubMed

    Hensler, M E; Frojmovic, M; Taylor, R G; Hantgan, R R; Lewis, J C

    1992-09-01

    Platelet exposure to agonists results in rapid morphologic changes paralleled by fibrinogen binding and platelet aggregation. The current study used standardized stereology in conjunction with immunogold electron microscopy to correlate the initial morphologic changes with fibrinogen receptor localization on the surfaces of ADP-activated human platelets. A 45% increase in platelet circumference was observed after 3 seconds of activation (P = 0.001). Virtually all of this increase was due to a 13-fold increase in projection membrane, and the projections observed by stereo microscopy at this time were mostly blunt. Both blunt and long projections also accounted for the increase in platelet-platelet contacts at 10 seconds of activation. Immunogold electron microscopy using the monoclonal antibodies P2 and AP-2 against the fibrinogen receptor, glycoprotein IIb/IIIa (GP IIb/IIIa), showed relatively equivalent immunogold densities on projections compared with cell body during 30 seconds of activation. The activation-dependent anti-GP IIb/IIIa monoclonal antibody, 7E3, showed an immunogold density 37% greater on projections compared with cell body (P = 0.0001). Colocalization studies using 7E3 with a polyclonal antifibrinogen antibody showed bound fibrinogen in close proximity to the GP IIb/IIIa localized by 7E3 on projections. These studies support an important role for platelet projections during the earliest stages of fibrinogen binding and ADP-induced aggregation.

  18. Nucleotides Acting at P2Y Receptors: Connecting Structure and Function

    PubMed Central

    Paoletta, Silvia; Katritch, Vsevolod; Wu, Beili; Gao, Zhan-Guo; Zhao, Qiang; Stevens, Raymond C.; Kiselev, Evgeny

    2015-01-01

    Eight G protein–coupled P2Y receptor (P2YR) subtypes are important physiologic mediators. The human P2YRs are fully activated by ATP (P2Y2 and P2Y11), ADP (P2Y1, P2Y12, and P2Y13), UTP (P2Y2 and P2Y4), UDP (P2Y6 and P2Y14), and UDP glucose (P2Y14). Their structural elucidation is progressing rapidly. The X-ray structures of three ligand complexes of the Gi-coupled P2Y12R and two of the Gq-coupled P2Y1Rs were recently determined and will be especially useful in structure-based ligand design at two P2YR subfamilies. These high-resolution structures, which display unusual binding site features, complement mutagenesis studies for probing ligand recognition and activation. The structural requirements for nucleotide agonist recognition at P2YRs are relatively permissive with respect to the length of the phosphate moiety, but less so with respect to base recognition. Nucleotide-like antagonists and partial agonists are also known for P2Y1, P2Y2, P2Y4, and P2Y12Rs. Each P2YR subtype has the ability to be activated by structurally bifunctional agonists, such as dinucleotides, typically, dinucleoside triphosphates or tetraphosphates, and nucleoside polyphosphate sugars (e.g., UDP glucose) as well as the more conventional mononucleotide agonists. A range of dinucleoside polyphosphates, from triphosphates to higher homologs, occurs naturally. Earlier modeling predictions of the P2YRs were not very accurate, but recent findings have provided much detailed structural insight into this receptor family to aid in the rational design of new drugs. PMID:25837834

  19. Succinate reverses in-vitro platelet inhibition by acetylsalicylic acid and P2Y receptor antagonists.

    PubMed

    Spath, Brigitte; Hansen, Arne; Bokemeyer, Carsten; Langer, Florian

    2012-01-01

    High on-treatment platelet reactivity has been associated with adverse cardiovascular events in patients receiving anti-platelet agents, but the molecular mechanisms underlying this phenomenon remain incompletely understood. Succinate, a citric acid cycle intermediate, is released into the circulation under conditions of mitochondrial dysfunction due to hypoxic organ damage, including sepsis, stroke, and myocardial infarction. Because the G protein-coupled receptor (GPCR) for succinate, SUCNR1 (GPR91), is present on human platelets, we hypothesized that succinate-mediated platelet stimulation may counteract the pharmacological effects of cyclooxygenase-1 and ADP receptor antagonists. To test this hypothesis in a controlled in-vitro study, washed platelets from healthy donors were treated with acetylsalicylic acid (ASA) or small-molecule P2Y(1) or P2Y(12) inhibitors and subsequently analyzed by light transmittance aggregometry using arachidonic acid (AA), ADP and succinate as platelet agonists. Aggregation in response to succinate alone was highly variable with only 29% of donors showing a (mostly delayed) platelet response. In contrast, succinate reproducibly and concentration-dependently (10-1000 µM) enhanced platelet aggregation in response to low concentrations of exogenous ADP. Furthermore, while succinate alone had no effect in the presence of platelet inhibitors, responsiveness of platelets to ADP after pretreatment with P2Y(1) or P2Y(12) antagonists was fully restored, when platelets were co-stimulated with 100 µM succinate. Similarly, succinate completely (at 1000 µM) or partially (at 100 µM) reversed the inhibitory effect of ASA on AA-induced platelet aggregation. In contrast, succinate failed to restore platelet responsiveness in the presence of both ASA and the P2Y(12) antagonist, suggesting that concomitant signaling via different GPCRs was required. Essentially identical results were obtained, when flow cytometric analysis of surface CD62P

  20. From eggs to hearts: what is the link between cyclic ADP-ribose and ryanodine receptors?

    PubMed

    Venturi, Elisa; Pitt, Samantha; Galfré, Elena; Sitsapesan, Rebecca

    2012-04-01

    It was first proposed that cyclic ADP-ribose (cADPR) could activate ryanodine receptors (RyR) in 1991. Following a subsequent report that cADPR could activate cardiac RyR (RyR2) reconstituted into artificial membranes and stimulate Ca(2+) -release from isolated cardiac SR, there has been a steadily mounting stockpile of publications proclaiming the physiological and pathophysiological importance of cADPR in the cardiovascular system. It was only 2 years earlier, in 1989, that cADPR was first identified as the active metabolite of nicotinamide adenine dinucleotide (NAD), responsible for triggering the release of Ca(2+) from crude homogenates of sea urchin eggs. Twenty years later, can we boast of being any closer to unraveling the mechanisms by which cADPR modulates intracellular Ca(2+) -release? This review sets out to examine the mechanisms underlying the effects of cADPR and ask whether cADPR is an important signaling molecule in the heart. PMID:21176119

  1. The Impact of Type 2 Diabetes on the Efficacy of ADP Receptor Blockers in Patients with Acute ST Elevation Myocardial Infarction: A Pilot Prospective Study

    PubMed Central

    Fedor, Marián; Kovář, František; Galajda, Peter; Bolek, Tomáš; Stančiaková, Lucia; Fedorová, Jana; Staško, Ján; Kubisz, Peter; Mokáň, Marián

    2016-01-01

    Background. The aim of this study was to validate the impact of type 2 diabetes (T2D) on the platelet reactivity in patients with acute ST elevation myocardial infarction (STEMI) treated with adenosine diphosphate (ADP) receptor blockers. Methods. A pilot prospective study was performed. Totally 67 patients were enrolled. 21 patients had T2D. Among all study population, 33 patients received clopidogrel and 34 patients received prasugrel. The efficacy of ADP receptor blocker therapy had been tested in two time intervals using light transmission aggregometry with specific inducer and vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) flow cytometry assay. Results. There were no significant differences in platelet aggregability among T2D and nondiabetic (ND) group. The platelet reactivity index of VASP-P did not differ significantly between T2D and ND group (59.4 ± 30.9% versus 60.0 ± 25.2% and 33.9 ± 25.3% versus 38.6 ± 29.3% in second testing). The number of ADP receptor blocker nonresponders did not differ significantly between T2D and ND patients. The time interval from ADP receptor blocker loading dosing to the blood sampling was similar in T2D and ND patients in both examinations. Conclusion. This prospective study did not confirm the higher platelet reactivity and higher prevalence of ADP receptor blocker nonresponders in T2D acute STEMI patients. PMID:27493970

  2. C-type lectin like receptor 2 (CLEC-2) signals independently of lipid raft microdomains in platelets.

    PubMed

    Manne, Bhanu Kanth; Badolia, Rachit; Dangelmaier, Carol A; Kunapuli, Satya P

    2015-01-15

    C-type lectin like receptor 2 (CLEC-2) has been reported to activate platelets through a lipid raft-dependent manner. Secreted ADP potentiates CLEC-2-mediated platelet aggregation. We have investigated whether the decrease in CLEC-2-mediated platelet aggregation, previously reported in platelets with disrupted rafts, is a result of the loss of agonist potentiation by ADP. We disrupted platelet lipid rafts with methyl-β-cyclodextrin (MβCD) and measured signaling events downstream of CLEC-2 activation. Lipid raft disruption decreases platelet aggregation induced by CLEC-2 agonists. The inhibition of platelet aggregation by the disruption of lipid rafts was rescued by the exogenous addition of epinephrine but not 2-methylthioadenosine diphosphate (2MeSADP), which suggests that lipid raft disruption effects P2Y12-mediated Gi activation but not Gz. Phosphorylation of Syk (Y525/526) and PLCγ2 (Y759), were not affected by raft disruption in CLEC-2 agonist-stimulated platelets. Furthermore, tyrosine phosphorylation of the CLEC-2 hemi-ITAM was not effected when MβCD disrupts lipid rafts. Lipid rafts do not directly contribute to CLEC-2 receptor activation in platelets. The effects of disruption of lipid rafts in in vitro assays can be attributed to inhibition of ADP feedback that potentiates CLEC-2 signaling.

  3. The proposed channel-enzyme transient receptor potential melastatin 2 does not possess ADP ribose hydrolase activity

    PubMed Central

    Iordanov, Iordan; Mihályi, Csaba; Tóth, Balázs; Csanády, László

    2016-01-01

    Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca2+-permeable cation channel essential for immunocyte activation, insulin secretion, and postischemic cell death. TRPM2 is activated by ADP ribose (ADPR) binding to its C-terminal cytosolic NUDT9-homology (NUDT9H) domain, homologous to the soluble mitochondrial ADPR pyrophosphatase (ADPRase) NUDT9. Reported ADPR hydrolysis classified TRPM2 as a channel-enzyme, but insolubility of isolated NUDT9H hampered further investigations. Here we developed a soluble NUDT9H model using chimeric proteins built from complementary polypeptide fragments of NUDT9H and NUDT9. When expressed in E.coli, chimeras containing up to ~90% NUDT9H sequence remained soluble and were affinity-purified. In ADPRase assays the conserved Nudix-box sequence of NUDT9 proved essential for activity (kcat~4-9s-1), that of NUDT9H did not support catalysis. Replacing NUDT9H in full-length TRPM2 with soluble chimeras retained ADPR-dependent channel gating (K1/2~1-5 μM), confirming functionality of chimeric domains. Thus, TRPM2 is not a 'chanzyme'. Chimeras provide convenient soluble NUDT9H models for structural/biochemical studies. DOI: http://dx.doi.org/10.7554/eLife.17600.001 PMID:27383051

  4. The proposed channel-enzyme transient receptor potential melastatin 2 does not possess ADP ribose hydrolase activity.

    PubMed

    Iordanov, Iordan; Mihályi, Csaba; Tóth, Balázs; Csanády, László

    2016-01-01

    Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca(2+)-permeable cation channel essential for immunocyte activation, insulin secretion, and postischemic cell death. TRPM2 is activated by ADP ribose (ADPR) binding to its C-terminal cytosolic NUDT9-homology (NUDT9H) domain, homologous to the soluble mitochondrial ADPR pyrophosphatase (ADPRase) NUDT9. Reported ADPR hydrolysis classified TRPM2 as a channel-enzyme, but insolubility of isolated NUDT9H hampered further investigations. Here we developed a soluble NUDT9H model using chimeric proteins built from complementary polypeptide fragments of NUDT9H and NUDT9. When expressed in E.coli, chimeras containing up to ~90% NUDT9H sequence remained soluble and were affinity-purified. In ADPRase assays the conserved Nudix-box sequence of NUDT9 proved essential for activity (kcat~4-9s(-1)), that of NUDT9H did not support catalysis. Replacing NUDT9H in full-length TRPM2 with soluble chimeras retained ADPR-dependent channel gating (K1/2~1-5 μM), confirming functionality of chimeric domains. Thus, TRPM2 is not a 'chanzyme'. Chimeras provide convenient soluble NUDT9H models for structural/biochemical studies. PMID:27383051

  5. ADP Signaling in Vascular Endothelial Cells

    PubMed Central

    Hess, Connie Ng; Kou, Ruqin; Johnson, Rosalyn P.; Li, Gordon K.; Michel, Thomas

    2009-01-01

    ADP responses underlie therapeutic approaches to many cardiovascular diseases, and ADP receptor antagonists are in widespread clinical use. The role of ADP in platelet biology has been extensively studied, yet ADP signaling pathways in endothelial cells remain incompletely understood. We found that ADP promoted phosphorylation of the endothelial isoform of nitric-oxide synthase (eNOS) at Ser1179 and Ser635 and dephosphorylation at Ser116 in cultured endothelial cells. Although eNOS activity was stimulated by both ADP and ATP, only ADP signaling was significantly inhibited by the P2Y1 receptor antagonist MRS 2179 or by knockdown of P2Y1 using small interfering RNA (siRNA). ADP activated the small GTPase Rac1 and promoted endothelial cell migration. siRNA-mediated knockdown of Rac1 blocked ADP-dependent eNOS Ser1179 and Ser635 phosphorylation, as well as eNOS activation. We analyzed pathways known to regulate eNOS, including phosphoinositide 3-kinase/Akt, ERK1/2, Src, and calcium/calmodulin-dependent kinase kinase-β (CaMKKβ) using the inhibitors wortmannin, PD98059, PP2, and STO-609, respectively. None of these inhibitors altered ADP-modulated eNOS phosphorylation. In contrast, siRNA-mediated knockdown of AMP-activated protein kinase (AMPK) inhibited ADP-dependent eNOS Ser635 phosphorylation and eNOS activity but did not affect eNOS Ser1179 phosphorylation. Importantly, the AMPK enzyme inhibitor compound C had no effect on ADP-stimulated eNOS activity, despite completely blocking AMPK activity. CaMKKβ knockdown suppressed ADP-stimulated eNOS activity, yet inhibition of CaMKKβ kinase activity using STO-609 failed to affect eNOS activation by ADP. These data suggest that the expression, but not the kinase activity, of AMPK and CaMKKβ is necessary for ADP signaling to eNOS. PMID:19783664

  6. Multiple receptor conformation docking, dock pose clustering and 3D QSAR studies on human poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors.

    PubMed

    Fatima, Sabiha; Jatavath, Mohan Babu; Bathini, Raju; Sivan, Sree Kanth; Manga, Vijjulatha

    2014-10-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) functions as a DNA damage sensor and signaling molecule. It plays a vital role in the repair of DNA strand breaks induced by radiation and chemotherapeutic drugs; inhibitors of this enzyme have the potential to improve cancer chemotherapy or radiotherapy. Three-dimensional quantitative structure activity relationship (3D QSAR) models were developed using comparative molecular field analysis, comparative molecular similarity indices analysis and docking studies. A set of 88 molecules were docked into the active site of six X-ray crystal structures of poly(ADP-ribose)polymerase-1 (PARP-1), by a procedure called multiple receptor conformation docking (MRCD), in order to improve the 3D QSAR models through the analysis of binding conformations. The docked poses were clustered to obtain the best receptor binding conformation. These dock poses from clustering were used for 3D QSAR analysis. Based on MRCD and QSAR information, some key features have been identified that explain the observed variance in the activity. Two receptor-based QSAR models were generated; these models showed good internal and external statistical reliability that is evident from the [Formula: see text], [Formula: see text] and [Formula: see text]. The identified key features enabled us to design new PARP-1 inhibitors. PMID:25046176

  7. Multiple receptor conformation docking, dock pose clustering and 3D QSAR studies on human poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors.

    PubMed

    Fatima, Sabiha; Jatavath, Mohan Babu; Bathini, Raju; Sivan, Sree Kanth; Manga, Vijjulatha

    2014-10-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) functions as a DNA damage sensor and signaling molecule. It plays a vital role in the repair of DNA strand breaks induced by radiation and chemotherapeutic drugs; inhibitors of this enzyme have the potential to improve cancer chemotherapy or radiotherapy. Three-dimensional quantitative structure activity relationship (3D QSAR) models were developed using comparative molecular field analysis, comparative molecular similarity indices analysis and docking studies. A set of 88 molecules were docked into the active site of six X-ray crystal structures of poly(ADP-ribose)polymerase-1 (PARP-1), by a procedure called multiple receptor conformation docking (MRCD), in order to improve the 3D QSAR models through the analysis of binding conformations. The docked poses were clustered to obtain the best receptor binding conformation. These dock poses from clustering were used for 3D QSAR analysis. Based on MRCD and QSAR information, some key features have been identified that explain the observed variance in the activity. Two receptor-based QSAR models were generated; these models showed good internal and external statistical reliability that is evident from the [Formula: see text], [Formula: see text] and [Formula: see text]. The identified key features enabled us to design new PARP-1 inhibitors.

  8. P2Y Receptors Sensitize Mouse and Human Colonic Nociceptors

    PubMed Central

    Hockley, James R. F.; Tranter, Michael M.; McGuire, Cian; Boundouki, George; Cibert-Goton, Vincent; Thaha, Mohamed A.; Blackshaw, L. Ashley; Michael, Gregory J.; Baker, Mark D.; Knowles, Charles H.; Winchester, Wendy J.

    2016-01-01

    Activation of visceral nociceptors by inflammatory mediators contributes to visceral hypersensitivity and abdominal pain associated with many gastrointestinal disorders. Purine and pyrimidine nucleotides (e.g., ATP and UTP) are strongly implicated in this process following their release from epithelial cells during mechanical stimulation of the gut, and from immune cells during inflammation. Actions of ATP are mediated through both ionotropic P2X receptors and metabotropic P2Y receptors. P2X receptor activation causes excitation of visceral afferents; however, the impact of P2Y receptor activation on visceral afferents innervating the gut is unclear. Here we investigate the effects of stimulating P2Y receptors in isolated mouse colonic sensory neurons, and visceral nociceptor fibers in mouse and human nerve-gut preparations. Additionally, we investigate the role of Nav1.9 in mediating murine responses. The application of UTP (P2Y2 and P2Y4 agonist) sensitized colonic sensory neurons by increasing action potential firing to current injection and depolarizing the membrane potential. The application of ADP (P2Y1, P2Y12, and P2Y13 agonist) also increased action potential firing, an effect blocked by the selective P2Y1 receptor antagonist MRS2500. UTP or ADP stimulated afferents, including mouse and human visceral nociceptors, in nerve-gut preparations. P2Y1 and P2Y2 transcripts were detected in 80% and 56% of retrogradely labeled colonic neurons, respectively. Nav1.9 transcripts colocalized in 86% of P2Y1-positive and 100% of P2Y2-positive colonic neurons, consistent with reduced afferent fiber responses to UTP and ADP in Nav1.9−/− mice. These data demonstrate that P2Y receptor activation stimulates mouse and human visceral nociceptors, highlighting P2Y-dependent mechanisms in the generation of visceral pain during gastrointestinal disease. SIGNIFICANCE STATEMENT Chronic visceral pain is a debilitating symptom of many gastrointestinal disorders. The activation of

  9. The ATP-gated P2X1 receptor plays a pivotal role in activation of aspirin-treated platelets by thrombin and epinephrine.

    PubMed

    Grenegård, Magnus; Vretenbrant-Oberg, Karin; Nylander, Martina; Désilets, Stéphanie; Lindström, Eva G; Larsson, Anders; Ramström, Ida; Ramström, Sofia; Lindahl, Tomas L

    2008-07-01

    Human platelets express protease-activated receptor 1 (PAR1) and PAR4 but limited data indicate for differences in signal transduction. We studied the involvement of PAR1 and PAR4 in the cross-talk between thrombin and epinephrine. The results show that epinephrine acted via alpha(2A)-adrenergic receptors to provoke aggregation, secretion, and Ca(2+) mobilization in aspirin-treated platelets pre-stimulated with subthreshold concentrations of thrombin. Incubating platelets with antibodies against PAR4 or the PAR4-specific inhibitor pepducin P4pal-i1 abolished the aggregation. Furthermore, platelets pre-exposed to the PAR4-activating peptide AYPGKF, but not to the PAR1-activating peptide SFLLRN, were aggregated by epinephrine, whereas both AYPGKF and SFLLRN synergized with epinephrine in the absence of aspirin. The roles of released ATP and ADP were elucidated by using antagonists of the purinergic receptors P2X(1), P2Y(1), and P2Y(12) (i.e. NF449, MRS2159, MRS2179, and cangrelor). Intriguingly, ATP, but not ADP, was required for the epinephrine/thrombin-induced aggregation. In Western blot analysis, a low concentration of AYPGKF, but not SFLLRN, stimulated phosphorylation of Akt on serine 473. Moreover, the phosphatidyl inositide 3-kinase inhibitor LY294002 antagonized the effect of epinephrine combined with thrombin or AYPGKF. Thus, in aspirin-treated platelets, PAR4, but not PAR1, interacts synergistically with alpha(2A)-adrenergic receptors, and the PI3-kinase/Akt pathway is involved in this cross-talk. Furthermore, in PAR4-pretreated platelets, epinephrine caused dense granule secretion, and subsequent signaling from the ATP-gated P2X(1)-receptor and the alpha(2A)-adrenergic receptor induced aggregation. These results suggest a new mechanism that has ATP as a key element and circumvents the action of aspirin on epinephrine-facilitated PAR4-mediated platelet activation. PMID:18480058

  10. A Potential Mechanism of High-Dose Ticagrelor in Modulating Platelet Activity and Atherosclerosis Mediated by Thymic Stromal Lymphopoietin Receptor

    PubMed Central

    Mao, Yi; Peng, Yudong; Zeng, Qiutang; Cheng, Longxian; Wang, Boyuan; Mao, Xiaobo; Meng, Kai; Liu, Yuzhou; Lian, Yitian; Li, Dazhu

    2015-01-01

    Abnormal expression of thymic stromal lymphopoietin (TSLP) and its receptor (TSLPR) was found in patients with acute coronary syndrome. Ticagrelor, an oral platelet ADP P2Y12 receptor antagonist, is widely used in these patients. The aim of this study was to verify whether different doses of ticagrelor regulated plaque progression and platelet activity by modulating TSLP/TSLPR. Seventy-five ApoE-/- mice were randomly divided into five groups: (1) high-cholesterol diet (HCD, n = 15); (2) HCD plus ticagrelor 25 mg/kg/d (T1, n = 15); (3) HCD plus ticagrelor 50 mg/kg/d (T2, n = 15); (4) HCD plus ticagrelor 100 mg/kg/d (T3, n = 15); and (5) a normal diet group (ND, n = 15). At day 0 and at week 16, blood lipids and serum TSLP levels, expression of TSLPR, CD62, and CD63, platelet aggregation, platelet ATP release, PI3K/Akt signaling pathway, and plaque morphology were assessed. HCD increased TSLPR expression and atherosclerosis progression but high-dose ticagrelor (100 mg/kg) moderated this trend. TSLPR was positively correlated with Akt1, platelet aggregation, corrected plaque area, and vulnerability index in the T3 group (P<0.01). In conclusion, low-dose ticagrelor only inhibited platelet activity. Besides this inhibition, high-dose ticagrelor modulated platelet activity and atherosclerosis mediated by TSLPR, potentially through the PI3K/Akt signal pathway. PMID:26517374

  11. Single KATP channel opening in response to stimulation of AMPA/kainate receptors is mediated by Na+ accumulation and submembrane ATP and ADP changes

    PubMed Central

    Mollajew, R; Toloe, J; Mironov, S L

    2013-01-01

    Excessive stimulation of glutamatergic receptors (GluRs) can overexcite neurons. This can be dampened by KATP channels linking metabolic and neuronal activities, but the cross-talk has not yet been examined on the single channel level. In the brainstem and hippocampal neurons, GluR agonists augmented the open state probability (Popen) of KATP channels with relative efficacy: kainate ≍ AMPA > NMDA > t-ACPD. Inhibition of calcium influx and chelation of intracellular calcium did not modify the effects. Kainate did not augment production of reactive oxygen species measured with roGFP1. H2O2 slightly increased Popen, but GluR effects were not modified. GluR actions were abolished in Na+-free solutions and after blockade of Na+-K+-ATPase. KATP channels in open-cell patch-clamp measurements were inhibited by ATP, stimulated by ADP, and kainate was effective only in the presence of ATP. GluR stimulation enhanced ATP consumption that decreased submembrane ATP levels, whereas metabolic poisoning diminished bulk ATP. Modelling showed strong ATP depletion and ADP accumulation near the membrane, and both effects contributed to Popen increases after GluR stimulation. Kainate and hypoxia activated KATP channels in the functional brainstem slices. Inhibition of aerobic ATP production and GluR stimulation were about equally effective in KATP channel opening during hypoxia. Induction of seizure-like activity in hippocampal slices with Mg2+-free solutions was accompanied by ATP decrease and KATP channel opening. We propose that KATP channels and GluRs are functionally coupled that can regulate long-lasting changes of neuronal activity in the CNS neurons. PMID:23507878

  12. Comparative pharmacokinetics and pharmacodynamics of platelet adenosine diphosphate receptor antagonists and their clinical implications.

    PubMed

    Floyd, Christopher N; Passacquale, Gabriella; Ferro, Albert

    2012-07-01

    Over the last two decades or more, anti-platelet therapy has become established as a cornerstone in the treatment of patients with ischaemic cardiovascular disease, since such drugs effectively reduce arterial thrombotic events. The original agent used in this context was aspirin (acetylsalicylic acid) but, with the advent of adenosine diphosphate (ADP) receptor antagonists, the use of dual anti-platelet therapy has resulted in further improvement in cardiovascular outcomes when compared with aspirin alone. The first group of platelet ADP receptor antagonists to be developed was the thienopyridine class, which comprise inactive pro-drugs that require in vivo metabolism to their active metabolites before exerting their inhibitory effect on the P2Y(12) receptor. Clopidogrel has been the principal ADP receptor antagonist in use over the past decade, but is limited by variability in its in vivo inhibition of platelet aggregation (IPA). The pharmacokinetics of clopidogrel are unpredictable due to their vulnerability to multiple independent factors including genetic polymorphisms. Expression of the 3435T/T genetic variant encoding the MDR1 gene for the P-glycoprotein efflux transporter results in a significantly reduced maximum drug concentration and area under the plasma concentration-time curve as intestinal absorption of clopidogrel is reduced; and the expression of the mutant *2 allele of CYP2C19 results in similar pharmacokinetic effects as the two cytochrome P450 (CYP)-mediated steps required for the production of the active metabolite of clopidogrel are impaired. These variable pharmacokinetics lead to erratic pharmacodynamics and cannot reliably be overcome with increased dosing. Both prasugrel, a third-generation thienopyridine, and ticagrelor, a cyto-pentyl-triazolo-pyrimidine, have more predictable pharmacokinetics and enhanced pharmacodynamics than clopidogrel. Neither appears to be affected by the same genetic polymorphisms as clopidogrel; prasugrel requires

  13. Differential transactivation by orphan nuclear receptor NOR1 and its fusion gene product EWS/NOR1: possible involvement of poly(ADP-ribose) polymerase I, PARP-1.

    PubMed

    Ohkura, Naganari; Nagamura, Yuko; Tsukada, Toshihiko

    2008-10-15

    In extraskeletal myxoid chondrosarcoma, a chromosomal translocation creates a gene fusion between EWS and an orphan nuclear receptor, NOR1. The resulting fusion protein EWS/NOR1 has been believed to lead to malignant transformation by functioning as a transactivator for NOR1-target genes. By comparing the gene expression profiles of NOR1- and EWS/NOR1-overexpressing cells, we found that they largely shared up-regulated genes, but no significant correlation was observed with respect to the transactivation levels of each gene. In addition, the proteins associated with NOR1 and EWS/NOR1 were mostly the same in these cells. The results suggest that these proteins differentially transactivate overlapping target genes through a similar transcriptional machinery. To clarify the mechanisms underlying the transcriptional divergence between NOR1 and EWS/NOR1, we searched for alternatively associated proteins, and identified poly(ADP-ribose) polymerase I (PARP-1) as an NOR1-specific binding protein. Consistent with its binding properties, PARP-1 acted as a transcriptional repressor of NOR1, but not EWS/NOR1, in a luciferase reporter assay employing PARP-1(-/-) fibroblasts. Interestingly, suppressive activity of PARP-1 was observed in a DNA response element-specific manner, and in a subtype-specific manner toward the NR4A family (Nur77, Nurr1, and NOR1), suggesting that PARP-1 plays a role in the diversity of transcriptional regulation mediated by the NR4A family in normal cells. Altogether, our findings suggest that NOR1 and EWS/NOR1 regulate overlapping target genes differently by utilizing associated proteins, including PARP-1; and that EWS/NOR1 may acquire oncogenic activities by avoiding (or gaining) transcription factor-specific modulation by the associated proteins.

  14. Modulation of farnesoid X receptor results in post-translational modification of poly (ADP-ribose) polymerase 1 in the liver

    SciTech Connect

    Zhu, Yan; Li, Guodong; Dong, Yafeng; Zhou, Helen H.; Kong, Bo; Aleksunes, Lauren M.; Richardson, Jason R.; Li, Fei; Guo, Grace L.

    2013-01-15

    The farnesoid X receptor (FXR) is a bile acid-activated transcription factor belonging to the nuclear receptor superfamily. FXR deficiency in mice results in cholestasis, metabolic disorders, and tumorigenesis in liver and intestine. FXR is known to contribute to pathogenesis by regulating gene transcription; however, changes in the post-transcriptional modification of proteins associated with FXR modulation have not been determined. In the current study, proteomic analysis of the livers of wild-type (WT) and FXR knockout (FXR-KO) mice treated with a FXR synthetic ligand or vehicle was performed. The results identified five proteins as novel FXR targets. Since FXR deficiency in mice leads to liver tumorigenesis, poly (ADP-ribose) polymerase family, member 1 (Parp1) that is important for DNA repair, was validated in the current study by quantitative real-time PCR, and 1- and 2-dimensional gel electrophoresis/western blot. The results showed that Parp1 mRNA levels were not altered by FXR genetic status or by agonist treatment. However, total Parp1 protein levels were increased in FXR-KO mice as early as 3 month old. Interestingly, total Parp1 protein levels were increased in WT mice in an age-dependent manner (from 3 to 18 months), but not in FXR-KO mice. Finally, activation of FXR in WT mice resulted in reduction of phosporylated Parp1 protein in the liver without affecting total Parp1 protein levels. In conclusion, this study reveals that FXR genetic status and agonist treatment affects basal levels and phosphorylation state of Parp1, respectively. These alterations, in turn, may be associated with the hepatobiliary alterations observed in FXR-KO mice and participate in FXR agonist-induced protection in the liver. -- Highlights: ► Proteomic analysis identified novel FXR targets. ► FXR modification altered post-translational modification of the Parp1 protein. ► Altered Parp1 function may contribute to mechanisms of FXR regulation of liver functions.

  15. Arg333 and Arg334 in the COOH terminus of the human P2Y1 receptor are crucial for Gq coupling.

    PubMed

    Ding, Zhongren; Tuluc, Florin; Bandivadekar, Kavita R; Zhang, Lili; Jin, Jianguo; Kunapuli, Satya P

    2005-03-01

    The P2Y(1) ADP receptor activates G(q) and causes increases in intracellular Ca(2+) concentration through stimulation of PLC. In this study, we investigated the role of the amino acid residues in the COOH terminus of the human P2Y(1) receptor in G(q) activation. Stimulation of Chinese hamster ovary (CHO-K1) cells stably expressing the wild-type human P2Y(1) receptor (P2Y(1)-WT cells), P2Y(1)-DeltaR340-L373, or P2Y(1)-DeltaD356-L373 with 2-methylthio-ADP (2-MeSADP) caused inositol phosphate production. In contrast, cells expressing P2Y(1)-DeltaT330-L373, a mutant lacking the entire COOH terminus, completely lost their response to 2-MeSADP. Similar data were obtained by using these cell lines and measuring Ca(2+) mobilization upon stimulation with 2-MeSADP, indicating that the 10 amino acids (330TFRRRLSRAT339) in the COOH terminus of the human P2Y(1) receptor are essential for G(q) coupling. Radioligand binding demonstrated that both the P2Y(1)-WT and P2Y(1)-DeltaT330-L373-expressing cells have almost equal binding of [(3)H]MRS2279, a P2Y(1) receptor antagonist, indicating that COOH-terminal truncation did not drastically affect the conformation of the receptor. CHO-K1 cells expressing a chimeric P2Y(12) receptor with the P2Y(1) COOH terminus failed to elicit G(q) functional responses, indicating that the P2Y(1) COOH terminus is essential but not sufficient for G(q) activation. Finally, cells expressing a double-mutant P2Y(1) receptor (R333A/R334A) in the conserved BBXXB region of the COOH terminus of the G(q)-activating P2Y receptors completely lost their functional ability to activate G(q). We conclude that the two arginine residues (R333R334) in the COOH terminus of the human P2Y(1) receptor are essential for G(q) coupling.

  16. Impact of aspirin dose on adenosine diphosphate-mediated platelet activities. Results of an in vitro pilot investigation.

    PubMed

    Tello-Montoliu, Antonio; Thano, Estela; Rollini, Fabiana; Patel, Ronakkumar; Wilson, Ryan E; Muñiz-Lozano, Ana; Franchi, Francesco; Darlington, Andrew; Desai, Bhaloo; Guzman, Luis A; Bass, Theodore A; Angiolillo, Dominick J

    2013-10-01

    Different aspirin dosing regimens have been suggested to impact outcomes when used in combination with adenosine diphosphate (ADP) P2Y12 receptor antagonists. Prior investigations have shown that not only aspirin, but also potent ADP P2Y12 receptor blockade can inhibit thromboxane A2-mediated platelet activation. The impact of aspirin dosing on ADP mediated platelet activities is unknown and represents the aim of this in vitro pilot pharmacodynamic (PD) investigation. Twenty-six patients with stable coronary artery disease on aspirin 81 mg/day and P2Y12 naïve were enrolled. PD assessments were performed at baseline, while patients were on 81 mg/day aspirin and after switching to 325 mg/day for 7 ± 2 days with and without escalating concentrations (vehicle, 1, 3, and 10 μM) of prasugrel's active metabolite (P-AM). PD assays included flow cytometric assessment of VASP to define the platelet reactivity index (PRI) and the Multiplate Analyzer (MEA) using multiple agonists [ADP, ADP + prostaglandin (PGE1), arachidonic acid (AA), and collagen]. Escalating P-AM concentrations showed incremental platelet P2Y12 inhibition measured by VASP-PRI (p<0.001). However, there were no differences according to aspirin dosing regimen at any P-AM concentration (vehicle: p=0.899; 1 μM: p=0.888; 3 μM: p=0.524; 10 μM: p=0.548). Similar findings were observed in purinergic markers assessed by MEA (ADP and ADP+PGE1). P-AM addition significantly reduced AA and collagen induced platelet aggregation (p<0.001 for all measures), irrespective of aspirin dose. In conclusion, aspirin dosing does not appear to affect PD measures of ADP-mediated platelet reactivity irrespective of the degree of P2Y12 receptor blockade. P2Y12 receptor blockade modulates platelet reactivity mediated by alternative activators. PMID:23884248

  17. Historical Lessons in Translational Medicine: Cyclooxygenase Inhibition and P2Y12 Antagonism

    PubMed Central

    Fitzgerald, Desmond J.; FitzGerald, Garret A.

    2013-01-01

    The development of drugs that inhibit platelets has been driven by a combination of clinical insights, fundamental science and sheer luck. The process has evolved as the days of stumbling upon therapeutic gems like aspirin have long passed and have been replaced by an arduous process where a drug is designed to target a specific protein implicated in a well-characterized pathophysiological process. Or so we would like to believe. The development of antiplatelet therapy illustrates the importance of understanding the mechanisms of disease and the pharmacology of the compounds we develop, coupled with careful clinical experimentation and observation. And yes, still, a fair bit of luck. PMID:23287454

  18. ADP's ABCs of Training

    ERIC Educational Resources Information Center

    Weinstein, Margery

    2010-01-01

    When a company's core competence is processing data, it is sometimes easy to lose sight of the obvious--the information right under its nose. In the case of Automatic Data Processing, Inc. (ADP), a business outsourcing company specializing in human resources, payroll, tax, and benefits administrations solutions, that is not a problem. Through…

  19. 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

  20. 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

  1. ADP computer security classification program

    SciTech Connect

    Augustson, S.J.

    1984-01-01

    CG-ADP-1, the Automatic Data Processing Security Classification Guide, provides for classification guidance (for security information) concerning the protection of Department of Energy (DOE) and DOE contractor Automatic Data Processing (ADP) systems which handle classified information. Within the DOE, ADP facilities that process classified information provide potentially lucrative targets for compromise. In conjunction with the security measures required by DOE regulations, necessary precautions must be taken to protect details of those ADP security measures which could aid in their own subversion. Accordingly, the basic principle underlying ADP security classification policy is to protect information which could be of significant assistance in gaining unauthorized access to classified information being processed at an ADP facility. Given this policy, classification topics and guidelines are approved for implementation. The basic program guide, CG-ADP-1 is broad in scope and based upon it, more detailed local guides are sometimes developed and approved for specific sites. Classification topics are provided for system features, system and security management, and passwords. Site-specific topics can be addressed in local guides if needed.

  2. Minocycline blocks asthma-associated inflammation in part by interfering with the T cell receptor-nuclear factor κB-GATA-3-IL-4 axis without a prominent effect on poly(ADP-ribose) polymerase.

    PubMed

    Naura, Amarjit S; Kim, Hogyoung; Ju, Jihang; Rodriguez, Paulo C; Jordan, Joaquin; Catling, Andrew D; Rezk, Bashir M; Abd Elmageed, Zakaria Y; Pyakurel, Kusma; Tarhuni, Abdelmetalab F; Abughazleh, Mohammad Q; Errami, Youssef; Zerfaoui, Mourad; Ochoa, Augusto C; Boulares, A Hamid

    2013-01-18

    Minocycline protects against asthma independently of its antibiotic function and was recently reported as a potent poly(ADP-ribose) polymerase (PARP) inhibitor. In an animal model of asthma, a single administration of minocycline conferred excellent protection against ovalbumin-induced airway eosinophilia, mucus hypersecretion, and Th2 cytokine production (IL-4/IL-5/IL-12(p70)/IL-13/GM-CSF) and a partial protection against airway hyperresponsiveness. These effects correlated with pronounced reduction in lung and sera allergen-specific IgE. A reduction in poly(ADP-ribose) immunoreactivity in the lungs of minocycline-treated/ovalbumin-challenged mice correlated with decreased oxidative DNA damage. The effect of minocycline on PARP may be indirect, as the drug failed to efficiently block direct PARP activation in lungs of N-methyl-N'-nitro-N-nitroso-guanidine-treated mice or H(2)O(2)-treated cells. Minocycline blocked allergen-specific IgE production in B cells potentially by modulating T cell receptor (TCR)-linked IL-4 production at the mRNA level but not through a modulation of the IL-4-JAK-STAT-6 axis, IL-2 production, or NFAT1 activation. Restoration of IL-4, ex vivo, rescued IgE production by minocycline-treated/ovalbumin-stimulated B cells. IL-4 blockade correlated with a preferential inhibition of the NF-κB activation arm of TCR but not GSK3, Src, p38 MAPK, or ERK1/2. Interestingly, the drug promoted a slightly higher Src and ERK1/2 phosphorylation. Inhibition of NF-κB was linked to a complete blockade of TCR-stimulated GATA-3 expression, a pivotal transcription factor for IL-4 expression. Minocycline also reduced TNF-α-mediated NF-κB activation and expression of dependent genes. These results show a potentially broad effect of minocycline but that it may block IgE production in part by modulating TCR function, particularly by inhibiting the signaling pathway, leading to NF-κB activation, GATA-3 expression, and subsequent IL-4 production.

  3. Platelet receptors and patient responses: The contributions of Professor Stan Heptinstall to platelet research.

    PubMed

    Clemetson, Kenneth J

    2015-01-01

    Stan Heptinstall's contributions to platelet research covered organising meetings at the national and European level as well as starting and maintaining the journal "Platelets". The major part of his research addressed problems of inhibition of platelet receptors and the effects of this on patient health. In particular, the effects of P2Y12 inhibitors on patients with acute cardiovascular problems were a major focus. Other studies included the effects of feverfew (Tanacetum parthenium) extracts on platelets, of direct anti-IIb/IIIa receptor (αIIbβ3) inhibitors and of prostanoids on platelet function. Recently, methods for assessing the effectiveness of platelet inhibition were investigated.

  4. RASA3 is a critical inhibitor of RAP1-dependent platelet activation

    PubMed Central

    Stefanini, Lucia; Paul, David S.; Robledo, Raymond F.; Chan, E. Ricky; Getz, Todd M.; Campbell, Robert A.; Kechele, Daniel O.; Casari, Caterina; Piatt, Raymond; Caron, Kathleen M.; Mackman, Nigel; Weyrich, Andrew S.; Parrott, Matthew C.; Boulaftali, Yacine; Adams, Mark D.; Peters, Luanne L.; Bergmeier, Wolfgang

    2015-01-01

    The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the GEF CalDAG-GEFI and an unknown regulator that operates downstream of the adenosine diphosphate (ADP) receptor, P2Y12, a target of antithrombotic therapy. Here, we provide evidence that the GAP, RASA3, inhibits platelet activation and provides a link between P2Y12 and activation of the RAP1 signaling pathway. In mice, reduced expression of RASA3 led to premature platelet activation and markedly reduced the life span of circulating platelets. The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of the gene encoding CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Moreover, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Together, our results indicate that RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling and suggest that P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation at sites of vascular injury. These findings provide insight into the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders. PMID:25705885

  5. Identification and Characterization of Novel Variations in Platelet G-Protein Coupled Receptor (GPCR) Genes in Patients Historically Diagnosed with Type 1 von Willebrand Disease

    PubMed Central

    Leo, Vincenzo C.; Sabi, Essa; Cunningham, Margaret R.; Eikenboom, Jeroen C.; Lethagen, Stefan; Schneppenheim, Reinhard; Goodeve, Anne C.; Watson, Steve P.; Mundell, Stuart J.; Daly, Martina E.

    2015-01-01

    The clinical expression of type 1 von Willebrand disease may be modified by co-inheritance of other mild bleeding diatheses. We previously showed that mutations in the platelet P2Y12 ADP receptor gene (P2RY12) could contribute to the bleeding phenotype in patients with type 1 von Willebrand disease. Here we investigated whether variations in platelet G protein-coupled receptor genes other than P2RY12 also contributed to the bleeding phenotype. Platelet G protein-coupled receptor genes P2RY1, F2R, F2RL3, TBXA2R and PTGIR were sequenced in 146 index cases with type 1 von Willebrand disease and the potential effects of identified single nucleotide variations were assessed using in silico methods and heterologous expression analysis. Seven heterozygous single nucleotide variations were identified in 8 index cases. Two single nucleotide variations were detected in F2R; a novel c.-67G>C transversion which reduced F2R transcriptional activity and a rare c.1063C>T transition predicting a p.L355F substitution which did not interfere with PAR1 expression or signalling. Two synonymous single nucleotide variations were identified in F2RL3 (c.402C>G, p.A134 =; c.1029 G>C p.V343 =), both of which introduced less commonly used codons and were predicted to be deleterious, though neither of them affected PAR4 receptor expression. A third single nucleotide variation in F2RL3 (c.65 C>A; p.T22N) was co-inherited with a synonymous single nucleotide variation in TBXA2R (c.6680 C>T, p.S218 =). Expression and signalling of the p.T22N PAR4 variant was similar to wild-type, while the TBXA2R variation introduced a cryptic splice site that was predicted to cause premature termination of protein translation. The enrichment of single nucleotide variations in G protein-coupled receptor genes among type 1 von Willebrand disease patients supports the view of type 1 von Willebrand disease as a polygenic disorder. PMID:26630678

  6. 45 CFR 95.621 - ADP reviews.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... CFR part 74 and the conditions of this subpart and to determine the efficiency, economy and... claim provisions of 45 CFR part 95, subpart A; and (iv) The service agreement was not previously... of Federal ADP systems and information processing. (2) ADP Security Program. State ADP...

  7. 45 CFR 95.621 - ADP reviews.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... all ADP systems used by State and local governments to administer programs covered under 45 CFR part... 45 Public Welfare 1 2012-10-01 2012-10-01 false ADP reviews. 95.621 Section 95.621 Public Welfare....621 ADP reviews. The Department will conduct periodic onsite surveys and reviews of State and...

  8. 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.

  9. Molecular modeling of the human P2Y14 receptor: A template for structure-based design of selective agonist ligands.

    PubMed

    Trujillo, Kevin; Paoletta, Silvia; Kiselev, Evgeny; Jacobson, Kenneth A

    2015-07-15

    The P2Y14 receptor (P2Y14R) is a Gi protein-coupled receptor that is activated by uracil nucleotides UDP and UDP-glucose. The P2Y14R structure has yet to be solved through X-ray crystallography, but the recent agonist-bound crystal structure of the P2Y12R provides a potentially suitable template for its homology modeling for rational structure-based design of selective and high-affinity ligands. In this study, we applied ligand docking and molecular dynamics refinement to a P2Y14R homology model to qualitatively explain structure-activity relationships of previously published synthetic nucleotide analogues and to probe the quality of P2Y14R homology modeling as a template for structure-based design. The P2Y14R model supports the hypothesis of a conserved binding mode of nucleotides in the three P2Y12-like receptors involving functionally conserved residues. We predict phosphate group interactions with R253(6.55), K277(7.35), Y256(6.58) and Q260(6.62), nucleobase (anti-conformation) π-π stacking with Y102(3.33) and the role of F191(5.42) as a means for selectivity among P2Y12-like receptors. The glucose moiety of UDP-glucose docked in a secondary subpocket at the P2Y14R homology model. Thus, P2Y14R homology modeling may allow detailed prediction of interactions to facilitate the design of high affinity, selective agonists as pharmacological tools to study the P2Y14R.

  10. 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.

  11. 45 CFR 95.621 - ADP reviews.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... the efficiency, economy and effectiveness of the equipment or service. (e) State Agency Maintenance of... appropriate ADP security requirements based on recognized industry standards or standards governing security... all ADP systems used by State and local governments to administer programs covered under 45 CFR...

  12. 45 CFR 95.621 - ADP reviews.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... the efficiency, economy and effectiveness of the equipment or service. (e) State Agency Maintenance of... appropriate ADP security requirements based on recognized industry standards or standards governing security... all ADP systems used by State and local governments to administer programs covered under 45 CFR...

  13. 45 CFR 95.621 - ADP reviews.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... Department of Health and Human Services GENERAL ADMINISTRATION GENERAL ADMINISTRATION-GRANT PROGRAMS (PUBLIC...) Physical security of ADP resources; (B) Equipment security to protect equipment from theft and unauthorized...; (G) Emergency preparedness; and, (H) Designation of an Agency ADP Security Manager. (iii)...

  14. The association of thromboxane A2 receptor with lipid rafts is a determinant for platelet functional responses.

    PubMed

    Moscardó, A; Vallés, J; Latorre, A; Santos, M T

    2014-08-25

    We have investigated the presence of thromboxane A2 (TXA2) receptor associated with lipid rafts in human platelets and the regulation of platelet function in response to TXA2 receptor agonists when lipid rafts are disrupted by cholesterol extraction. Platelet aggregation with TXA2 analogs U46619 and IBOP was almost blunted in cholesterol-depleted platelets, as well as αIIbβ3 integrin activation and P-selectin exposure. Raft disruption also inhibited TXA2-induced cytosolic calcium increase and nucleotide release, ruling out an implication of P2Y12 receptor. An important proportion of TXA2 receptor (40%) was colocalized at lipid rafts. The presence of the TXA2 receptor associated with lipid rafts in platelets is important for functional platelet responses to TXA2.

  15. Characterization of a new peptide agonist of the protease-activated receptor-1.

    PubMed

    Mao, Yingying; Jin, Jianguo; Kunapuli, Satya P

    2008-01-15

    A new peptide (TFRRRLSRATR), derived from the c-terminal of human platelet P2Y(1) receptor, was synthesized and its biological function was evaluated. This peptide activated platelets in a concentration-dependent manner, causing shape change, aggregation, secretion and calcium mobilization. Of the several receptor antagonists tested, only BMS200261, a protease activated receptor 1 (PAR-1) specific antagonist, totally abolished the peptide-induced platelet aggregation, secretion and calcium mobilization. The TFRRR-peptide-pretreated washed platelets failed to aggregate in response to SFLLRN (10 microM) but not to AYPGKF (500 microM). In addition, in mouse platelets, peptide concentrations up to 600 microM failed to cause platelet activation, indicating that the TFRRR-peptide activated platelets through the PAR-1 receptor, rather than through the PAR-4 receptor. The shape change induced by 10 microM peptide was totally abolished by Y-27632, an inhibitor of p160(ROCK) which is a downstream mediator of G12/13 pathways. The TFRRR-peptide, YFLLRNP, and the physiological agonist thrombin selectively activated G12/13 pathways at low concentrations and began to activate both Gq and G12/13 pathways with increasing concentrations. Similar to SFLLRN, the TFRRR-peptide caused phosphorylation of Akt and Erk in a P2Y(12) receptor-dependent manner, and p-38 MAP kinase activation in a P2Y(12)-independent manner. The effects of this peptide are elicited by the first six amino acids (TFRRRL) whereas the remaining peptide (LSRATR), TFERRN, or TFEERN had no effects on platelets. We conclude that TFRRRL activates human platelets through PAR-1 receptors. PMID:17950254

  16. Purinergic Receptors in Thrombosis and Inflammation.

    PubMed

    Hechler, Béatrice; Gachet, Christian

    2015-11-01

    Under various pathological conditions, including thrombosis and inflammation, extracellular nucleotide levels may increase because of both active release and passive leakage from damaged or dying cells. Once in the extracellular compartment, nucleotides interact with plasma membrane receptors belonging to the P2 purinergic family, which are expressed by virtually all circulating blood cells and in most blood vessels. In this review, we focus on the specific role of the 3 platelet P2 receptors P2Y1, P2Y12, and P2X1 in hemostasis and arterial thrombosis. Beyond platelets, these 3 receptors, along with the P2Y2, P2Y6, and P2X7 receptors, constitute the main P2 receptors mediating the proinflammatory effects of nucleotides, which play important roles in various functions of circulating blood cells and cells of the vessel wall. Each of these P2 receptor subtypes specifically contributes to chronic or acute vascular inflammation and related diseases, such as atherosclerosis, restenosis, endotoxemia, and sepsis. The potential for therapeutic targeting of these P2 receptor subtypes is also discussed.

  17. Kinesin ATPase: Rate-limiting ADP release

    SciTech Connect

    Hackney, D.D.

    1988-09-01

    The ATPase rate of kinesin isolated from bovine brain by the method of S.A. Kuznetsov and V.I. Gelfand is stimulated 1000-fold by interaction with tubulin. The tubulin-stimulated reaction exhibits no extra incorporation of water-derived oxygens over a wide range of ATP and tubulin concentrations, indicating that P/sub i/ release is faster than the reversal of hydrolysis. ADP release, however, is slow for the basal reaction and its release is rate limiting as indicated by the very tight ADP binding (K/sub i/ < 5 nM), the retention of a stoichiometric level of bound ADP through ion-exchange chromatography and dialysis, and the reversible labeling of a bound ADP by (/sup 14/C)ATP at the steady-state ATPase rate as shown by centrifuge gel filtration and inaccessibility to pyruvate kinase. Tubulin accelerates the release of the bound ADP consistent with its activation of the net ATPase reaction. The detailed kinetics of ADP release in the presence of tubulin are biphasic indicating apparent heterogeneity with a fraction of the kinesin active sites being unaffected by tubulin.

  18. Kinesin ATPase: Rate-Limiting ADP Release

    NASA Astrophysics Data System (ADS)

    Hackney, David D.

    1988-09-01

    The ATPase rate of kinesin isolated from bovine brain by the method of S. A. Kuznetsov and V. I. Gelfand [(1986) Proc. Natl. Acad. Sci. USA 83, 8530-8534)] is stimulated 1000-fold by interaction with tubulin (turnover rate per 120-kDa peptide increases from ≈ 0.009 sec-1 to 9 sec-1). The tubulin-stimulated reaction exhibits no extra incorporation of water-derived oxygens over a wide range of ATP and tubulin concentrations, indicating that Pi release is faster than the reversal of hydrolysis. ADP release, however, is slow for the basal reaction and its release is rate limiting as indicated by the very tight ADP binding (Ki < 5 nM), the retention of a stoichiometric level of bound ADP through ion-exchange chromatography and dialysis, and the reversible labeling of a bound ADP by [14C]ATP at the steady-state ATPase rate as shown by centrifuge gel filtration and inaccessibility to pyruvate kinase. Tubulin accelerates the release of the bound ADP consistent with its activation of the net ATPase reaction. The detailed kinetics of ADP release in the presence of tubulin are biphasic indicating apparent heterogeneity with a fraction of the kinesin active sites being unaffected by tubulin.

  19. Apical targeting of the P2Y(4) receptor is directed by hydrophobic and basic residues in the cytoplasmic tail.

    PubMed

    DuBose, D Ross; Wolff, Samuel C; Qi, Ai-Dong; Naruszewicz, Izabela; Nicholas, Robert A

    2013-02-01

    The P2Y(4) receptor is selectively targeted to the apical membrane in polarized epithelial cell lines and has been shown to play a key role in intestinal chloride secretion. In this study, we delimit a 23 amino acid sequence within the P2Y(4) receptor C-tail that directs its apical targeting. Using a mutagenesis approach, we found that four hydrophobic residues near the COOH-terminal end of the signal are necessary for apical sorting, whereas two basic residues near the NH(2)-terminal end of the signal are involved to a lesser extent. Interestingly, mutation of the key hydrophobic residues results in a basolateral enrichment of the receptor construct, suggesting that the apical targeting sequence may prevent insertion or disrupt stability of the receptor at the basolateral membrane. The signal is not sequence specific, as an inversion of the 23 amino acid sequence does not disrupt apical targeting. We also show that the apical targeting sequence is an autonomous signal and is capable of redistributing the normally basolateral P2Y(12) receptor, suggesting that the apical signal is dominant over the basolateral signal in the main body of the P2Y(12) receptor. The targeting sequence is unique to the P2Y(4) receptor, and sequence alignments of the COOH-terminal tail of mammalian orthologs reveal that the hydrophobic residues in the targeting signal are highly conserved. These data define the novel apical sorting signal of the P2Y(4) receptor, which may represent a common mechanism for trafficking of epithelial transmembrane proteins.

  20. 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

  1. 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

  2. Effects of epinephrine on ADP-induced changes in platelet inositol phosphates

    SciTech Connect

    Vickers, J.D.; Keraly, C.L.; Kinlough-Rathbone, R.L.; Mustard, J.F.

    1986-03-01

    Epinephrine (EPI) does not aggregate rabbit platelets, but it does increase the labelling of inositol phosphate (IP) at 60s (21%, p < 0.05) in the presence of 20 mM Li/sup +/, in platelets prelabelled with (/sup 3/H) inositol. In contrast, 0.5 ..mu..M ADP which causes aggregation, increases the labelling of inositol bisphosphate (IP/sub 2/) by 30% (p < 0.01) at 10s and by 46% (p < 0.05) at 60s and IP by 26% (p < 0.05) at 60s. The combination of 0.5 ..mu..M ADP and 50 ..mu..M EPI causes more extensive aggregation and increases IP/sub 2/ by 154% (p < 0.01) and IP by 65% (p < 0.01) at 60s. The increase in IP/sub 2/ stimulated by ADP + EPI was greater than the increase caused by ADP (p < 0.05). The authors examined the effects of ..cap alpha..- and ..beta..-adrenergic receptor blockers on EPI + ADP-induced changes in the inositol phosphates. The ..beta..-adrenergic blocker Sotalol (50 ..mu..M), which had no effect by itself, enhanced the accumulation of IP/sub 2/ due to 0.2 ..mu..M ADP + 0.6 ..mu..M EPI by 70% (p < 0.01) at 60s, as well as aggregation. This is consistent with EPI inhibition mediated through stimulation of adenylate cyclase via the ..beta..-adrenergic receptor. The ..cap alpha..-adrenergic blocker phentolamine (50 ..mu..M), reduced aggregation stimulated by 0.5 ..mu..M ADP + 50 ..mu..M EPI, and reduced the accumulation of IP by 53% (p < 0.05) and IP/sub 2/ by 108% (0 < 0.05). These data are compatible with the hypothesis that the effect of EPI on ADP-induced aggregation involves IP/sub 2/ metabolism stimulated via the ..cap alpha..-adrenergic receptor.

  3. Raman gains of ADP and KDP crystals

    NASA Astrophysics Data System (ADS)

    Zhou, Hai-Liang; Zhang, Qing-Hua; Wang, Bo; Xu, Xin-Guang; Wang, Zheng-Ping; Sun, Xun; Zhang, Fang; Zhang, Li-Song; Liu, Bao-An; Chai, Xiang-Xu

    2015-04-01

    In this paper, the Raman gain coefficients of ammonium dihydrogen phosphate (ADP) and potassium dihydrogen phosphate (KDP) crystals are measured. By using a pump source of a 30-ps, 532-nm laser, the gain coefficients of ADP and KDP are 1.22 cm/GW, and 0.91 cm/GW, respectively. While for a 20-ps, 355-nm pump laser, the gain coefficients of these two crystals are similar, which are 1.95 cm/GW for ADP and 1.86 for KDP. The present results indicate that for ultra-violet frequency conversion, the problem of stimulated Raman scattering for ADP crystal will not be more serious than that for KDP crystal. Considering other advantages such the larger nonlinear optical coefficient, higher laser damage threshold, and lower noncritical phase-matching temperature, it can be anticipated that ADP will be a powerful competitor to KDP in large aperture, high energy third-harmonic generation or fourth-harmonic generation applications. Project supported by the National Natural Science Foundation of China (Grant Nos. 51323002 and 51402173), the Independent Innovation Foundation of Shandong University, China (Grant Nos. IIFSDU and 2012JC016), the Program for New Century Excellent Talents in University, China (Grant No. NCET-10-0552), the Fund from the Key Laboratory of Neutron Physics, China Academy of Engineering Physics (Grant No. 2014BB07), and the Natural Science Foundation for Distinguished Young Scholar of Shandong Province, China (Grant No. JQ201218).

  4. Antiplatelet therapy: thrombin receptor antagonists

    PubMed Central

    Tello-Montoliu, Antonio; Tomasello, Salvatore D; Ueno, Masafumi; Angiolillo, Dominick J

    2011-01-01

    Activated platelets stimulate thrombus formation in response to rupture of an atherosclerotic plaque or endothelial cell erosion, promoting atherothrombotic disease. Multiple pathways contribute to platelet activation. Aspirin, an irreversible inhibitor of thromboxane A2 synthesis, in combination with clopidogrel, an inhibitor of P2Y12 adenosine diphosphate platelet receptors, represent the current standard-of-care of antiplatelet therapy for patients with acute coronary syndrome and for those undergoing percutaneous coronary intervention. Although these agents have demonstrated significant clinical benefit, the increased risk of bleeding and the recurrence of thrombotic events represent substantial limitations. Thrombin is one of the most important platelet activators. The inhibition of protease-activated receptor 1 showed a good safety profile in preclinical studies. In fact, phase II studies with vorapaxar (SCH530348) and atopaxar (E5555) showed no increase of bleeding events in addition to the current standard-of-care of antiplatelet therapy. Although the results of phase III trials for both drugs are awaited, this family is a promising new addition to the current clinical practice for patients with atherothrombotic disease, not only as an alternative, but also as additional therapy. PMID:21906120

  5. 45 CFR 95.619 - Use of ADP systems.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... PROGRAMS (PUBLIC ASSISTANCE, MEDICAL ASSISTANCE AND STATE CHILDREN'S HEALTH INSURANCE PROGRAMS) Automatic... Conditions for Ffp § 95.619 Use of ADP systems. ADP systems designed, developed, or installed with FFP...

  6. Dielectric, thermal and mechanical properties of ADP doped PVA composites

    NASA Astrophysics Data System (ADS)

    Naik, Jagadish; Bhajantri, R. F.; Ravindrachary, V.; Rathod, Sunil G.; Sheela, T.; Naik, Ishwar

    2015-06-01

    Polymer composites of poly(vinyl alcohol) (PVA), doped with different concentrations of ammonium dihydrogen phosphate (ADP) has been prepared by solution casting. The formation of complexation between ADP and PVA was confirmed with the help of Fourier transforms infrared (FTIR) spectroscopy. Thermogravimetric analysis (TGA) shows thermal stability of the prepared composites. Impedance analyzer study revealed the increase in dielectric constant and loss with increase the ADP concentration and the strain rate of the prepared composites decreases with ADP concentration.

  7. ADP--A Must in the Secondary School

    ERIC Educational Resources Information Center

    Majernik, John A.

    1974-01-01

    The rationale for including automated data processing (ADP) in secondary schools is given. ADP instruction: prepares students for data processing employment and for advanced ADP study, aids all students preparing for business careers, aids students in choosing a career, provides consumer information, and adds realism to other classroom…

  8. 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

  9. The fate of P2Y-related orphan receptors: GPR80/99 and GPR91 are receptors of dicarboxylic acids.

    PubMed

    Gonzalez, Nathalie Suarez; Communi, Didier; Hannedouche, Sébastien; Boeynaems, Jean-Marie

    2004-12-01

    Several orphan G protein-coupled receptors are structurally close to the family of P2Y nucleotide receptors: GPR80/99 and GPR91 are close to P2Y(1/2/4/6/11) receptors, whereas GPR87, H963 and GPR34 are close to P2Y(12/13/14). Over the years, several laboratories have attempted without success to identify the ligands of those receptors. In early 2004, two papers have been published: One claiming that GPR80/99 is an AMP receptor, called P2Y(15), and the other one showing that GPR80/99 is a receptor for alpha-ketoglutarate, while GPR91 is a succinate receptor. The accompanying paper by Qi et al. entirely supports that GPR80/99 is an alpha-ketoglutarate receptor and not an AMP receptor. The closeness of dicarboxylic acid and P2Y nucleotide receptors might be linked to the negative charges of both types of ligands and the involvement of conserved Arg residues in their neutralization. PMID:18404396

  10. Oscillations in cytosolic free Ca2+ induced by ADP and ATP in single rat hepatocytes display differential sensitivity to application of phorbol ester.

    PubMed Central

    Dixon, C J; Cobbold, P H; Green, A K

    1995-01-01

    We have previously described differences in the oscillatory responses of cytosolic free Ca2+ concentration ([Ca2+]i) in hepatocytes to ADP and ATP, which we have interpreted as evidence that these two nucleotides are acting at distinct receptors. We show here that ADP- and ATP-induced oscillations are differentially sensitive to application of the phorbol ester 4 beta-phorbol 12,13-dibutyrate (PDB). ADP-induced [Ca2+]i oscillations are abolished by low concentrations of PDB (5-10 nM), whereas ATP-induced oscillations of long duration are refractory to PDB, even at greatly elevated concentrations (100 nM). The data illustrate a further difference in the actions of ADP and ATP, strengthening the argument that these agonists are not acting at the same receptor on rat hepatocytes. PMID:7619050

  11. Novel Oral P2Y12 Inhibitor Prasugrel vs. Clopidogrel in Patients with Acute Coronary Syndrome: Evidence Based on 6 Studies

    PubMed Central

    Jia, Min; Li, Zaibo; Chu, Hongtao; Li, Lin; Chen, Keyong

    2015-01-01

    Background Whether prasugrel can take the place of clopidogrel for patients with acute coronary syndrome (ACS) is not clear. The aim of this study was to perform a meta-analysis for systematically reviewing the evidence on prasugrel in comparison to clopidogrel in patients with ACS. Material/Methods Relevant prospective and retrospective studies were searched in databases. Six studies were finally included. Pooled risk ratios (RRs) and 95% confidence intervals (CIs) were calculated to assess all causes of death, myocardial infarction (MI), stroke, major bleeding, major/minor bleeding, and stent thrombosis (for PCI performed). Results Compared with clopidogrel, prasugrel had similar risks of all cause of death (Pooled RR: 0.83; 95% CI: 0.64–1.06, p=0.14, I2=55%), MI (Pooled RR: 0.86; 95% CI: 0.71–1.04, p=0.12) and stroke (pooled RR: 0.88; 95% CI: 0.70–1.10, p=0.25). However, prasugrel was associated with significantly higher risk of both major bleeding (Pooled RR: 1.19; 95% CI: 0.99–1.44, p=0.06, I2=0%) and the risk of total major and minor bleeding (Pooled RR: 1.30; 95% CI: 1.15–1.48, p<0.0001, I2=0%). For the patients who underwent percutaneous coronary intervention (PCI), prasugrel was associated with significantly lower risk of stent thrombosis (Pooled RR: 0.47; 95% CI: 0.34–0.61, p<0.00001, I2=0%). Conclusions Prasugrel has similar effects as clopidogrel in terms of all causes of death, MI, and stroke in ACS patients. For the patients who underwent PCI, prasugrel contributes to lower risk of stent thrombosis. However, prasugrel is associated with significantly higher risk of bleeding. For the patients with active pathological bleeding or a history of stroke and/or TIA, prasugrel should not be recommended. PMID:25893318

  12. 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.

  13. Presence of poly (ADP-ribose) polymerase and poly (ADP-ribose) glycohydrolase in the dinoflagellate Crypthecodinium cohnii.

    PubMed

    Werner, E; Sohst, S; Gropp, F; Simon, D; Wagner, H; Kröger, H

    1984-02-15

    Poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase have been detected in chromatin extracts from the dinoflagellate Crypthecodinium cohnii. Poly(ADP-ribose) glycohydrolase was detected by the liberation of ADP-ribose from poly(ADP-ribose). Poly(ADP-ribose) polymerase was proved by (a) demonstration of phosphoribosyl-AMP in the phosphodiesterase digest of the reaction product, (b) demonstration of ADP-ribose oligomers by fractionation of the reaction product on DEAE-Sephadex. The (ADP-ribose)-protein transfer is dependent on DNA; it is inhibited by nicotinamide, thymidine, theophylline and benzamide. The protein-(ADP-ribose bond is susceptible to 0.1 M NaOH (70%) and 0.4 M NH2OH (33%). Dinoflagellates, nucleated protists, are unique in that their chromatin lacks histones and shows a conformation like bacterial chromatin [Loeblich, A. R., III (1976) J. Protozool. 23, 13--28]; poly(ADP-ribose) polymerase, however, has been found only in eucaryotes. Thus our results suggest that histones were not relevant to the establishment of poly(ADP-ribose) during evolution.

  14. 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.

  15. 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

  16. Pancreatic β-Cell Death, Regeneration and Insulin Secretion: Roles of Poly(ADP-Ribose) Polymerase and Cyclic ADP-Ribose

    PubMed Central

    Takasawa, Shin; Okamoto, Hiroshi

    2002-01-01

    In the early 1980s, we proposed a unifying model for β-cell damage (The OKAMOTO model), in which poly(ADP-ribose) synthetase/ polymerase (PARP) activation plays an essential role in the consumption of NAD+, which leads to energy depletion and necrotic cell death. In 1984, we demonstrated that the administration of PARP inhibitors to 90% depancreatized rats induces islet regeneration. From the regenerating islet-derived cDNA library we isolated Reg (Regenerating Gene) and demonstrated that Reg protein induces βcell replication via the Reg receptor and ameliorates experimental diabetes. More recently, we showed that the combined addition of IL-6 and dexamethasone induces the Reg gene expression in β-cells and that PARP inhibitors enhance the expression. In 1993, we found that cyclic ADP-ribose (cADPR), a product synthesized from NAD+, is a second messenger for intracellular Ca+ mobilization for insulin secretion by glucose, and proposed a novel mechanism of insulin secretion, the CD38-cADPR signal system. Therefore, PARP inhibitors prevent β-cell necrosis, induce β-cell replication and maintain insulin secretion. In this paper, we would like to present a perspective view based on our studies concerning cell death, cell regeneration, and cell function, especially on insulin-producing pancreatic βcells, in the processes of which poly(ADPribose) synthetase/polymerase (PARP) and cyclic ADP-ribose (cADPR) are functioning. PMID:11991201

  17. Discovery of 2-(phenoxypyridine)-3-phenylureas as small molecule P2Y1 antagonists.

    PubMed

    Chao, Hannguang; Turdi, Huji; Herpin, Timothy F; Roberge, Jacques Y; Liu, Yalei; Schnur, Dora M; Poss, Michael A; Rehfuss, Robert; Hua, Ji; Wu, Qimin; Price, Laura A; Abell, Lynn M; Schumacher, William A; Bostwick, Jeffrey S; Steinbacher, Thomas E; Stewart, Anne B; Ogletree, Martin L; Huang, Christine S; Chang, Ming; Cacace, Angela M; Arcuri, Maredith J; Celani, Deborah; Wexler, Ruth R; Lawrence, R Michael

    2013-02-28

    Two distinct G protein-coupled purinergic receptors, P2Y1 and P2Y12, mediate ADP-driven platelet activation. The clinical effectiveness of P2Y12 blockade is well established. Recent preclinical data suggest that P2Y1 and P2Y12 inhibition provide equivalent antithrombotic efficacy, while targeting P2Y1 has the potential for reduced bleeding liability. In this account, the discovery of a 2-(phenoxypyridine)-3-phenylurea chemotype that inhibited ADP-mediated platelet aggregation in human blood samples is described. Optimization of this series led to the identification of compound 16, 1-(2-(2-tert-butylphenoxy)pyridin-3-yl)-3-4-(trifluoromethoxy)phenylurea, which demonstrated a 68 ± 7% thrombus weight reduction in an established rat arterial thrombosis model (10 mg/kg plus 10 mg/kg/h) while only prolonging cuticle and mesenteric bleeding times by 3.3- and 3.1-fold, respectively, in provoked rat bleeding time models. These results suggest that a P2Y1 antagonist could potentially provide a safe and efficacious antithrombotic profile. PMID:23368907

  18. 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.

  19. ADP Analysis project for the Human Resources Management Division

    NASA Technical Reports Server (NTRS)

    Tureman, Robert L., Jr.

    1993-01-01

    The ADP (Automated Data Processing) Analysis Project was conducted for the Human Resources Management Division (HRMD) of NASA's Langley Research Center. The three major areas of work in the project were computer support, automated inventory analysis, and an ADP study for the Division. The goal of the computer support work was to determine automation needs of Division personnel and help them solve computing problems. The goal of automated inventory analysis was to find a way to analyze installed software and usage on a Macintosh. Finally, the ADP functional systems study for the Division was designed to assess future HRMD needs concerning ADP organization and activities.

  20. Pravastatin and C reactive protein modulate protease- activated receptor-1 expression in vitro blood platelets.

    PubMed

    Chu, L-X; Zhou, S-X; Yang, F; Qin, Y-Q; Liang, Z-S; Mo, C-G; Wang, X-D; Xie, J; He, L-P

    2016-01-01

    Protease-activated receptor-1 (PAR-1) plays an important role in mediating activation of human platelets by thrombin. However, mechanism of statin in ADP-induced platelet PAR-1 expression is also unknown. Aggregometry, flow cytometry, immunoblotting and ELISA were used to determine role of pravastatin participating in ADP-induced platelet activation and PAR-1 expression. ADP stimulation significantly increased PAR-1 expression on platelets. PAR-1 antagonist SCH-79797 inhibited platelet aggregation as well as decreased platelet P-selectin expression induced by ADP. CRP inhibited PAR-1 expression induced by ADP in a concentration-dependent manner. Pravastatin treatment reduced PAR-1 expression in a concentration-dependent manner. Combination treatment of CRP and Pravastatin significantly reduced platelet PAR-1 expression induced by ADP. By western-blot analysis, pravastatin treatment did not influence total PAR-1 after ADP treatment. CRP decreased platelet total PAR-1 expression induced by ADP. Pravastatin and CRP reduced TXB2 formation by ADP significantly. CRP decreased thrombin fragment F1+2 level with ADP treatment. Pravastatin, in contrast, did not influence F1+2 level. Upon treatment with Pravastatin reduced platelet LOX-1 expression induced by ADP. In conclusion, PAR-1 served as a critical mechanism to relay platelet activation process induced by ADP. CRP and pravastatin reduce PAR-1 expression in platelet by ADP pathway. PMID:26950455

  1. Nudix hydrolases degrade protein-conjugated ADP-ribose

    PubMed Central

    Daniels, Casey M.; Thirawatananond, Puchong; Ong, Shao-En; Gabelli, Sandra B.; Leung, Anthony K. L.

    2015-01-01

    ADP-ribosylation refers to the transfer of the ADP-ribose group from NAD+ to target proteins post-translationally, either attached singly as mono(ADP-ribose) (MAR) or in polymeric chains as poly(ADP-ribose) (PAR). Though ADP-ribosylation is therapeutically important, investigation of this protein modification has been limited by a lack of proteomic tools for site identification. Recent work has demonstrated the potential of a tag-based pipeline in which MAR/PAR is hydrolyzed down to phosphoribose, leaving a 212 Dalton tag at the modification site. While the pipeline has been proven effective by multiple groups, a barrier to application has become evident: the enzyme used to transform MAR/PAR into phosphoribose must be purified from the rattlesnake Crotalus adamanteus venom, which is contaminated with proteases detrimental for proteomic applications. Here, we outline the steps necessary to purify snake venom phosphodiesterase I (SVP) and describe two alternatives to SVP—the bacterial Nudix hydrolase EcRppH and human HsNudT16. Importantly, expression and purification schemes for these Nudix enzymes have already been proven, with high-quality yields easily attainable. We demonstrate their utility in identifying ADP-ribosylation sites on Poly(ADP-ribose) Polymerase 1 (PARP1) with mass spectrometry and discuss a structure-based rationale for this Nudix subclass in degrading protein-conjugated ADP-ribose, including both MAR and PAR. PMID:26669448

  2. 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

  3. 42 CFR 457.230 - FFP for State ADP expenditures.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... (CONTINUED) STATE CHILDREN'S HEALTH INSURANCE PROGRAMS (SCHIPs) ALLOTMENTS AND GRANTS TO STATES General... ADP expenditures for the design, development, or installation of mechanized claims processing and... procedures regarding the availability of FFP for ADP expenditures are in 45 CFR part 74, 45 CFR part...

  4. 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

  5. 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.

  6. ADP is a vasodilator component from Lasiodora sp. mygalomorph spider venom.

    PubMed

    Horta, C C; Rezende, B A; Oliveira-Mendes, B B R; Carmo, A O; Capettini, L S A; Silva, J F; Gomes, M T; Chávez-Olórtegui, C; Bravo, C E S; Lemos, V S; Kalapothakis, E

    2013-09-01

    Members of the spider genus Lasiodora are widely distributed in Brazil, where they are commonly known as caranguejeiras. Lasiodora spider venom is slightly harmful to humans. The bite of this spider causes local pain, edema and erythema. However, Lasiodora sp. spider venom may be a source of important pharmacological tools. Our research group has described previously that Lasiodora sp. venom produces bradycardia in the isolated rat heart. In the present work, we sought to evaluate the vascular effect of Lasiodora sp. venom and to isolate the vasoactive compounds from the venom. The results showed that Lasiodora spider venom induced a concentration-dependent vasodilation in rat aortic rings, which was dependent on the presence of a functional endothelium and abolished by the nitric oxide synthase (NOS) inhibitor L-NAME. Western blot experiments revealed that the venom also increased endothelial NOS function by increasing phosphorylation of the Ser¹¹⁷⁷ residue. Assay-directed fractionation isolated a vasoactive fraction from Lasiodora sp. venom. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) assays identified a mixture of two compounds: adenosine diphosphate (ADP, approximately 90%) and adenosine monophosphate (AMP, approximately 10%). The vasodilator effects of Lasiodora sp. whole venom, as well as ADP, were significantly inhibited by suramin, which is a purinergic P2-receptor antagonist. Therefore, the results of the present work indicate that ADP is a main vasodilator component of Lasiodora sp. spider venom.

  7. 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

  8. Consensus and update on the definition of on-treatment platelet reactivity to adenosine diphosphate associated with ischemia and bleeding.

    PubMed

    Tantry, Udaya S; Bonello, Laurent; Aradi, Daniel; Price, Matthew J; Jeong, Young-Hoon; Angiolillo, Dominick J; Stone, Gregg W; Curzen, Nick; Geisler, Tobias; Ten Berg, Jurrien; Kirtane, Ajay; Siller-Matula, Jolanta; Mahla, Elisabeth; Becker, Richard C; Bhatt, Deepak L; Waksman, Ron; Rao, Sunil V; Alexopoulos, Dimitrios; Marcucci, Rossella; Reny, Jean-Luc; Trenk, Dietmar; Sibbing, Dirk; Gurbel, Paul A

    2013-12-17

    Dual antiplatelet therapy with aspirin and a P2Y12 receptor blocker is a key strategy to reduce platelet reactivity and to prevent thrombotic events in patients treated with percutaneous coronary intervention. In an earlier consensus document, we proposed cutoff values for high on-treatment platelet reactivity to adenosine diphosphate (ADP) associated with post-percutaneous coronary intervention ischemic events for various platelet function tests (PFTs). Updated American and European practice guidelines have issued a Class IIb recommendation for PFT to facilitate the choice of P2Y12 receptor inhibitor in selected high-risk patients treated with percutaneous coronary intervention, although routine testing is not recommended (Class III). Accumulated data from large studies underscore the importance of high on-treatment platelet reactivity to ADP as a prognostic risk factor. Recent prospective randomized trials of PFT did not demonstrate clinical benefit, thus questioning whether treatment modification based on the results of current PFT platforms can actually influence outcomes. However, there are major limitations associated with these randomized trials. In addition, recent data suggest that low on-treatment platelet reactivity to ADP is associated with a higher risk of bleeding. Therefore, a therapeutic window concept has been proposed for P2Y12 inhibitor therapy. In this updated consensus document, we review the available evidence addressing the relation of platelet reactivity to thrombotic and bleeding events. In addition, we propose cutoff values for high and low on-treatment platelet reactivity to ADP that might be used in future investigations of personalized antiplatelet therapy.

  9. Poly (ADP-ribose) polymerase inhibitor: an evolving paradigm in the treatment of prostate cancer.

    PubMed

    Zhang, Jingsong

    2014-01-01

    Recent phase I studies have reported single-agent activities of poly (ADP-ribose) polymerase (PARP) inhibitor in sporadic and in BRCA-mutant prostate cancers. Two of the most common genetic alterations in prostate cancer, ETS gene rearrangement and loss of PTEN, have been linked to increased sensitivity to PARP inhibitor in preclinical models. Emerging evidence also suggests that PARP1 plays an important role in mediating the transcriptional activities of androgen receptor (AR) and ETS gene rearrangement. In this article, the preclinical work and early-phase clinical trials in developing PARP inhibitor-based therapy as a new treatment paradigm for metastatic prostate cancer are reviewed.

  10. Imaging changes in the cytosolic ATP-to-ADP ratio.

    PubMed

    Tantama, Mathew; Yellen, Gary

    2014-01-01

    Adenosine triphosphate (ATP) is a central metabolite that plays fundamental roles as an energy transfer molecule, a phosphate donor, and a signaling molecule inside the cells. The phosphoryl group transfer potential of ATP provides a thermodynamic driving force for many metabolic reactions, and phosphorylation of both small metabolites and large proteins can serve as a regulatory modification. In the process of phosphoryl transfer from ATP, the diphosphate ADP is produced, and as a result, the ATP-to-ADP ratio is an important physiological control parameter. The ATP-to-ADP ratio is directly proportional to cellular energy charge and phosphorylation potential. Furthermore, several ATP-dependent enzymes and signaling proteins are regulated by ADP, and their activation profiles are a function of the ATP-to-ADP ratio. Finally, regeneration of ATP from ADP can serve as an important readout of energy metabolism and mitochondrial function. We, therefore, developed a genetically encoded fluorescent biosensor tuned to sense ATP-to-ADP ratios in the physiological range of healthy mammalian cells. Here, we present a protocol for using this biosensor to visualize energy status using live-cell fluorescence microscopy.

  11. Molecular cloning of ADP-glucose pyrophosphorylase from cyanobacteria

    SciTech Connect

    Kakefuda, G.; Yeeyung Charng; Iglesias, A.; McIntosh, L. )

    1991-05-01

    Bacterial and higher plant ADP-glucose pyrophosphorylase differ in structure (homotetramer vs heterotetramer respectively) and allosteric activator and inhibitor. However, highly conserved regions can be identified when sequence comparisons are made between ADP-glucose pyrophosphorylases from diverse species. The fructose 1,6 bisphosphate binding site (activator site) in E. coli is highly conserved in all species for which ADP-glucose pyrophosphorylase has been sequenced. A second conserved region, which is labeled by 8-azido-ATP, is also highly conserved in bacteria and higher plants. In previously cloned ADP-glucose pyrophosphorylases the two conserved regions are separated by approximately 80 amino acids. The authors have used these conserved amino acid sequences to design degenerate oligonucleotide primers for polymerase chain reaction amplification (PCR) of part of the ADP-glucose pyrophosphorylase geae. A predicted 240 bp fragment is amplified in PCR reactions using Anabaena sp. PCC 7120 and Synechocystis sp. PCC 6803 genomic DNA as template. The deduced amino acid sequence from the 240 bp Anabaena fragment shares 75 and 76% identity to that of the rice endosperm and spinach leaf ADP-glucose pyrophosphorylases respectively. The Anabaena amino acid sequence shares 42% identity in amino acid sequence to the E. coli enzyme. At the nucleotide level there is 66% identity of the Anabaena sequence to rice endosperm ADP-glucose pyrophosphorylase and 54% to the E. coli gene. The PCR amplified fragments are being used to screen respective Anabaena and Synechocystis genomic gene libraries.

  12. Ticagrelor--a new platelet aggregation inhibitor in patients with acute coronary syndromes. An improvement of other inhibitors?

    PubMed

    Kowalczyk, Mariusz; Banach, Maciej; Mikhailidis, Dimitri P; Hannam, Simon; Rysz, Jacek

    2009-12-01

    Antiplatelet agents play an essential role in the treatment of acute coronary syndrome (ACS). Thienopyridines are a class of drugs that function via inhibition of the adenosine diphosphate (ADP) P2Y12 platelet receptors. Currently, clopidogrel, a second generation thienopyridine, is the main drug of choice and the combination of aspirin and clopidogrel is administered orally for the treatment of ACS. Clopidogrel, is a pro-drug that needs to be metabolized in the liver and intestines to form active metabolites. Prasugrel, a third generation thienopyridine, was approved for use in Europe in February 2009, and is currently available in the United Kingdom. All thienopyridines however, have pharmacological limitations that lead to a search for more effective non-thienopyridine P2Y12 inhibitors. Promising results have been reported with ticagrelor, an oral first reversible, direct-acting inhibitor of the P2Y12 receptor. Ticagrelor is the first oral P2Y12 receptor binding antagonist that does not require metabolic activation. Furthermore, ticagrelor has at last 1 active metabolite, which has very similar pharmacokinetics to the parent compound. Therefore, ticagrelor has more rapid onset and more pronounced platelet inhibition than other antiplatelet agents. The safety and efficacy of ticagrelor compared with clopidogrel in ACS patient has been recently evaluated by the PLATelet inhibition and patient Outcomes (PLATO) trial. Ticagrelor compared with clopidogrel had a significantly greater reduction in the death rate from vascular causes, myocardial infarction, or stroke without major bleeding. There was however, an increase in non-procedure related bleeding, dyspnoea and ventricular pauses in the first week of treatment. Further studies on new antiplatelet agents are needed to establish a new "gold standard" antiplatelet therapy. PMID:19946242

  13. The NLRP3 inflammasome is activated by nanoparticles through ATP, ADP and adenosine

    PubMed Central

    Baron, L; Gombault, A; Fanny, M; Villeret, B; Savigny, F; Guillou, N; Panek, C; Le Bert, M; Lagente, V; Rassendren, F; Riteau, N; Couillin, I

    2015-01-01

    The NLR pyrin domain containing 3 (NLRP3) inflammasome is a major component of the innate immune system, but its mechanism of activation by a wide range of molecules remains largely unknown. Widely used nano-sized inorganic metal oxides such as silica dioxide (nano-SiO2) and titanium dioxide (nano-TiO2) activate the NLRP3 inflammasome in macrophages similarly to silica or asbestos micro-sized particles. By investigating towards the molecular mechanisms of inflammasome activation in response to nanoparticles, we show here that active adenosine triphosphate (ATP) release and subsequent ATP, adenosine diphosphate (ADP) and adenosine receptor signalling are required for inflammasome activation. Nano-SiO2 or nano-TiO2 caused a significant increase in P2Y1, P2Y2, A2A and/or A2B receptor expression, whereas the P2X7 receptor was downregulated. Interestingly, IL-1β secretion in response to nanoparticles is increased by enhanced ATP and ADP hydrolysis, whereas it is decreased by adenosine degradation or selective A2A or A2B receptor inhibition. Downstream of these receptors, our results show that nanoparticles activate the NLRP3 inflammasome via activation of PLC-InsP3 and/or inhibition of adenylate cyclase (ADCY)-cAMP pathways. Finally, a high dose of adenosine triggers inflammasome activation and IL-1β secretion through adenosine cellular uptake by nucleotide transporters and by its subsequent transformation in ATP by adenosine kinase. In summary, we show for the first time that extracellular adenosine activates the NLRP3 inflammasome by two ways: by interacting with adenosine receptors at nanomolar/micromolar concentrations and through cellular uptake by equilibrative nucleoside transporters at millimolar concentrations. These findings provide new molecular insights on the mechanisms of NLRP3 inflammasome activation and new therapeutic strategies to control inflammation. PMID:25654762

  14. 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.

  15. ADP study of the structure of the IUE halo

    NASA Technical Reports Server (NTRS)

    Massa, Derck

    1992-01-01

    Results of a two year ADP study of gas in the Galactic halo are presented. This is partly a summary of 2 papers which were published in referred journals and partly a discussion of work currently underway.

  16. 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

  17. Mono-ADP-ribosyltransferase as a potential pharmacological drug target in the GLP-1 based therapy of obesity and diabetes mellitus type 2.

    PubMed

    Bavec, Aljosa

    2013-01-01

    Glucagon-like peptide-1 (GLP-1) based therapy is well established for treating diabetes mellitus type 2. Moreover, GLP-1 receptor agonists influence weight loss, and have potential for treating obesity. GLP-1 receptor agonists should be administered in low doses, together with drugs that potentiate insulin release, to avoid some minor side effects. We have focused on incretin hormones, especially GLP-1 and its analogues. Here we discuss the effect of the third intracellular loop-derived peptide of GLP-1 receptor on intracellular mono-ADP-ribosyltransferase and its role in regulating the receptor. We suggest that this intracellular mono-ADP-ribosyltransferase could constitute a possible novel pharmacological target in the treatment of diabetes mellitus type 2 and obesity.

  18. Automated Data Processing (ADP) research and development

    SciTech Connect

    Dowla, F.U.; Kohlhepp, V.N.; Leach, R.R. Jr.

    1995-07-01

    Monitoring a comprehensive test ban treaty (CTBT) will require screening tens of thousands of seismic events each year. Reliable automated data analysis will be essential in keeping up with the continuous stream of events that a global monitoring network will detect. We are developing automated event location and identification algorithms by looking at the gaps and weaknesses in conventional ADP systems and by taking advantage of modem computational paradigms. Our research focus is on three areas: developing robust algorithms for signal feature extraction, integrating the analysis of critical measurements, and exploiting joint estimation techniques such as using data from acoustic, hydroacoustic, and seismic sensors. We identify several important problems for research and development; e.g., event location with approximate velocity models and event identification in the presence of outliers. We are employing both linear and nonlinear methods and advanced signal transform techniques to solve these event monitoring problems. Our goal is to increase event-interpretation throughput by employing the power and efficiency of modem computational techniques, and to improve the reliability of automated analysis by reducing the rates of false alarms and missed detections.

  19. 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

  20. 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

  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. Structural basis for lack of ADP-ribosyltransferase activity in poly(ADP-ribose) polymerase-13/zinc finger antiviral protein.

    PubMed

    Karlberg, Tobias; Klepsch, Mirjam; Thorsell, Ann-Gerd; Andersson, C David; Linusson, Anna; Schüler, Herwig

    2015-03-20

    The mammalian poly(ADP-ribose) polymerase (PARP) family includes ADP-ribosyltransferases with diphtheria toxin homology (ARTD). Most members have mono-ADP-ribosyltransferase activity. PARP13/ARTD13, also called zinc finger antiviral protein, has roles in viral immunity and microRNA-mediated stress responses. PARP13 features a divergent PARP homology domain missing a PARP consensus sequence motif; the domain has enigmatic functions and apparently lacks catalytic activity. We used x-ray crystallography, molecular dynamics simulations, and biochemical analyses to investigate the structural requirements for ADP-ribosyltransferase activity in human PARP13 and two of its functional partners in stress granules: PARP12/ARTD12, and PARP15/BAL3/ARTD7. The crystal structure of the PARP homology domain of PARP13 shows obstruction of the canonical active site, precluding NAD(+) binding. Molecular dynamics simulations indicate that this closed cleft conformation is maintained in solution. Introducing consensus side chains in PARP13 did not result in 3-aminobenzamide binding, but in further closure of the site. Three-dimensional alignment of the PARP homology domains of PARP13, PARP12, and PARP15 illustrates placement of PARP13 residues that deviate from the PARP family consensus. Introducing either one of two of these side chains into the corresponding positions in PARP15 abolished PARP15 ADP-ribosyltransferase activity. Taken together, our results show that PARP13 lacks the structural requirements for ADP-ribosyltransferase activity.

  3. Quantification of the Blood Platelet Reactivity in the ADP-Induced Model of Non-Lethal Pulmonary Thromboembolism in Mice with the Use of Laser Doppler Flowmetry

    PubMed Central

    Przygodzki, Tomasz; Talar, Marcin; Blazejczyk, Agnieszka; Kalchenko, Vyacheslav; Watala, Cezary

    2016-01-01

    Introduction The paper describes an alternative method for quantification of in vivo ADP-induced thromboembolism. The aim of the studies was to develop a method of quantification which would not require either extravasation or labelling of platelets. Our proposed approach is based on the monitoring of changes of blood flow with the use of laser Doppler flowmetry. Materials and Methods Mice of C57Bl strain were used in the study. ADP was injected to the vena cava and blood flow was monitored with the use of a laser Doppler flowmeter in the mesentery. Measurements in platelet-depleted mice, mice pretreated with cangrelor, an ADP receptor antagonist, and eptifibatide, a blocker of fibrinogen binding to GPIIbIIIa, were conducted as the proof-of-concept in the performed experiments. Intravital microscopy and ex vivo imaging of organs was performed to identify the sites of aggregate formation resulting from ADP injection. Results The injection of ADP resulted in a dose-dependent reduction of the blood flow in the mesentery. These responses were fully attributable to blood platelet aggregation, as shown by the lack of the effect in platelet-depleted mice, and significantly reduced responses in mice pretreated with cangrelor and eptifibatide. No platelet aggregate formation in mesenteric vessels was revealed by intravital microscopy, while ex vivo imaging showed accumulation of fluorescent labelled platelets in the lung. Conclusions Injection of ADP to the venous system results in the formation of platelet aggregates predominantly in the lung. This results in reversible blood flow cessation in peripheral blood vessels. The measurement of this blood flow cessation in the mesentery allows indirect measurement of ADP-induced pulmonary thromboembolism. We suggest that this approach can be useful for in vivo screening for antiplatelet drug candidates. PMID:26751810

  4. ADP1 affects plant architecture by regulating local auxin biosynthesis.

    PubMed

    Li, Ruixi; Li, Jieru; Li, Shibai; Qin, Genji; Novák, Ondřej; Pěnčík, Aleš; Ljung, Karin; Aoyama, Takashi; Liu, Jingjing; Murphy, Angus; Gu, Hongya; Tsuge, Tomohiko; Qu, Li-Jia

    2014-01-01

    Plant architecture is one of the key factors that affect plant survival and productivity. Plant body structure is established through the iterative initiation and outgrowth of lateral organs, which are derived from the shoot apical meristem and root apical meristem, after embryogenesis. Here we report that ADP1, a putative MATE (multidrug and toxic compound extrusion) transporter, plays an essential role in regulating lateral organ outgrowth, and thus in maintaining normal architecture of Arabidopsis. Elevated expression levels of ADP1 resulted in accelerated plant growth rate, and increased the numbers of axillary branches and flowers. Our molecular and genetic evidence demonstrated that the phenotypes of plants over-expressing ADP1 were caused by reduction of local auxin levels in the meristematic regions. We further discovered that this reduction was probably due to decreased levels of auxin biosynthesis in the local meristematic regions based on the measured reduction in IAA levels and the gene expression data. Simultaneous inactivation of ADP1 and its three closest homologs led to growth retardation, relative reduction of lateral organ number and slightly elevated auxin level. Our results indicated that ADP1-mediated regulation of the local auxin level in meristematic regions is an essential determinant for plant architecture maintenance by restraining the outgrowth of lateral organs. PMID:24391508

  5. Reprogramming cellular events by poly(ADP-ribose)-binding proteins

    PubMed Central

    Pic, Émilie; Ethier, Chantal; Dawson, Ted M.; Dawson, Valina L.; Masson, Jean-Yves; Poirier, Guy G.; Gagné, Jean-Philippe

    2013-01-01

    Poly(ADP-ribosyl)ation is a posttranslational modification catalyzed by the poly(ADP-ribose) polymerases (PARPs). These enzymes covalently modify glutamic, aspartic and lysine amino acid side chains of acceptor proteins by the sequential addition of ADP-ribose (ADPr) units. The poly(ADP-ribose) (pADPr) polymers formed alter the physico-chemical characteristics of the substrate with functional consequences on its biological activities. Recently, non-covalent binding to pADPr has emerged as a key mechanism to modulate and coordinate several intracellular pathways including the DNA damage response, protein stability and cell death. In this review, we describe the basis of non-covalent binding to pADPr that has led to the emerging concept of pADPr-responsive signaling pathways. This review emphasizes the structural elements and the modular strategies developed by pADPr-binding proteins to exert a fine-tuned control of a variety of pathways. Poly(ADP-ribosyl)ation reactions are highly regulated processes, both spatially and temporally, for which at least four specialized pADPr-binding modules accommodate different pADPr structures and reprogram protein functions. In this review, we highlight the role of well-characterized and newly discovered pADPr-binding modules in a diverse set of physiological functions. PMID:23268355

  6. Force-producing ADP state of myosin bound to actin

    PubMed Central

    Wulf, Sarah F.; Ropars, Virginie; Fujita-Becker, Setsuko; Oster, Marco; Hofhaus, Goetz; Trabuco, Leonardo G.; Pylypenko, Olena; Sweeney, H. Lee; Houdusse, Anne M.; Schröder, Rasmus R.

    2016-01-01

    Molecular motors produce force when they interact with their cellular tracks. For myosin motors, the primary force-generating state has MgADP tightly bound, whereas myosin is strongly bound to actin. We have generated an 8-Å cryoEM reconstruction of this state for myosin V and used molecular dynamics flexed fitting for model building. We compare this state to the subsequent state on actin (Rigor). The ADP-bound structure reveals that the actin-binding cleft is closed, even though MgADP is tightly bound. This state is accomplished by a previously unseen conformation of the β-sheet underlying the nucleotide pocket. The transition from the force-generating ADP state to Rigor requires a 9.5° rotation of the myosin lever arm, coupled to a β-sheet rearrangement. Thus, the structure reveals the detailed rearrangements underlying myosin force generation as well as the basis of strain-dependent ADP release that is essential for processive myosins, such as myosin V. PMID:26976594

  7. Niacin status, NAD distribution and ADP-ribose metabolism.

    PubMed

    Kirkland, James B

    2009-01-01

    Dietary niacin deficiency, and pharmacological excesses of nicotinic acid or nicotinamide, have dramatic effects on cellular NAD pools, ADP-ribose metabolism, tissue function and health. ADP-ribose metabolism is providing new targets for pharmacological intervention, and it is important to consider how the supply of vitamin B3 may directly influence ADP-ribosylation reactions, or create interactions with other drugs designed to influence these pathways. In addition to its redox roles, NAD+ is used as a substrate for mono-, poly- and cyclic ADP-ribose formation. During niacin deficiency, not all of these processes can be maintained, and dramatic changes in tissue function and clinical condition take place. Conversely, these reactions may be differentially enhanced by pharmacological intakes of vitamin B3, and potentially by changing expression of specific NAD generating enzymes. A wide range of metabolic changes can take place following pharmacological supplementation of nicotinic acid or nicotinamide. As niacin status decreases towards a deficient state, the function of other types of pharmaceutical agents may be modified, including those that target ADP-ribosylation reactions, apoptosis and inflammation. This article will explore what is known and yet to be learned about the response of tissues, cells and subcellular compartments to excessive and limiting supplies of niacin, and will discuss the etiology of the resulting pathologies.

  8. Two disparate ligand binding sites in the human P2Y1 receptor

    PubMed Central

    Zhang, Dandan; Gao, Zhan-Guo; Zhang, Kaihua; Kiselev, Evgeny; Crane, Steven; Wang, Jiang; Paoletta, Silvia; Yi, Cuiying; Ma, Limin; Zhang, Wenru; Han, Gye Won; Liu, Hong; Cherezov, Vadim; Katritch, Vsevolod; Jiang, Hualiang; Stevens, Raymond C.; Jacobson, Kenneth A.; Zhao, Qiang; Wu, Beili

    2015-01-01

    In response to adenosine 5′-diphosphate, the P2Y1 receptor (P2Y1R) facilitates platelet aggregation, and thus serves as an important antithrombotic drug target. Here we report the crystal structures of the human P2Y1R in complex with a nucleotide antagonist MRS2500 at 2.7Å resolution, and with a non-nucleotide antagonist BPTU at 2.2Å resolution. The structures reveal two distinct ligand binding sites, providing atomic details of P2Y1R’s unique ligand binding modes. MRS2500 recognizes a binding site within the seven transmembrane bundle of P2Y1R, which, however, is different in shape and location from the nucleotide binding site in previously determined P2Y12R structure. BPTU binds to an allosteric pocket on the external receptor interface with the lipid bilayer, making it the first structurally characterized selective G protein-coupled receptor (GPCR) ligand located entirely outside of the helical bundle. These high-resolution insights into P2Y1R should enable discovery of new orthosteric and allosteric antithrombotic drugs with reduced adverse effects. PMID:25822790

  9. Modulation of urokinase plasminogen activator system by poly(ADP-ribose)polymerase-1 inhibition.

    PubMed

    Madunić, Josip; Antica, Mariastefania; Cvjetko, Petra; Požgaj, Lidija; Matulić, Maja

    2016-08-01

    The urokinase plasminogen activator (uPA) system is a complex regulator of extracellular proteolysis which is involved in various physiological and pathological processes. The major components of this system are the serine protease uPA, two inhibitors PAI-1 and PAI-2, and the receptor uPAR. It has been previously shown by several groups that the uPA system has an important role in cancer progression and therefore its possible prognostic and therapeutic value has been evaluated. The aim of this study is to tackle the role of poly(ADP-ribosyl)ation in the induction of uPA activity in a glioblastoma cell line, A1235. This cell line is sensitive to alkylation damage and is a model for drug treatment. The components of the uPA system and the level of DNA damage were analyzed after alkylation agent treatment in combination with poly(ADP-ribose)polymerase-1 (PARP-1) inhibition. Here we show that the increase in uPA activity results from the net balance change between uPA and its inhibitor at mRNA level. Further, PARP-1 inhibition exerts its influence on uPA activity through DNA damage increase. Involvement of several signaling pathways, as well as cell specific regulation influencing the uPA system are discussed.

  10. Impact of Dabigatran versus Phenprocoumon on ADP Induced Platelet Aggregation in Patients with Atrial Fibrillation with or without Concomitant Clopidogrel Therapy (the Dabi-ADP-1 and Dabi-ADP-2 Trials)

    PubMed Central

    Martischnig, Amadea M.; Mehilli, Julinda; Pollak, Janina; Petzold, Tobias; Fiedler, Anette K.; Mayer, Katharina; Schulz-Schüpke, Stefanie; Sibbing, Dirk; Massberg, Steffen; Kastrati, Adnan; Sarafoff, Nikolaus

    2015-01-01

    Background. A relevant number of patients receive triple therapy with clopidogrel, aspirin, and oral anticoagulation. Clopidogrel's efficacy on ADP induced platelet function may be influenced by concomitant antithrombotic therapies. Data regarding the effect of dabigatran on platelet function is limited to in vitro studies and healthy individuals. Methods. The “Dabi-ADP-1” and “Dabi-ADP-2” trials randomized patients with atrial fibrillation to either dabigatran or phenprocoumon for a 2-week period. In Dabi-ADP-1 (n = 70) patients with clopidogrel therapy were excluded and in Dabi-ADP-2 (n = 46) patients had to be treated concomitantly with clopidogrel. The primary endpoint was ADP-induced platelet aggregation between dabigatran and phenprocoumon at 14 days. Secondary endpoints were ADPtest HS-, TRAP-, and COL-induced platelet aggregation. Results. There was no significant difference regarding the primary endpoint between both groups in either trial (Dabi-ADP-1: Dabigatran: 846 [650–983] AU × min versus phenprocoumon: 839 [666–1039] AU × min, P = 0.90 and Dabi-ADP-2: 326 [268–462] versus 350 [214–535], P = 0.70) or regarding the secondary endpoints, ADPtest HS-, TRAP-, and COL-induced platelet aggregation. Conclusion. Dabigatran as compared to phenprocoumon has no impact on ADP-induced platelet aggregation in atrial fibrillation patients neither with nor without concomitant clopidogrel therapy. PMID:26229963

  11. 2´,3´-Dialdehyde of ATP, ADP, and adenosine inhibit HIV-1 reverse transcriptase and HIV-1 replication.

    PubMed

    Schachter, Julieta; Valadao, Ana Luiza Chaves; Aguiar, Renato Santana; Barreto-de-Souza, Victor; Rossi, Atila Duque; Arantes, Pablo Ricardo; Verli, Hugo; Quintana, Paula Gabriela; Heise, Norton; Tanuri, Amilcar; Bou-Habib, Dumith Chequer; Persechini, Pedro Muanis

    2014-01-01

    The 2´3´-dialdehyde of ATP or oxidized ATP (oATP) is a compound known for specifically making covalent bonds with the nucleotide-binding site of several ATP-binding enzymes and receptors. We investigated the effects of oATP and other oxidized purines on HIV-1 infection and we found that this compound inhibits HIV-1 and SIV infection by blocking early steps of virus replication. oATP, oxidized ADP (oADP), and oxidized Adenosine (oADO) impact the natural activity of endogenous reverse transcriptase enzyme (RT) in cell free virus particles and are able to inhibit viral replication in different cell types when added to the cell cultures either before or after infection. We used UFLC-UV to show that both oADO and oATP can be detected in the cell after being added in the extracellular medium. oATP also suppresses RT activity and replication of the HIV-1 resistant variants M184V and T215Y. We conclude that oATP, oADP and oADO display anti HIV-1 activity that is at in least in part due to inhibitory activity on HIV-1 RT.

  12. Crystal structure of potato tuber ADP-glucose pyrophosphorylase

    PubMed Central

    Jin, Xiangshu; Ballicora, Miguel A; Preiss, Jack; Geiger, James H

    2005-01-01

    ADP-glucose pyrophosphorylase catalyzes the first committed and rate-limiting step in starch biosynthesis in plants and glycogen biosynthesis in bacteria. It is the enzymatic site for regulation of storage polysaccharide accumulation in plants and bacteria, being allosterically activated or inhibited by metabolites of energy flux. We report the first atomic resolution structure of ADP-glucose pyrophosphorylase. Crystals of potato tuber ADP-glucose pyrophosphorylase α subunit were grown in high concentrations of sulfate, resulting in the sulfate-bound, allosterically inhibited form of the enzyme. The N-terminal catalytic domain resembles a dinucleotide-binding Rossmann fold and the C-terminal domain adopts a left-handed parallel β helix that is involved in cooperative allosteric regulation and a unique oligomerization. We also report structures of the enzyme in complex with ATP and ADP-glucose. Communication between the regulator-binding sites and the active site is both subtle and complex and involves several distinct regions of the enzyme including the N-terminus, the glucose-1-phosphate-binding site, and the ATP-binding site. These structures provide insights into the mechanism for catalysis and allosteric regulation of the enzyme. PMID:15692569

  13. 7 CFR 272.10 - ADP/CIS Model Plan.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 4 2014-01-01 2014-01-01 false ADP/CIS Model Plan. 272.10 Section 272.10 Agriculture Regulations of the Department of Agriculture (Continued) FOOD AND NUTRITION SERVICE, DEPARTMENT OF AGRICULTURE... other State agencies that have similar characteristics such as whether they are urban or rural,...

  14. ADP correspondence system: Unsolicited proposal evaluation tracking application

    NASA Technical Reports Server (NTRS)

    Greene, W. A.; Goodwin, D. J.

    1976-01-01

    A complete description of a correspondence control system, designed to be used by non-ADP clerical personnel is provided. In addition to operating instructions, sufficient design and conceptual information is provided to allow use or adaption of the system in related applications. The complete COBOL program and documentation are available.

  15. Crystal structure of potato tuber ADP-glucose pyrophosphorylase.

    PubMed

    Jin, Xiangshu; Ballicora, Miguel A; Preiss, Jack; Geiger, James H

    2005-02-23

    ADP-glucose pyrophosphorylase catalyzes the first committed and rate-limiting step in starch biosynthesis in plants and glycogen biosynthesis in bacteria. It is the enzymatic site for regulation of storage polysaccharide accumulation in plants and bacteria, being allosterically activated or inhibited by metabolites of energy flux. We report the first atomic resolution structure of ADP-glucose pyrophosphorylase. Crystals of potato tuber ADP-glucose pyrophosphorylase alpha subunit were grown in high concentrations of sulfate, resulting in the sulfate-bound, allosterically inhibited form of the enzyme. The N-terminal catalytic domain resembles a dinucleotide-binding Rossmann fold and the C-terminal domain adopts a left-handed parallel beta helix that is involved in cooperative allosteric regulation and a unique oligomerization. We also report structures of the enzyme in complex with ATP and ADP-glucose. Communication between the regulator-binding sites and the active site is both subtle and complex and involves several distinct regions of the enzyme including the N-terminus, the glucose-1-phosphate-binding site, and the ATP-binding site. These structures provide insights into the mechanism for catalysis and allosteric regulation of the enzyme.

  16. Abiogenic Photophosphorylation of ADP to ATP Sensitized by Flavoproteinoid Microspheres

    NASA Astrophysics Data System (ADS)

    Kolesnikov, Michael P.; Telegina, Taisiya A.; Lyudnikova, Tamara A.; Kritsky, Mikhail S.

    2008-06-01

    A model for abiogenic photophosphorylation of ADP by orthophosphate to yield ATP was studied. The model is based on the photochemical activity of flavoproteinoid microspheres that are formed by aggregation in an aqueous medium of products of thermal condensation of a glutamic acid, glycine and lysine mixture (8:3:1) and contain, along with amino acid polymers (proteinoids), abiogenic isoalloxazine (flavin) pigments. Irradiation of aqueous suspensions of microspheres with blue visible light or ultraviolet in the presence of ADP and orthophosphate resulted in ATP formation. The yield of ATP in aerated suspensions was 10 20% per one mol of starting ADP. Deaeration reduced the photophosphorylating activity of microspheres five to 10 times. Treatment of aerated microsphere suspensions with superoxide dismutase during irradiation partially suppressed ATP formation. Deaerated microspheres restored completely their photophosphorylating activity after addition of hydrogen peroxide to the suspension. The photophosphorylating activity of deaerated suspensions of flavoproteinoid microspheres was also recovered by introduction of Fe3+-cytochrome c, an electron acceptor alternative to oxygen. On the basis of the results obtained, a chemical mechanism of phosphorylation is proposed in which the free radical form of reduced flavin sensitizer left( {{text{FlH}}^ bullet } right) and ADP are involved.

  17. American Diploma Project (ADP) End-of-Course Exams: 2010 Annual Report

    ERIC Educational Resources Information Center

    Achieve, Inc., 2010

    2010-01-01

    To assess the raised expectations of college and career readiness for all students, a group of American Diploma Project (ADP) Network states formed the ADP Assessment Consortium in 2005. The Consortium created Algebra I and II end-of-course exams, based in large part on Achieve's ADP mathematics benchmarks, which would provide an honest assessment…

  18. Development of second generation peptides modulating cellular adiponectin receptor responses

    PubMed Central

    Otvos, Laszlo; Knappe, Daniel; Hoffmann, Ralf; Kovalszky, Ilona; Olah, Julia; Hewitson, Tim D.; Stawikowska, Roma; Stawikowski, Maciej; Cudic, Predrag; Lin, Feng; Wade, John D.; Surmacz, Eva; Lovas, Sandor

    2014-01-01

    The adipose tissue participates in the regulation of energy homeostasis as an important endocrine organ that secretes a number of biologically active adipokines, including adiponectin. Recently we developed and characterized a first-in-class peptide-based adiponectin receptor agonist by using in vitro and in vivo models of glioblastoma and breast cancer (BC). In the current study, we further explored the effects of peptide ADP355 in additional cellular models and found that ADP355 inhibited chronic myeloid leukemia (CML) cell proliferation and renal myofibroblast differentiation with mid-nanomolar IC50 values. According to molecular modeling calculations, ADP355 was remarkably flexible in the global minimum with a turn present in the middle of the peptide. Considering these structural features of ADP355 and the fact that adiponectin normally circulates as multimeric complexes, we developed and tested the activity of a linear branched dimer (ADP399). The dimer exhibited approximately 20-fold improved cellular activity inhibiting K562 CML and MCF-7 cell growth with high pM—low nM relative IC50 values. Biodistribution studies suggested superior tissue dissemination of both peptides after subcutaneous administration relative to intraperitoneal inoculation. After screening of a 397-member adiponectin active site library, a novel octapeptide (ADP400) was designed that counteracted 10–1000 nM ADP355- and ADP399-mediated effects on CML and BC cell growth at nanomolar concentrations. ADP400 induced mitogenic effects in MCF-7 BC cells perhaps due to antagonizing endogenous adiponectin actions or acting as an inverse agonist. While the linear dimer agonist ADP399 meets pharmacological criteria of a contemporary peptide drug lead, the peptide showing antagonist activity (ADP400) at similar concentrations will be an important target validation tool to study adiponectin functions. PMID:25368867

  19. Development of second generation peptides modulating cellular adiponectin receptor responses

    NASA Astrophysics Data System (ADS)

    Otvos, Laszlo; Knappe, Daniel; Hoffmann, Ralf; Kovalszky, Ilona; Olah, Julia; Hewitson, Tim; Stawikowska, Roma; Stawikowski, Maciej; Cudic, Predrag; Lin, Feng; Wade, John; Surmacz, Eva; Lovas, Sandor

    2014-10-01

    The adipose tissue participates in the regulation of energy homeostasis as an important endocrine organ that secretes a number of biologically active adipokines, including adiponectin. Recently we developed and characterized a first-in-class peptide-based adiponectin receptor agonist by using in vitro and in vivo models of glioblastoma and breast cancer (BC). In the current study, we further explored the effects of peptide ADP355 in additional cellular models and found that ADP355 inhibited chronic myeloid leukemia (CML) cell proliferation and renal myofibroblast differentiation with mid-nanomolar IC50 values. According to molecular modeling calculations, ADP355 was remarkably flexible in the global minimum with a turn present in the middle of the peptide. Considering these structural features of ADP355 and the fact that adiponectin normally circulates as multimeric complexes, we developed and tested the activity of a linear branched dimer (ADP399). The dimer exhibited approximately 20-fold improved cellular activity inhibiting K562 CML and MCF-7 cell growth with high pM - low nM relative IC50 values. Biodistribution studies suggested superior tissue dissemination of both peptides after subcutaneous administration relative to intraperitoneal inoculation. After screening of a 397-member adiponectin active site library, a novel octapeptide (ADP400) was designed that counteracted 10-1000 nM ADP355- and ADP399-mediated effects on CML and BC cell growth at nanomolar concentrations. ADP400 induced mitogenic effects in MCF-7 BC cells perhaps due to antagonizing endogenous adiponectin actions or acting as an inverse agonist. While the linear dimer agonist ADP399 meets pharmacological criteria of a contemporary peptide drug lead, the peptide showing antagonist activity (ADP400) at similar concentrations will be an important target validation tool to study adiponectin functions.

  20. Targeting renal purinergic signalling for the treatment of lithium-induced nephrogenic diabetes insipidus.

    PubMed

    Kishore, B K; Carlson, N G; Ecelbarger, C M; Kohan, D E; Müller, C E; Nelson, R D; Peti-Peterdi, J; Zhang, Y

    2015-06-01

    Lithium still retains its critical position in the treatment of bipolar disorder by virtue of its ability to prevent suicidal tendencies. However, chronic use of lithium is often limited by the development of nephrogenic diabetes insipidus (NDI), a debilitating condition. Lithium-induced NDI is due to resistance of the kidney to arginine vasopressin (AVP), leading to polyuria, natriuresis and kaliuresis. Purinergic signalling mediated by extracellular nucleotides (ATP/UTP), acting via P2Y receptors, opposes the action of AVP on renal collecting duct (CD) by decreasing the cellular cAMP and thus AQP2 protein levels. Taking a cue from this phenomenon, we discovered the potential involvement of ATP/UTP-activated P2Y2 receptor in lithium-induced NDI in rats and showed that P2Y2 receptor knockout mice are significantly resistant to Li-induced polyuria, natriuresis and kaliuresis. Extension of these studies revealed that ADP-activated P2Y12 receptor is expressed in the kidney, and its irreversible blockade by the administration of clopidogrel bisulphate (Plavix(®)) ameliorates Li-induced NDI in rodents. Parallel in vitro studies showed that P2Y12 receptor blockade by the reversible antagonist PSB-0739 sensitizes CD to the action of AVP. Thus, our studies unravelled the potential beneficial effects of targeting P2Y2 or P2Y12 receptors to counter AVP resistance in lithium-induced NDI. If established in further studies, our findings may pave the way for the development of better and safer methods for the treatment of NDI by bringing a paradigm shift in the approach from the current therapies that predominantly counter the anti-AVP effects to those that enhance the sensitivity of the kidney to AVP action.

  1. A human pseudoautosomal gene encodes the ANT3 ADP/ATP translocase and escapes X-inactivation

    SciTech Connect

    Slim, R. Institut Gustave Roussy, Villejuif ); Levilliers, J.; Petit, C. ); Luedecke, H.J.; Horsthemke, B. ); Claussen, U. ); Nguyen, V.C. ); Gough, N.M. )

    1993-04-01

    The authors report that the human ANT3 ADP/ATP translocase gene is [alpha] pseudoautosomal gene located proximal to the GM-CSF receptor a chain gene (CSF2RA). An ANT3-homologous locus, likely corresponding to a pseudogene, maps to chromosome 9. The ANT3 gene is transcribed from the centromere to the telomere and contains in its first intron a CpG island mapped 1300 kb from the telomere. This gene is transcribed from the Y chromosome and from the active and inactive X chromosomes. This gene thus escapes X-inactivation as predicted for genes belonging to the pseudoautosomal region. 61 refs., 5 figs., 1 tab.

  2. Structure and function of the ARH family of ADP-ribose-acceptor hydrolases

    PubMed Central

    Mashimo, Masato; Kato, Jiro; Moss, Joel

    2014-01-01

    ADP-ribosylation is a post-translational protein modification, in which ADP-ribose is transferred from nicotinamide adenine dinucleotide (NAD+) to specific acceptors, thereby altering their activities. The ADP-ribose transfer reactions are divided into mono- and poly-(ADP-ribosyl)ation. Cellular ADP-ribosylation levels are tightly regulated by enzymes that transfer ADP-ribose to acceptor proteins (e.g. ADP-ribosyltransferases, poly-(ADP-ribose) polymerases (PARP)) and those that cleave the linkage between ADP-ribose and acceptor (e.g. ADP-ribosyl-acceptor hydrolases (ARH), poly-(ADP-ribose) glycohydrolases (PARG)), thereby constituting an ADP-ribosylation cycle. This review summarizes current findings related to the ARH family of proteins. This family comprises three members (ARH1-3) with similar size (39 kDa) and amino acid sequence. ARH1 catalyzes the hydrolysis of the N-glycosidic bond of mono-(ADP-ribosyl)ated arginine. ARH3 hydrolyzes poly-(ADP-ribose) (PAR) and O-acetyl-ADP-ribose. The different substrate specificities of ARH1 and ARH3 contribute to their unique roles in the cell. Based on a phenotype analysis of ARH1−/− and ARH3−/− mice, ARH1 is involved in the action by bacterial toxins as well as in tumorigenesis. ARH3 participates in the degradation of PAR that is synthesized by PARP1 in response to oxidative stress-induced DNA damage; this hydrolytic reaction suppresses PAR-mediated cell death, a pathway termed parthanatos. PMID:24746921

  3. ADP-Ribose Activates the TRPM2 Channel from the Sea Anemone Nematostella vectensis Independently of the NUDT9H Domain.

    PubMed

    Kühn, Frank J P; Kühn, Cornelia; Winking, Mathis; Hoffmann, Daniel C; Lückhoff, Andreas

    2016-01-01

    The human redox-sensitive Transient receptor potential melastatin type 2 (hTRPM2) channel contains the C-terminal Nudix hydrolase domain NUDT9H which most likely binds ADP-ribose. During oxidative stress, the intracellular release of ADP-ribose triggers the activation of hTRPM2. The TRPM2 orthologue from Nematostella vectensis (nv) is also stimulated by ADP-ribose but not by the oxidant hydrogen peroxide. For further clarification of the structure-function relationships of these two distantly related channel orthologues, we performed whole-cell as well as single channel patch-clamp recordings, Ca2+-imaging and Western blot analysis after heterologous expression of wild-type and mutated channels in HEK-293 cells. We demonstrate that the removal of the entire NUDT9H domain does not disturb the response of nvTRPM2 to ADP-ribose. The deletion, however, created channels that were activated by hydrogen peroxide, as did mutations within the NUDT9H domain of nvTRPM2 that presumably suppress its enzymatic function. The same findings were obtained with the nvTRPM2 channel when the NUDT9H domain was replaced by the corresponding sequences of the original hNUDT9 enzyme. Whenever the enzyme domain was mutated to presumably inactive variants, channel activation by hydrogen peroxide could be achieved. Moreover, we found strong evidences for ADPRase activity of the isolated NUDT9H domain of nvTRPM2 in co-expression experiments with the C-terminally truncated nvTRPM2 channel. Thus, there is a clear correlation between the loss of enzymatic activity and the capability of nvTRPM2 to respond to oxidative stress. In striking contrast, the channel function of the hTRPM2 orthologue, in particular its sensitivity to ADP-ribose, was abrogated by already small changes of the NUDT9H domain. These findings establish nvTRPM2 as a channel gated by ADP-ribose through a novel mechanism. We conclude that the endogenous NUDT9H domain does not directly affect ADP-ribose-dependent gating of the nv

  4. ADP-Ribose Activates the TRPM2 Channel from the Sea Anemone Nematostella vectensis Independently of the NUDT9H Domain

    PubMed Central

    Kühn, Frank J. P.; Kühn, Cornelia; Winking, Mathis; Hoffmann, Daniel C.; Lückhoff, Andreas

    2016-01-01

    The human redox-sensitive Transient receptor potential melastatin type 2 (hTRPM2) channel contains the C-terminal Nudix hydrolase domain NUDT9H which most likely binds ADP-ribose. During oxidative stress, the intracellular release of ADP-ribose triggers the activation of hTRPM2. The TRPM2 orthologue from Nematostella vectensis (nv) is also stimulated by ADP-ribose but not by the oxidant hydrogen peroxide. For further clarification of the structure-function relationships of these two distantly related channel orthologues, we performed whole-cell as well as single channel patch-clamp recordings, Ca2+-imaging and Western blot analysis after heterologous expression of wild-type and mutated channels in HEK-293 cells. We demonstrate that the removal of the entire NUDT9H domain does not disturb the response of nvTRPM2 to ADP-ribose. The deletion, however, created channels that were activated by hydrogen peroxide, as did mutations within the NUDT9H domain of nvTRPM2 that presumably suppress its enzymatic function. The same findings were obtained with the nvTRPM2 channel when the NUDT9H domain was replaced by the corresponding sequences of the original hNUDT9 enzyme. Whenever the enzyme domain was mutated to presumably inactive variants, channel activation by hydrogen peroxide could be achieved. Moreover, we found strong evidences for ADPRase activity of the isolated NUDT9H domain of nvTRPM2 in co-expression experiments with the C-terminally truncated nvTRPM2 channel. Thus, there is a clear correlation between the loss of enzymatic activity and the capability of nvTRPM2 to respond to oxidative stress. In striking contrast, the channel function of the hTRPM2 orthologue, in particular its sensitivity to ADP-ribose, was abrogated by already small changes of the NUDT9H domain. These findings establish nvTRPM2 as a channel gated by ADP-ribose through a novel mechanism. We conclude that the endogenous NUDT9H domain does not directly affect ADP-ribose-dependent gating of the nv

  5. Proximal ADP-ribose Hydrolysis in Trypanosomatids is Catalyzed by a Macrodomain

    PubMed Central

    Haikarainen, Teemu; Lehtiö, Lari

    2016-01-01

    ADP-ribosylation is a ubiquitous protein modification utilized by both prokaryotes and eukaryotes for several cellular functions, such as DNA repair, proliferation, and cell signaling. Higher eukaryotes, such as humans, utilize various enzymes to reverse the modification and to regulate ADP-ribose dependent signaling. In contrast, some lower eukaryotes, including trypanosomatids, lack many of these enzymes and therefore have a much more simplified ADP-ribose metabolism. Here we identified and characterized ADP-ribose hydrolases from Trypanosoma brucei and Trypanosoma cruzi, which are homologous to human O-acetyl-ADP-ribose deacetylases MacroD1 and MacroD2. The enzymes are capable for hydrolysis of protein linked ADP-ribose and a product of sirtuin-mediated lysine deacetylation, O-acetyl-ADP-ribose. Crystal structures of the trypanosomatid macrodomains revealed a conserved catalytic site with distinct differences to human MacroD1 and MacroD2. PMID:27064071

  6. 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

  7. The 1994 NASA/USRA/ADP Design Projects

    NASA Technical Reports Server (NTRS)

    Cruse, Thomas; Richardson, Joseph; Tryon, Robert

    1994-01-01

    The NASA/USRA/ADP Design Projects from Vanderbilt University, Department of Mechanical Engineering (1994) are enclosed in this final report. Design projects include: (1) Protein Crystal Growth, both facilities and methodology; (2) ACES Deployable Space Boom; (3) Hybrid Launch System designs for both manned and unmanned systems; (4) LH2 Fuel Tank design (SSTO); (5) SSTO design; and (6) Pressure Tank Feed System design.

  8. 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.

  9. 26 CFR 1.401(k)-2 - ADP test.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...)(2)(iv) (as it appeared in the April 1, 2007, edition of 26 CFR part 1). (v) Distribution. Within 12... determined under § 1.401(k)-2(b)(2)(vi) (as it appeared in the April 1, 2007, edition of 26 CFR Part 1). (C... 26 Internal Revenue 5 2010-04-01 2010-04-01 false ADP test. 1.401(k)-2 Section 1.401(k)-2...

  10. 26 CFR 1.401(k)-2 - ADP test.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... determined under § 1.401(k)-2(b)(2)(vi) (as it appeared in the April 1, 2007, edition of 26 CFR Part 1). (C...)(2)(iv) (as it appeared in the April 1, 2007, edition of 26 CFR part 1). (v) Distribution. Within 12... 26 Internal Revenue 5 2014-04-01 2014-04-01 false ADP test. 1.401(k)-2 Section 1.401(k)-2...

  11. 26 CFR 1.401(k)-2 - ADP test.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... determined under § 1.401(k)-2(b)(2)(vi) (as it appeared in the April 1, 2007, edition of 26 CFR Part 1). (C...)(2)(iv) (as it appeared in the April 1, 2007, edition of 26 CFR part 1). (v) Distribution. Within 12... 26 Internal Revenue 5 2013-04-01 2013-04-01 false ADP test. 1.401(k)-2 Section 1.401(k)-2...

  12. 26 CFR 1.401(k)-2 - ADP test.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... determined under § 1.401(k)-2(b)(2)(vi) (as it appeared in the April 1, 2007, edition of 26 CFR Part 1). (C...)(2)(iv) (as it appeared in the April 1, 2007, edition of 26 CFR part 1). (v) Distribution. Within 12... 26 Internal Revenue 5 2011-04-01 2011-04-01 false ADP test. 1.401(k)-2 Section 1.401(k)-2...

  13. 26 CFR 1.401(k)-2 - ADP test.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... determined under § 1.401(k)-2(b)(2)(vi) (as it appeared in the April 1, 2007, edition of 26 CFR Part 1). (C...)(2)(iv) (as it appeared in the April 1, 2007, edition of 26 CFR part 1). (v) Distribution. Within 12... 26 Internal Revenue 5 2012-04-01 2011-04-01 true ADP test. 1.401(k)-2 Section 1.401(k)-2...

  14. 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

  15. Submaximal ADP-stimulated respiration is impaired in ZDF rats and recovered by resveratrol.

    PubMed

    Smith, Brennan K; Perry, Christopher G R; Herbst, Eric A F; Ritchie, Ian R; Beaudoin, Marie-Soleil; Smith, Jeffrey C; Neufer, P Darrell; Wright, David C; Holloway, Graham P

    2013-12-01

    Mitochondrial dysfunction and reactive oxygen species (ROS) have been implicated in the aetiology of skeletal muscle insulin resistance, although there is considerable controversy regarding these concepts. Mitochondrial function has been traditionally assessed in the presence of saturating ADP, but ATP turnover and the resultant ADP is thought to limit respiration in vivo. Therefore, we investigated the potential link between submaximal ADP-stimulated respiration rates, ROS generation and skeletal muscle insulin sensitivity in a model of type 2 diabetes mellitus, the ZDF rat. Utilizing permeabilized muscle fibres we observed that submaximal ADP-stimulated respiration rates (250-2000 μm ADP) were lower in ZDF rats than in lean controls, which coincided with decreased adenine nucleotide translocase 2 (ANT2) protein content. This decrease in submaximal ADP-stimulated respiration occurred in the absence of a decrease in electron transport chain function. Treating ZDF rats with resveratrol improved skeletal muscle insulin resistance and this was associated with elevated submaximal ADP-stimulated respiration rates as well as an increase in ANT2 protein content. These results coincided with a greater ability of ADP to attenuate mitochondrial ROS emission and an improvement in cellular redox balance. Together, these data suggest that mitochondrial dysfunction is present in skeletal muscle insulin resistance when assessed at submaximal ADP concentrations and that ADP dynamics may influence skeletal muscle insulin sensitivity through alterations in the propensity for mitochondrial ROS emission.

  16. Diurnal dynamic behavior of microglia in response to infected bacteria through the UDP-P2Y6 receptor system

    PubMed Central

    Takayama, Fumiko; Hayashi, Yoshinori; Wu, Zhou; Liu, Yicong; Nakanishi, Hiroshi

    2016-01-01

    It has long been believed that microglia morphologically transform into the activated state by retracting their long processes and consuming pathogens when bacteria infect into the brain parenchyma. In the present study, however, we showed for the first time that murine cortical microglia extend their processes towards focally injected Porphyromonas gingivalis. This P. gingivalis-induced microglial process extension was significantly increased during the light (sleeping) phase than the dark (waking) phase. In contrast, focally injected ATP-induced microglial process extension was significantly increased during the dark phase than the light phase. Furthermore, in contrast to the P2Y12 receptor-mediated mechanism of ATP-induced microglial process extension, the P. gingivalis-mediated microglial process extension was mediated by P2Y6 receptors. The infection of bacteria such as P. gingivalis to the brain parenchyma may induce the secretion of UDP from microglia at the site of infection, which in turn induces the process extension of the neighboring microglia. PMID:27445174

  17. Comparative single-molecule and ensemble myosin enzymology: sulfoindocyanine ATP and ADP derivatives.

    PubMed Central

    Oiwa, K; Eccleston, J F; Anson, M; Kikumoto, M; Davis, C T; Reid, G P; Ferenczi, M A; Corrie, J E; Yamada, A; Nakayama, H; Trentham, D R

    2000-01-01

    Single-molecule and macroscopic reactions of fluorescent nucleotides with myosin have been compared. The single-molecule studies serve as paradigms for enzyme-catalyzed reactions and ligand-receptor interactions analyzed as individual stochastic processes. Fluorescent nucleotides, called Cy3-EDA-ATP and Cy5-EDA-ATP, were derived by coupling the dyes Cy3.29.OH and Cy5.29.OH (compounds XI and XIV, respectively, in, Bioconjug. Chem. 4:105-111)) with 2'(3')-O-[N-(2-aminoethyl)carbamoyl]ATP (EDA-ATP). The ATP(ADP) analogs were separated into their respective 2'- and 3'-O-isomers, the interconversion rate of which was 30[OH(-)] s(-1) (0.016 h(-1) at pH 7.1) at 22 degrees C. Macroscopic studies showed that 2'(3')-O-substituted nucleotides had properties similar to those of ATP and ADP in their interactions with myosin, actomyosin, and muscle fibers, although the ATP analogs did not relax muscle as well as ATP did. Significant differences in the fluorescence intensity of Cy3-nucleotide 2'- and 3'-O-isomers in free solution and when they interacted with myosin were evident. Single-molecule studies using total internal reflection fluorescence microscopy showed that reciprocal mean lifetimes of the nucleotide analogs interacting with myosin filaments were one- to severalfold greater than predicted from macroscopic data. Kinetic and equilibrium data of nucleotide-(acto)myosin interactions derived from single-molecule microscopy now have a biochemical and physiological framework. This is important for single-molecule mechanical studies of motor proteins. PMID:10827983

  18. Proteomic investigation of phosphorylation sites in poly(ADP-ribose) polymerase-1 and poly(ADP-ribose) glycohydrolase.

    PubMed

    Gagné, Jean-Philippe; Moreel, Xavier; Gagné, Pierre; Labelle, Yves; Droit, Arnaud; Chevalier-Paré, Mélissa; Bourassa, Sylvie; McDonald, Darin; Hendzel, Michael J; Prigent, Claude; Poirier, Guy G

    2009-02-01

    Phosphorylation is a very common post-translational modification event known to modulate a wide range of biological responses. Beyond the regulation of protein activity, the interrelation of phosphorylation with other post-translational mechanisms is responsible for the control of diverse signaling pathways. Several observations suggest that phosphorylation of poly(ADP-ribose) polymerase-1 (PARP-1) regulates its activity. There is also accumulating evidence to suggest the establishment of phosphorylation-dependent assembly of PARP-1-associated multiprotein complexes. Although it is relatively straightforward to demonstrate phosphorylation of a defined target, identification of the actual amino acids involved still represents a technical challenge for many laboratories. With the use of a combination of bioinformatics-based predictions tools for generic and kinase-specific phosphorylation sites, in vitro phosphorylation assays and mass spectrometry analysis, we investigated the phosphorylation profile of PARP-1 and poly(ADP-ribose) glycohydrolase (PARG), two major enzymes responsible for poly(ADP-ribose) turnover. Mass spectrometry analysis revealed the phosphorylation of several serine/threonine residues within important regulatory domains and motifs of both enzymes. With the use of in vivo microirradiation-induced DNA damage, we show that altered phosphorylation at specific sites can modify the dynamics of assembly and disassembly of PARP-1 at sites of DNA damage. By documenting and annotating a collection of known and newly identified phosphorylation sites, this targeted proteomics study significantly advances our understanding of the roles of phosphorylation in the regulation of PARP-1 and PARG.

  19. Practical Experience of Discharge Measurement in Flood Conditions with ADP

    NASA Astrophysics Data System (ADS)

    Vidmar, A.; Brilly, M.; Rusjan, S.

    2009-04-01

    Accurate discharge estimation is important for an efficient river basin management and especially for flood forecasting. The traditional way of estimating the discharge in hydrological practice is to measure the water stage and to convert the recorded water stage values into discharge by using the single-valued rating curve .Relationship between the stage and discharge values of the rating curve for the extreme events are usually extrapolated by using different mathematical methods and are not directly measured. Our practice shows that by using the Accoustic Doppler Profiler (ADP) instrument we can record the actual relation between the water stage and the flow velocity at the occurrence of flood waves very successfully. Measurement in flood conditions it is not easy task, because of high water surface velocity and large amounts of sediments in the water and floating objects on the surface like branches, bushes, trees, piles and others which can also easily damage ADP instrument. We made several measurements in such extreme events on the Sava River down to the nuclear power plant Kr\\vsko where we have install fixed cable way. During the several measurement with traditional "moving-boat" measurement technique a mowing bed phenomenon was clearly seen. Measuring flow accurately using ADP that uses the "moving-boat" technique, the system needs a reference against which to relate water velocities to. This reference is river bed and must not move. During flood events we detected difficulty finding a static bed surface to which to relate water velocities. This is caused by motion of the surface layer of bed material or also sediments suspended in the water near bed very densely. So these traditional »moving-boat« measurement techniques that we normally use completely fail. Using stationary measurement method to making individual velocity profile measurements, using an Acoustic Doppler Profiler (ADP), at certain time at fixed locations across the width of a stream gave

  20. 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

  1. 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.

  2. How to test the effect of aspirin and clopidogrel in patients on dual antiplatelet therapy?

    PubMed

    Bagoly, Zsuzsa; Homoródi, Nóra; Kovács, Emese Gyöngyvér; Sarkady, Ferenc; Csiba, László; Édes, István; Muszbek, László

    2016-01-01

    Dual antiplatelet therapy with clopidogrel and aspirin is frequently used for the prevention of recurrent ischemic events. Various laboratory methods are used to detect the effect of these drugs administered in monotherapy, however their value in dual therapy has not been explored. Here, we determined which methods used for testing the effect of clopidogrel or aspirin are influenced by the other antiplatelet agent. One arm of the study included 53 ischemic stroke patients being on clopidogrel monotherapy showing effective inhibition of the P2Y12 ADP receptor. Laboratory tests routinely used for the detection of aspirin resistance (arachidonic acid (AA)-induced platelet aggregation/secretion, AA-induced thromboxane B2 (TXB2) production in platelet-rich plasma and VerifyNow Aspirin assay) were carried out on samples obtained from these patients. The other arm of the study involved 52 patients with coronary artery disease being on aspirin monotherapy. Methods used for testing the effect of clopidogrel (ADP-induced platelet aggregation and secretion, flow cytometric analysis of vasodilator-stimulated phosphoprotein (VASP) phosphorylation and a newly developed P2Y12-specific platelet aggregation (ADP[PGE1] test)) were performed on samples obtained from these patients. Clopidogrel monotherapy significantly inhibited AA-induced platelet aggregation and secretion, moreover, AA-induced TXB2 production was also significantly decreased. VASP phosphorylation and AA-induced platelet aggregation showed fair correlation in patients taking clopidogrel only. Clopidogrel did not inhibit the VerifyNow Aspirin test significantly. Aspirin monotherapy influenced ADP-induced platelet aggregation and secretion, but did not have an effect on VASP phosphorylation and on the ADP[PGE1] platelet aggregation test.

  3. Kinetics of a self-amplifying substrate cycle: ADP-ATP cycling assay.

    PubMed Central

    Valero, E; Varón, R; García-Carmona, F

    2000-01-01

    A kinetic study of an ATP-ADP amplification cyclic system involving the enzymes adenylate kinase, pyruvate kinase and L-lactate dehydrogenase has been made. The stoichiometry of the cycle is 2:1, because two molecules of ADP are synthesized from one each of ATP and AMP, and one molecule of ADP is converted back into one of ATP at each turn of the cycle. This results in a continuous exponential increase in the concentrations of ATP and ADP in the reaction medium, according to the equations obtained. This is therefore a substrate cycle that amplifies itself, the cycling rate increasing continuously with time. The background signal of the reagent was reduced by using apyrase to degrade ATP and ADP in the reagent, permitting detection limits as low as 16 pmol of ATP and/or ADP in a continuous spectrophotometric assay. PMID:10926849

  4. Seasonal cycles of mitochondrial ADP sensitivity and oxidative capacities in trout oxidative muscle.

    PubMed

    Guderley, H; St Pierre, J

    1999-10-01

    Mitochondria from red myotomal muscle of rainbow trout, Oncorhynchus mykiss, showed seasonal cycles of their maximal rates of substrate oxidation (nmol.min-1 mg-1 mitochondrial protein) and their apparent ADP affinity (Kmapp), as well as in the thermal sensitivity of these properties. Increases in the maximal capacity of pyruvate oxidation were sufficient to compensate for seasonal changes in temperature, except during the winter months when rates at habitat temperature were depressed relative to other periods. The ADP affinity of isolated mitochondria was highest during cold months. Thus, the Kmapp for ADP at habitat temperature showed less seasonal variation than the ADP Kmapp at a given temperature. A loss in ADP affinity with decreasing temperature occurred through much of the year, and only was definitively suppressed in December and July. Both the ADP affinity and the maximal oxidative capacities of muscle mitochondria seem to be regulated parameters. PMID:10595316

  5. Structures of the Human Poly (ADP-Ribose) Glycohydrolase Catalytic Domain Confirm Catalytic Mechanism and Explain Inhibition by ADP-HPD Derivatives

    PubMed Central

    Tucker, Julie A.; Bennett, Neil; Brassington, Claire; Durant, Stephen T.; Hassall, Giles; Holdgate, Geoff; McAlister, Mark; Nissink, J. Willem M.; Truman, Caroline; Watson, Martin

    2012-01-01

    Poly(ADP-ribose) glycohydrolase (PARG) is the only enzyme known to catalyse hydrolysis of the O-glycosidic linkages of ADP-ribose polymers, thereby reversing the effects of poly(ADP-ribose) polymerases. PARG deficiency leads to cell death whilst PARG depletion causes sensitisation to certain DNA damaging agents, implicating PARG as a potential therapeutic target in several disease areas. Efforts to develop small molecule inhibitors of PARG activity have until recently been hampered by a lack of structural information on PARG. We have used a combination of bio-informatic and experimental approaches to engineer a crystallisable, catalytically active fragment of human PARG (hPARG). Here, we present high-resolution structures of the catalytic domain of hPARG in unliganded form and in complex with three inhibitors: ADP-ribose (ADPR), adenosine 5′-diphosphate (hydroxymethyl)pyrrolidinediol (ADP-HPD) and 8-n-octyl-amino-ADP-HPD. Our structures confirm conservation of overall fold amongst mammalian PARG glycohydrolase domains, whilst revealing additional flexible regions in the catalytic site. These new structures rationalise a body of published mutational data and the reported structure-activity relationship for ADP-HPD based PARG inhibitors. In addition, we have developed and used biochemical, isothermal titration calorimetry and surface plasmon resonance assays to characterise the binding of inhibitors to our PARG protein, thus providing a starting point for the design of new inhibitors. PMID:23251397

  6. 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

  7. The rise and fall of poly(ADP-ribose): An enzymatic perspective.

    PubMed

    Pascal, John M; Ellenberger, Tom

    2015-08-01

    Human cells respond to DNA damage with an acute and transient burst in production of poly(ADP-ribose), a posttranslational modification that expedites damage repair and plays a pivotal role in cell fate decisions. Poly(ADP-ribose) polymerases (PARPs) and glycohydrolase (PARG) are the key set of enzymes that orchestrate the rise and fall in cellular levels of poly(ADP-ribose). In this perspective, we focus on recent structural and mechanistic insights into the enzymes involved in poly(ADP-ribose) production and turnover, and we highlight important questions that remain to be answered.

  8. Bovine spermatozoa incorporate 32Pi into ADP by an unknown pathway.

    PubMed

    Cheetham, J A; Lardy, H A

    1992-03-15

    Intact ejaculated bovine sperm incorporate 32Pi into ADP to a specific activity two to three times higher than into ATP. This contrasts with other cell types where ATP specific activity is higher than that of ADP. Predominant labeling of ADP may be partially due to compartmentation of ATP, but removal of cytosolic ATP does not change the relative labeling of ADP and ATP. Dilution of extracellular 32Pi following labeling resulted in loss of 70% of label from ADP but only 50% loss from gamma-ATP at 26 min. ADP was labeled in the absence of detectable ATP in the presence of rotenone plus antimycin. Fractionation of ejaculated sperm yielded midpieces that are depleted of adenylate kinase and have coupled respiration. ATP was labeled with 32Pi, but ADP was not in midpieces. Evidence for mitochondrial substrate level phosphorylation-supported incorporation of 32Pi into nucleotides was observed for intact sperm incubated with pyruvate and inhibitors of oxidative phosphorylation, but this activity did not occur in midpieces and does not appear to explain disproportionate labeling of ADP. We conclude that labeling of ADP in intact and permeabilized cells occurs by two pathways; one involves adenylate kinase, and the other is an unknown pathway which may be independent of ATP. PMID:1544901

  9. The transport mechanism of the mitochondrial ADP/ATP carrier.

    PubMed

    Kunji, Edmund R S; Aleksandrova, Antoniya; King, Martin S; Majd, Homa; Ashton, Valerie L; Cerson, Elizabeth; Springett, Roger; Kibalchenko, Mikhail; Tavoulari, Sotiria; Crichton, Paul G; Ruprecht, Jonathan J

    2016-10-01

    The mitochondrial ADP/ATP carrier imports ADP from the cytosol and exports ATP from the mitochondrial matrix, which are key transport steps for oxidative phosphorylation in eukaryotic organisms. The transport protein belongs to the mitochondrial carrier family, a large transporter family in the inner membrane of mitochondria. It is one of the best studied members of the family and serves as a paradigm for the molecular mechanism of mitochondrial carriers. Structurally, the carrier consists of three homologous domains, each composed of two transmembrane α-helices linked with a loop and short α-helix on the matrix side. The transporter cycles between a cytoplasmic and matrix state in which a central substrate binding site is alternately accessible to these compartments for binding of ADP or ATP. On both the cytoplasmic and matrix side of the carrier are networks consisting of three salt bridges each. In the cytoplasmic state, the matrix salt bridge network is formed and the cytoplasmic network is disrupted, opening the central substrate binding site to the intermembrane space and cytosol, whereas the converse occurs in the matrix state. In the transport cycle, tighter substrate binding in the intermediate states allows the interconversion of conformations by lowering the energy barrier for disruption and formation of these networks, opening and closing the carrier to either side of the membrane in an alternating way. Conversion between cytoplasmic and matrix states might require the simultaneous rotation of three domains around a central translocation pathway, constituting a unique mechanism among transport proteins. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.

  10. Discovery of novel poly(ADP-ribose) glycohydrolase inhibitors by a quantitative assay system using dot-blot with anti-poly(ADP-ribose)

    SciTech Connect

    Okita, Naoyuki; Ashizawa, Daisuke; Ohta, Ryo; Abe, Hideaki; Tanuma, Sei-ichi

    2010-02-19

    Poly(ADP-ribosyl)ation, which is mainly regulated by poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG), is a unique protein modification involved in cellular responses such as DNA repair and replication. PARG hydrolyzes glycosidic linkages of poly(ADP-ribose) synthesized by PARP and liberates ADP-ribose residues. Recent studies have suggested that inhibitors of PARG are able to be potent anti-cancer drug. In order to discover the potent and specific Inhibitors of PARG, a quantitative and high-throughput screening assay system is required. However, previous PARG assay systems are not appropriate for high-throughput screening because PARG activity is measured by radioactivities of ADP-ribose residues released from radioisotope (RI)-labeled poly(ADP-ribose). In this study, we developed a non-RI and quantitative assay system for PARG activity based on dot-blot assay using anti-poly(ADP-ribose) and nitrocellulose membrane. By our method, the maximum velocity (V{sub max}) and the michaelis constant (k{sub m}) of PARG reaction were 4.46 {mu}M and 128.33 {mu}mol/min/mg, respectively. Furthermore, the IC50 of adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), known as a non-competitive PARG inhibitor, was 0.66 {mu}M. These kinetics values were similar to those obtained by traditional PARG assays. By using our assay system, we discovered two novel PARG inhibitors that have xanthene scaffold. Thus, our quantitative and convenient method is useful for a high-throughput screening of PARG specific inhibitors.

  11. Abscisic acid signaling through cyclic ADP-ribose in plants

    SciTech Connect

    Wu, Yan; Kuzma, J.; Marechal, E.

    1997-12-19

    Abscisic acid (ABA) is the primary hormone that mediates plant responses to stresses such as cold, drought, and salinity. Single-cell microinjection experiments in tomato were used to identify possible intermediates involved in ABA signal transduction. Cyclic ADP-ribose (cADPR) was identified as a signaling molecule in the ABA response and was shown to exert its effects by way of calcium. Bioassay experiments showed that the amounts of cADPR in Arabidopsis thaliana plants increased in response to ABA treatment and before ABA-induced gene expression.

  12. Molecular bases of catalysis and ADP-ribose preference of human Mn2+-dependent ADP-ribose/CDP-alcohol diphosphatase and conversion by mutagenesis to a preferential cyclic ADP-ribose phosphohydrolase.

    PubMed

    Cabezas, Alicia; Ribeiro, João Meireles; Rodrigues, Joaquim Rui; López-Villamizar, Iralis; Fernández, Ascensión; Canales, José; Pinto, Rosa María; Costas, María Jesús; Cameselle, José Carlos

    2015-01-01

    Among metallo-dependent phosphatases, ADP-ribose/CDP-alcohol diphosphatases form a protein family (ADPRibase-Mn-like) mainly restricted, in eukaryotes, to vertebrates and plants, with preferential expression, at least in rodents, in immune cells. Rat and zebrafish ADPRibase-Mn, the only biochemically studied, are phosphohydrolases of ADP-ribose and, somewhat less efficiently, of CDP-alcohols and 2´,3´-cAMP. Furthermore, the rat but not the zebrafish enzyme displays a unique phosphohydrolytic activity on cyclic ADP-ribose. The molecular basis of such specificity is unknown. Human ADPRibase-Mn showed similar activities, including cyclic ADP-ribose phosphohydrolase, which seems thus common to mammalian ADPRibase-Mn. Substrate docking on a homology model of human ADPRibase-Mn suggested possible interactions of ADP-ribose with seven residues located, with one exception (Cys253), either within the metallo-dependent phosphatases signature (Gln27, Asn110, His111), or in unique structural regions of the ADPRibase-Mn family: s2s3 (Phe37 and Arg43) and h7h8 (Phe210), around the active site entrance. Mutants were constructed, and kinetic parameters for ADP-ribose, CDP-choline, 2´,3´-cAMP and cyclic ADP-ribose were determined. Phe37 was needed for ADP-ribose preference without catalytic effect, as indicated by the increased ADP-ribose Km and unchanged kcat of F37A-ADPRibase-Mn, while the Km values for the other substrates were little affected. Arg43 was essential for catalysis as indicated by the drastic efficiency loss shown by R43A-ADPRibase-Mn. Unexpectedly, Cys253 was hindering for cADPR phosphohydrolase, as indicated by the specific tenfold gain of efficiency of C253A-ADPRibase-Mn with cyclic ADP-ribose. This allowed the design of a triple mutant (F37A+L196F+C253A) for which cyclic ADP-ribose was the best substrate, with a catalytic efficiency of 3.5´104 M-1s-1 versus 4´103 M-1s-1 of the wild type.

  13. Molecular Bases of Catalysis and ADP-Ribose Preference of Human Mn2+-Dependent ADP-Ribose/CDP-Alcohol Diphosphatase and Conversion by Mutagenesis to a Preferential Cyclic ADP-Ribose Phosphohydrolase

    PubMed Central

    Cabezas, Alicia; Ribeiro, João Meireles; Rodrigues, Joaquim Rui; López-Villamizar, Iralis; Fernández, Ascensión; Canales, José; Pinto, Rosa María; Costas, María Jesús; Cameselle, José Carlos

    2015-01-01

    Among metallo-dependent phosphatases, ADP-ribose/CDP-alcohol diphosphatases form a protein family (ADPRibase-Mn-like) mainly restricted, in eukaryotes, to vertebrates and plants, with preferential expression, at least in rodents, in immune cells. Rat and zebrafish ADPRibase-Mn, the only biochemically studied, are phosphohydrolases of ADP-ribose and, somewhat less efficiently, of CDP-alcohols and 2´,3´-cAMP. Furthermore, the rat but not the zebrafish enzyme displays a unique phosphohydrolytic activity on cyclic ADP-ribose. The molecular basis of such specificity is unknown. Human ADPRibase-Mn showed similar activities, including cyclic ADP-ribose phosphohydrolase, which seems thus common to mammalian ADPRibase-Mn. Substrate docking on a homology model of human ADPRibase-Mn suggested possible interactions of ADP-ribose with seven residues located, with one exception (Cys253), either within the metallo-dependent phosphatases signature (Gln27, Asn110, His111), or in unique structural regions of the ADPRibase-Mn family: s2s3 (Phe37 and Arg43) and h7h8 (Phe210), around the active site entrance. Mutants were constructed, and kinetic parameters for ADP-ribose, CDP-choline, 2´,3´-cAMP and cyclic ADP-ribose were determined. Phe37 was needed for ADP-ribose preference without catalytic effect, as indicated by the increased ADP-ribose Km and unchanged kcat of F37A-ADPRibase-Mn, while the Km values for the other substrates were little affected. Arg43 was essential for catalysis as indicated by the drastic efficiency loss shown by R43A-ADPRibase-Mn. Unexpectedly, Cys253 was hindering for cADPR phosphohydrolase, as indicated by the specific tenfold gain of efficiency of C253A-ADPRibase-Mn with cyclic ADP-ribose. This allowed the design of a triple mutant (F37A+L196F+C253A) for which cyclic ADP-ribose was the best substrate, with a catalytic efficiency of 3.5´104 M-1s-1 versus 4´103 M-1s-1 of the wild type. PMID:25692488

  14. Diversity and Impact of Rare Variants in Genes Encoding the Platelet G Protein-Coupled Receptors

    PubMed Central

    Jones, Matthew L.; Norman, Jane E.; Morgan, Neil V.; Mundell, Stuart J.; Lordkipanidzé, Marie; Lowe, Gillian C.; Daly, Martina E.; Simpson, Michael A.; Drake, Sian; Watson, Steve P.

    2015-01-01

    Summary Platelet responses to activating agonists are influenced by common population variants within or near G protein-coupled receptor (GPCR) genes that affect receptor activity. However, the impact of rare GPCR gene variants is unknown. We describe the rare single nucleotide variants (SNVs) in the coding and splice regions of 18 GPCR genes in 7,595 exomes from the 1,000-genomes and Exome Sequencing Project databases and in 31 cases with inherited platelet function disorders (IPFDs). In the population databases, the GPCR gene target regions contained 740 SNVs (318 synonymous, 410 missense, 7 stop gain and 6 splice region) of which 70% had global minor allele frequency (MAF) < 0.05%. Functional annotation using six computational algorithms, experimental evidence and structural data identified 156/740 (21%) SNVs as potentially damaging to GPCR function, most commonly in regions encoding the transmembrane and C-terminal intracellular receptor domains. In 31 index cases with IPFDs (Gi-pathway defect n=15; secretion defect n=11; thromboxane pathway defect n=3 and complex defect n=2) there were 256 SNVs in the target regions of 15 stimulatory platelet GPCRs (34 unique; 12 with MAF<1% and 22 with MAF ≥ 1%). These included rare variants predicting R122H, P258T and V207A substitutions in the P2Y12 receptor that were annotated as potentially damaging, but only partially explained the platelet function defects in each case. Our data highlight that potentially damaging variants in platelet GPCR genes have low individual frequencies, but are collectively abundant in the population. Potentially damaging variants are also present in pedigrees with IPFDs and may contribute to complex laboratory phenotypes. PMID:25567036

  15. 8-Bromo-cyclic inosine diphosphoribose: towards a selective cyclic ADP-ribose agonist

    PubMed Central

    Kirchberger, Tanja; Moreau, Christelle; Wagner, Gerd K.; Fliegert, Ralf; Siebrands, Cornelia C.; Nebel, Merle; Schmid, Frederike; Harneit, Angelika; Odoardi, Francesca; Flügel, Alexander; Potter, Barry V. L.; Guse, Andreas H.

    2009-01-01

    cADPR (cyclic ADP-ribose) is a universal Ca2+ mobilizing second messenger. In T-cells cADPR is involved in sustained Ca2+ release and also in Ca2+ entry. Potential mechanisms for the latter include either capacitative Ca2+ entry, secondary to store depletion by cADPR, or direct activation of the non-selective cation channel TRPM2 (transient receptor potential cation channel, subfamily melastatin, member 2). Here we characterize the molecular target of the newly-described membrane-permeant cADPR agonist 8-Br-N1-cIDPR (8-bromo-cyclic IDP-ribose). 8-Br-N1-cIDPR evoked Ca2+ signalling in the human T-lymphoma cell line Jurkat and in primary rat T-lymphocytes. Ca2+ signalling induced by 8-Br-N1-cIDPR consisted of Ca2+ release and Ca2+ entry. Whereas Ca2+ release was sensitive to both the RyR (ryanodine receptor) blocker RuRed (Ruthenium Red) and the cADPR antagonist 8-Br-cADPR (8-bromo-cyclic ADP-ribose), Ca2+ entry was inhibited by the Ca2+ entry blockers Gd3+ (gadolinium ion) and SKF-96365, as well as by 8-Br-cADPR. To unravel a potential role for TRPM2 in sustained Ca2+ entry evoked by 8-Br-N1-cIDPR, TRPM2 was overexpressed in HEK (human embryonic kidney)-293 cells. However, though activation by H2O2 was enhanced dramatically in those cells, Ca2+ signalling induced by 8-Br-N1-cIDPR was almost unaffected. Similarly, direct analysis of TRPM2 currents did not reveal activation or co-activation of TRPM2 by 8-Br-N1-cIDPR. In summary, the sensitivity to the Ca2+ entry blockers Gd3+ and SKF-96365 is in favour of the concept of capacitative Ca2+ entry, secondary to store depletion by 8-Br-N1-cIDPR. Taken together, 8-Br-N1-cIDPR appears to be the first cADPR agonist affecting Ca2+ release and secondary Ca2+ entry, but without effect on TRPM2. PMID:19492987

  16. Differential activities of cellular and viral macro domain proteins in binding of ADP-ribose metabolites.

    PubMed

    Neuvonen, Maarit; Ahola, Tero

    2009-01-01

    Macro domain is a highly conserved protein domain found in both eukaryotes and prokaryotes. Macro domains are also encoded by a set of positive-strand RNA viruses that replicate in the cytoplasm of animal cells, including coronaviruses and alphaviruses. The functions of the macro domain are poorly understood, but it has been suggested to be an ADP-ribose-binding module. We have here characterized three novel human macro domain proteins that were found to reside either in the cytoplasm and nucleus [macro domain protein 2 (MDO2) and ganglioside-induced differentiation-associated protein 2] or in mitochondria [macro domain protein 1 (MDO1)], and compared them with viral macro domains from Semliki Forest virus, hepatitis E virus, and severe acute respiratory syndrome coronavirus, and with a yeast macro protein, Poa1p. MDO2 specifically bound monomeric ADP-ribose with a high affinity (K(d)=0.15 microM), but did not bind poly(ADP-ribose) efficiently. MDO2 also hydrolyzed ADP-ribose-1'' phosphate, resembling Poa1p in all these properties. Ganglioside-induced differentiation-associated protein 2 did not show affinity for ADP-ribose or its derivatives, but instead bound poly(A). MDO1 was generally active in these reactions, including poly(A) binding. Individual point mutations in MDO1 abolished monomeric ADP-ribose binding, but not poly(ADP-ribose) binding; in poly(ADP-ribose) binding assays, the monomer did not compete against polymer binding. The viral macro proteins bound poly(ADP-ribose) and poly(A), but had a low affinity for monomeric ADP-ribose. Thus, the viral proteins do not closely resemble any of the human proteins in their biochemical functions. The differential activity profiles of the human proteins implicate them in different cellular pathways, some of which may involve RNA rather than ADP-ribose derivatives.

  17. Frequency doubling of copper lasers using temperature-tuned ADP

    SciTech Connect

    Molander, W.A.

    1994-03-01

    The ability to generate high average power uv at 255 nm by frequency doubling the green line (510.6 nm) of copper lasers would greatly extend the utility of copper lasers. Material processing and microlithography are two areas of interest. The frequency-doubled copper laser could replace the KrF excimer laser, which has a similar wavelength (248 nm), in some applications. The frequency-doubled copper laser has a narrow linewidth and excellent beam quality at a competitive cost. Other attractive features are high reliability, low operating costs, and the absence of toxic gases. This paper will report recent progress in high-efficiency, high-average-power harmonic generation of the copper laser green line using noncritical phase matching in ADP. Frequency doubling of the yellow line (578.2 nm) and sum-frequency mixing of the two lines are also of interest. These processes, however, cannot be phase-matched in ADP and, therefore, will not be discussed here. The results reported and the issues identified here would be important in these other processes and also in many other frequency conversion schemes in the uv such as 4{omega} conversion of Nd{sup 3+}:YAG lasers.

  18. 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.

  19. Nitrogen Control of Atrazine Utilization in Pseudomonas sp. Strain ADP

    PubMed Central

    García-González, Vicente; Govantes, Fernando; Shaw, Liz J.; Burns, Richard G.; Santero, Eduardo

    2003-01-01

    Pseudomonas sp. strain ADP uses the herbicide atrazine as the sole nitrogen source. We have devised a simple atrazine degradation assay to determine the effect of other nitrogen sources on the atrazine degradation pathway. The atrazine degradation rate was greatly decreased in cells grown on nitrogen sources that support rapid growth of Pseudomonas sp. strain ADP compared to cells cultivated on growth-limiting nitrogen sources. The presence of atrazine in addition to the nitrogen sources did not stimulate degradation. High degradation rates obtained in the presence of ammonium plus the glutamine synthetase inhibitor MSX and also with an Nas− mutant derivative grown on nitrate suggest that nitrogen regulation operates by sensing intracellular levels of some key nitrogen-containing metabolite. Nitrate amendment in soil microcosms resulted in decreased atrazine mineralization by the wild-type strain but not by the Nas− mutant. This suggests that, although nitrogen repression of the atrazine catabolic pathway may have a strong impact on atrazine biodegradation in nitrogen-fertilized soils, the use of selected mutant variants may contribute to overcoming this limitation. PMID:14660340

  20. 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

  1. 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.

  2. Effect of heat shock on poly(ADP-ribose) synthetase and DNA repair in Drosophila cells

    SciTech Connect

    Nolan, N.L.; Kidwell, W.R.

    1982-04-01

    Poly(ADP-ribose) synthetase, a chromatin-bound enzyme which attaches polyanionic chains of ADP-ribose to nuclear proteins, was found to be temperature sensitive in intact Drosophila melanogaster cells. The synthetase was completely inactivated by heat-shocking the cells at 37/sup 0/C for 5 min, a condition which had no appreciable effect on the subsequent growth of Drosophila cells at their physiological temperature. The heat-shock effect on synthetase was reversible; enzyme activity began to reappear about 2 hr post heat shock. During the 2-hr interval when poly(ADP-ribose) synthetase was absent, the cells were competent in repair of ..gamma..-ray-induced DNA strand breaks as shown by DNA sedimentation studies on alkaline sucrose gradients. It is thus concluded that poly(ADP-ribose) synthesis is unnecessary for repair of DNA strand breaks introduced by irradiation. The same conclusion was reached from the fact that two inhibitors of poly(ADP-ribose) synthetase 3-aminobenzamide and 5-methylnicotinamide, failed to block repair of ..gamma..-ray-induced DNA chain breaks even though both inhibitors reduced the amount of poly(ADP-ribose) synthesized in cells by 50-75%. Although it was found that the repair of DNA strand breaks is independent of poly(ADP-ribose) synthesis, irradiation does activate the synthetase in control cells, as shown by radioimmunoassay of poly(ADP-ribose) levels.

  3. 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.

  4. 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

  5. ADP1 affects abundance and endocytosis of PIN-FORMED proteins in Arabidopsis.

    PubMed

    Li, Jieru; Li, Ruixi; Jiang, Zhaoyun; Gu, Hongya; Qu, Li-Jia

    2015-01-01

    Auxin, as a vital plant hormone, regulates almost every aspect of plant growth and development. We previously identified a dominant mutant, adp1-D, displaying loss of apical dominance. We also demonstrated that down-regulation of local auxin biosynthesis in adp1-D was responsible for the bushy phenotype of this mutant. Consistent with the reduction of local auxin biosynthesis, we recently discovered that protein abundance of PIN1, PIN3, and PIN7 was reduced in adp1-D without accompanying transcription level changes. Additionally, subcellular analysis revealed that over-expression of ADP1 inhibited endocytosis of PIN proteins. Taken together, we conclude that ADP1 regulates plant architecture through the fine-tuning of local auxin biosynthesis and through post-transcriptional regulation of auxin transporters. PMID:25482774

  6. 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.

  7. Ethanol-induced changes in poly (ADP ribose) polymerase and neuronal developmental gene expression.

    PubMed

    Gavin, David P; Kusumo, Handojo; Sharma, Rajiv P; Guizzetti, Marina

    2016-11-01

    Prenatal alcohol exposure has profound effects on neuronal growth and development. Poly-ADP Ribose Polymerase (PARP) enzymes are perhaps unique in the field of epigenetics in that they directly participate in histone modifications, transcription factor modifications, DNA methylation/demethylation and are highly inducible by ethanol. It was our hypothesis that ethanol would induce PARP enzymatic activity leading to alterations in neurodevelopmental gene expression. Mouse E18 cortical neurons were treated with ethanol, PARP inhibitors, and nuclear hormone receptor transcription factor PPARγ agonists and antagonists. Subsequently, we measured PARP activity and changes in Bdnf, OKSM (Oct4, Klf4, Sox2, c-Myc), DNA methylating/demethylating factors, and Pparγ mRNA expression, promoter 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC), and PPARγ promoter binding. We found that ethanol reduced Bdnf4, 9a, and Klf4 mRNA expression, and increased c-Myc expression. These changes were reversed with a PARP inhibitor. In agreement with its role in DNA demethylation PARP inhibition increased 5MC levels at the c-Myc promoter. In addition, we found that inhibition of PARP enzymatic activity increased PPARγ promoter binding, and this corresponded to increased Bdnf and Klf4 mRNA expression. Our results suggest that PARP participates in DNA demethylation and reduces PPARγ promoter binding. The current study underscores the importance of PARP in ethanol-induced changes to neurodevelopmental gene expression. PMID:27497606

  8. Ethanol-induced changes in poly (ADP ribose) polymerase and neuronal developmental gene expression.

    PubMed

    Gavin, David P; Kusumo, Handojo; Sharma, Rajiv P; Guizzetti, Marina

    2016-11-01

    Prenatal alcohol exposure has profound effects on neuronal growth and development. Poly-ADP Ribose Polymerase (PARP) enzymes are perhaps unique in the field of epigenetics in that they directly participate in histone modifications, transcription factor modifications, DNA methylation/demethylation and are highly inducible by ethanol. It was our hypothesis that ethanol would induce PARP enzymatic activity leading to alterations in neurodevelopmental gene expression. Mouse E18 cortical neurons were treated with ethanol, PARP inhibitors, and nuclear hormone receptor transcription factor PPARγ agonists and antagonists. Subsequently, we measured PARP activity and changes in Bdnf, OKSM (Oct4, Klf4, Sox2, c-Myc), DNA methylating/demethylating factors, and Pparγ mRNA expression, promoter 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC), and PPARγ promoter binding. We found that ethanol reduced Bdnf4, 9a, and Klf4 mRNA expression, and increased c-Myc expression. These changes were reversed with a PARP inhibitor. In agreement with its role in DNA demethylation PARP inhibition increased 5MC levels at the c-Myc promoter. In addition, we found that inhibition of PARP enzymatic activity increased PPARγ promoter binding, and this corresponded to increased Bdnf and Klf4 mRNA expression. Our results suggest that PARP participates in DNA demethylation and reduces PPARγ promoter binding. The current study underscores the importance of PARP in ethanol-induced changes to neurodevelopmental gene expression.

  9. 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.

  10. Crosstalk between poly(ADP-ribose) polymerase and sirtuin enzymes

    PubMed Central

    Cantó, Carles; Sauve, Anthony A.; Bai, Peter

    2013-01-01

    Poly(ADP-ribose) polymerases (PARPs) are NAD+ dependent enzymes that were identified as DNA repair proteins, however, today it seems clear that PARPs are responsible for a plethora of biological functions. Sirtuins (SIRTs) are NAD+-dependent deacetylase enzymes involved in the same biological processes as PARPs raising the question whether PARP and SIRT enzymes may interact with each other in physiological and pathophysiological conditions. Hereby we review the current understanding of the SIRT-PARP interplay in regard to the biochemical nature of the interaction (competition for the common NAD+ substrate, mutual posttranslational modifications and direct transcriptional effects) and the physiological, or pathophysiological consequences of the interactions (metabolic events, oxidative stress response, genomic stability and ageing). Finally, we give an overview of the possibilities of pharmacological intervention to modulate PARP and SIRT enzymes either directly, or through modulating NAD+ homeostasis. PMID:23357756

  11. Crosstalk between poly(ADP-ribose) polymerase and sirtuin enzymes.

    PubMed

    Cantó, Carles; Sauve, Anthony A; Bai, Peter

    2013-12-01

    Poly(ADP-ribose) polymerases (PARPs) are NAD(+) dependent enzymes that were identified as DNA repair proteins, however, today it seems clear that PARPs are responsible for a plethora of biological functions. Sirtuins (SIRTs) are NAD(+)-dependent deacetylase enzymes involved in the same biological processes as PARPs raising the question whether PARP and SIRT enzymes may interact with each other in physiological and pathophysiological conditions. Hereby we review the current understanding of the SIRT-PARP interplay in regard to the biochemical nature of the interaction (competition for the common NAD(+) substrate, mutual posttranslational modifications and direct transcriptional effects) and the physiological or pathophysiological consequences of the interactions (metabolic events, oxidative stress response, genomic stability and aging). Finally, we give an overview of the possibilities of pharmacological intervention to modulate PARP and SIRT enzymes either directly, or through modulating NAD(+) homeostasis.

  12. Higher cytoplasmic and nuclear poly(ADP-ribose) polymerase expression in familial than in sporadic breast cancer.

    PubMed

    Klauke, Marie-Luise; Hoogerbrugge, Nicoline; Budczies, Jan; Bult, Peter; Prinzler, Judith; Radke, Cornelia; van Krieken, J Han J M; Dietel, Manfred; Denkert, Carsten; Müller, Berit Maria

    2012-10-01

    Poly(ADP-ribose) polymerase 1 (PARP) is a key element of the single-base excision pathway for repair of DNA single-strand breaks. To compare the cytoplasmic and nuclear poly(ADP-ribose) expression between familial (BRCA1, BRCA2, or non BRCA1/2) and sporadic breast cancer, we investigated 39 sporadic and 39 familial breast cancer cases. The two groups were matched for hormone receptor status and human epidermal growth factor receptor 2 status. Additionally, they were matched by grading with a maximum difference of ±1 degree (e.g., G2 instead of G3). Cytoplasmic PARP (cPARP) expression was significantly higher in familial compared to sporadic breast cancer (P = 0.008, chi-squared test for trends) and a high nuclear PARP expression (nPARP) was significantly more frequently observed in familial breast cancer (64 %) compared with sporadic breast cancer (36 %) (P = 0.005, chi-squared test). The overall PARP expression was significantly higher in familial breast cancer (P = 0.042, chi-squared test). In familial breast cancer, a combination of high cPARP and high nPARP expression is the most common (33 %), whereas in sporadic breast cancer, a combination of low cPARP and intermediate nPARP expression is the most common (39 %). Our results show that the overall PARP expression in familial breast cancer is higher than in sporadic breast cancer which might suggest they might respond better to treatment with PARP inhibitors.

  13. 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.

  14. Purinergic receptors contribute to early mesangial cell transformation and renal vessel hypertrophy during angiotensin II-induced hypertension

    PubMed Central

    Graciano, Miguel L.; Nishiyama, Akira; Jackson, Keith; Seth, Dale M.; Ortiz, Rudy M.; Prieto-Carrasquero, Minolfa C.; Kobori, Hiroyuki; Navar, L. Gabriel

    2008-01-01

    Chronic ANG II infusions lead to increases in intrarenal ANG II levels, hypertension, and tissue injury. Increased blood pressure also elicits increases in renal interstitial fluid (RIF) ATP concentrations that stimulate cell proliferation. We evaluated the contribution of purinergic receptor activation to ANG II-induced renal injury in rats by treating with clopidogrel, a P2Y12 receptor blocker, or with PPADS, a nonselective P2 receptor blocker. α-Actin expression in mesangial cells, afferent arteriolar wall thickness (AAWT), cortical cell proliferation, and macrophage infiltration were used as early markers of renal injury. Clopidogrel and PPADS did not alter blood pressure, renin or kidney ANG II content. α-Actin expression increased from control of 0.6 ± 0.4% of mesangial area to 6.3 ± 1.9% in ANG II-infused rats and this response was prevented by clopidogrel (0.4 ± 0.2%) and PPADS. The increase in AAWT from 4.7 ± 0.1 to 6.0 ± 0.1 mm in ANG II rats was also prevented by clopidogrel (4.8 ± 0.1 mm) and PPADS. ANG II infusion led to interstitial macrophage infiltration (105 ± 16 vs. 62 ± 4 cell/mm2) and tubular proliferation (71 ± 15 vs. 20 ± 4 cell/mm2) and these effects were prevented by clopidogrel (52 ± 4 and 36 ± 3 cell/mm2) and PPADS. RIF ATP levels were higher in ANG II-infused rats than in control rats (11.8 ± 1.9 vs. 5.6 ± 0.6 nmol/l, P < 0.05). The results suggest that activation of vascular and glomerular purinergic P2 receptors may contribute to the mesangial cell transformation, renal inflammation, and vascular hypertrophy observed in ANG II-dependent hypertension. PMID:17989111

  15. Comparative studies on antibodies to poly(ADP-ribose) in rabbits and patients with systemic lupus erythematosus.

    PubMed Central

    Kanai, Y; Sugimura, T

    1981-01-01

    Immunochemical studies were made on the antibodies induced in rabbits against poly(ADP-ribose) and naturally-occurring antibodies in patients with systemic lupus erythematosus. Antibodies against poly(ADP-ribose) could also be induced in rabbits by oligo(ADP-ribose) associated with rat liver histones and by a complex of poly(ADP-ribose) with methylated bovine serum albumin (MBSA). The two types of antibody were inhibited to the same extent by poly(ADP-ribose). However, the antibody induced by oligo(ADP-ribose) associated with histones was inhibited by oligo(ADP-ribose) with an average chain length if 4 ADP-ribosyl units and by phosphoribosyl adenosine monophosphate (PR-AMP) but not by mono ADP-ribose, whereas that induced by poly(ADP-ribose) was practically not inhibited by these related compounds even in excess amounts. The sera of ten cases of systemic lupus erythematosus showing high antibody activity against poly(ADP-ribose) were also examined immunochemically. It was found that the antibodies of three patients showed a similar inhibitory pattern to that of antibody induced in rabbits by oligo(ADP-ribose) associated with histones, those of three patients showed a similar pattern to that of antibody produced in rabbits by poly(ADP-ribose), and the remainder did not show either pattern. These findings suggest that oligo(ADP-ribose) associated with histones may serve as antigen to elicit naturally-occuring antibodies to poly(ADP-ribose) in patients with systemic lupus erythematosus. PMID:7251045

  16. Proteome-wide identification of the endogenous ADP-ribosylome of mammalian cells and tissue

    PubMed Central

    Martello, Rita; Leutert, Mario; Jungmichel, Stephanie; Bilan, Vera; Larsen, Sara C.; Young, Clifford; Hottiger, Michael O.; Nielsen, Michael L.

    2016-01-01

    Although protein ADP-ribosylation is involved in diverse biological processes, it has remained a challenge to identify ADP-ribose acceptor sites. Here, we present an experimental workflow for sensitive and unbiased analysis of endogenous ADP-ribosylation sites, capable of detecting more than 900 modification sites in mammalian cells and mouse liver. In cells, we demonstrate that Lys residues, besides Glu, Asp and Arg residues, are the dominant in vivo targets of ADP-ribosylation during oxidative stress. In normal liver tissue, we find Arg residues to be the predominant modification site. The cellular distribution and biological processes that involve ADP-ribosylated proteins are different in cultured cells and liver tissue, in the latter of which the majority of sites were found to be in cytosolic and mitochondrial protein networks primarily associated with metabolism. Collectively, we describe a robust methodology for the assessment of the role of ADP-ribosylation and ADP-ribosyltransferases in physiological and pathological states. PMID:27686526

  17. Structure of the Escherichia coli heptosyltransferase WaaC: binary complexes with ADP and ADP-2-deoxy-2-fluoro heptose.

    PubMed

    Grizot, Sylvestre; Salem, Michèle; Vongsouthi, Vanida; Durand, Lionel; Moreau, François; Dohi, Hirofumi; Vincent, Stéphane; Escaich, Sonia; Ducruix, Arnaud

    2006-10-20

    Lipopolysaccharides constitute the outer leaflet of the outer membrane of Gram-negative bacteria and are therefore essential for cell growth and viability. The heptosyltransferase WaaC is a glycosyltransferase (GT) involved in the synthesis of the inner core region of LPS. It catalyzes the addition of the first L-glycero-D-manno-heptose (heptose) molecule to one 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residue of the Kdo2-lipid A molecule. Heptose is an essential component of the LPS core domain; its absence results in a truncated lipopolysaccharide associated with the deep-rough phenotype causing a greater susceptibility to antibiotic and an attenuated virulence for pathogenic Gram-negative bacteria. Thus, WaaC represents a promising target in antibacterial drug design. Here, we report the structure of WaaC from the Escherichia coli pathogenic strain RS218 alone at 1.9 A resolution, and in complex with either ADP or the non-cleavable analog ADP-2-deoxy-2-fluoro-heptose of the sugar donor at 2.4 A resolution. WaaC adopts the GT-B fold in two domains, characteristic of one glycosyltransferase structural superfamily. The comparison of the three different structures shows that WaaC does not undergo a domain rotation, characteristic of the GT-B family, upon substrate binding, but allows the substrate analog and the reaction product to adopt remarkably distinct conformations inside the active site. In addition, both binary complexes offer a close view of the donor subsite and, together with results from site-directed mutagenesis studies, provide evidence for a model of the catalytic mechanism.

  18. Insights into the mechanism of ADP action on flagellar motility derived from studies on bull sperm.

    PubMed

    Lesich, Kathleen A; Pelle, Dominic W; Lindemann, Charles B

    2008-07-01

    Adenosine diphosphate (ADP) is known to have interesting effects on flagellar motility. Permeabilized and reactivated bull sperm exhibit a marked reduction in beating frequency and a greatly increased beat amplitude in the presence of 1-4 mM ADP. In this study we examined the force production of sperm reactivated with 0.1 mM ATP with and without 1 mM ADP and found that there is little or no resulting change in the stalling force produced by a bull sperm flagella in response to ADP. Because bull sperm bend to a higher curvature after ADP treatment we explored the possibility that ADP-treated sperm flagella are more flexible. We measured the stiffness of 50 muM sodium vanadate treated bull sperm in the presence of 4 mM ADP, but found no change in the passive flagellar stiffness. When we analyzed the torque that develops in ADP-treated sperm at the point of beat reversal we found that the torque developed by the flagellum is significantly increased. Our torque estimates also allow us to calculate the transverse force (t-force) acting on the flagellum at the point of beat direction reversal. We find that the t-force at the switch-point of the beat is increased significantly in the ADP treated condition, averaging 0.7 +/- 0.29 nN/microm in 0.1 mM ATP and increasing to 2.9 +/- 1.2 nN/microm in 0.1 mM ATP plus 4 mM ADP. This suggests that ADP is exerting its effect on the beat by increasing the tenacity of dynein attachment at the B-subtubule. This could be a direct result of a regulatory effect of ADP on the binding affinity of dynein for the B-subtubule of the outer doublets. This result could also help to explain a number of previous experimental observations, as discussed. PMID:18375503

  19. Insights into the Mechanism of ADP Action on Flagellar Motility Derived from Studies on Bull Sperm

    PubMed Central

    Lesich, Kathleen A.; Pelle, Dominic W.; Lindemann, Charles B.

    2008-01-01

    Adenosine diphosphate (ADP) is known to have interesting effects on flagellar motility. Permeabilized and reactivated bull sperm exhibit a marked reduction in beating frequency and a greatly increased beat amplitude in the presence of 1–4 mM ADP. In this study we examined the force production of sperm reactivated with 0.1 mM ATP with and without 1 mM ADP and found that there is little or no resulting change in the stalling force produced by a bull sperm flagella in response to ADP. Because bull sperm bend to a higher curvature after ADP treatment we explored the possibility that ADP-treated sperm flagella are more flexible. We measured the stiffness of 50 μM sodium vanadate treated bull sperm in the presence of 4 mM ADP, but found no change in the passive flagellar stiffness. When we analyzed the torque that develops in ADP-treated sperm at the point of beat reversal we found that the torque developed by the flagellum is significantly increased. Our torque estimates also allow us to calculate the transverse force (t-force) acting on the flagellum at the point of beat direction reversal. We find that the t-force at the switch-point of the beat is increased significantly in the ADP treated condition, averaging 0.7 ± 0.29 nN/μm in 0.1 mM ATP and increasing to 2.9 ± 1.2 nN/μm in 0.1 mM ATP plus 4 mM ADP. This suggests that ADP is exerting its effect on the beat by increasing the tenacity of dynein attachment at the B-subtubule. This could be a direct result of a regulatory effect of ADP on the binding affinity of dynein for the B-subtubule of the outer doublets. This result could also help to explain a number of previous experimental observations, as discussed. PMID:18375503

  20. Poly(ADP-ribose) glycohydrolase and poly(ADP-ribose)-interacting protein Hrp38 regulate pattern formation during Drosophila eye development.

    PubMed

    Ji, Yingbiao; Jarnik, Michael; Tulin, Alexei V

    2013-09-10

    Drosophila Hrp38, a homolog of human hnRNP A1, has been shown to regulate splicing, but its function can be modified by poly(ADP-ribosyl)ation. Notwithstanding such findings, our understanding of the roles of poly(ADP-ribosyl)ated Hrp38 on development is limited. Here, we have demonstrated that Hrp38 is essential for fly eye development based on a rough-eye phenotype with disorganized ommatidia observed in adult escapers of the hrp38 mutant. We also observed that poly(ADP-ribose) glycohydrolase (Parg) loss-of-function, which caused increased Hrp38 poly(ADP-ribosyl)ation, also resulted in the rough-eye phenotype with disrupted ommatidial lattice and reduced number of photoreceptor cells. In addition, ectopic expression of DE-cadherin, which is required for retinal morphogenesis, fully rescued the rough-eye phenotype of the hrp38 mutant. Similarly, Parg mutant eye clones had decreased expression level of DE-cadherin with orientation defects, which is reminiscent of DE-cadherin mutant eye phenotype. Therefore, our results suggest that Hrp38 poly(ADP-ribosyl)ation controls eye pattern formation via regulation of DE-cadherin expression, a finding which has implications for understanding the pathogenic mechanisms of Hrp38-related Fragile X syndrome and PARP1-related retinal degeneration diseases.

  1. The Level of AdpA Directly Affects Expression of Developmental Genes in Streptomyces coelicolor ▿ †

    PubMed Central

    Wolański, Marcin; Donczew, Rafał; Kois-Ostrowska, Agnieszka; Masiewicz, Paweł; Jakimowicz, Dagmara; Zakrzewska-Czerwińska, Jolanta

    2011-01-01

    AdpA is a key regulator of morphological differentiation in Streptomyces. In contrast to Streptomyces griseus, relatively little is known about AdpA protein functions in Streptomyces coelicolor. Here, we report for the first time the translation accumulation profile of the S. coelicolor adpA (adpASc) gene; the level of S. coelicolor AdpA (AdpASc) increased, reaching a maximum in the early stage of aerial mycelium formation (after 36 h), and remained relatively stable for the next several hours (48 to 60 h), and then the signal intensity decreased considerably. AdpASc specifically binds the adpASc promoter region in vitro and in vivo, suggesting that its expression is autoregulated; surprisingly, in contrast to S. griseus, the protein presumably acts as a transcriptional activator. We also demonstrate a direct influence of AdpASc on the expression of several genes whose products play key roles in the differentiation of S. coelicolor: STI, a protease inhibitor; RamR, an atypical response regulator that itself activates expression of the genes for a small modified peptide that is required for aerial growth; and ClpP1, an ATP-dependent protease. The diverse influence of AdpASc protein on the expression of the analyzed genes presumably results mainly from different affinities of AdpASc protein to individual promoters. PMID:21926228

  2. [Assessment of platelet function in man].

    PubMed

    Gaussem, Pascale

    2006-01-01

    Assessment of platelet function was primarily designed to explore patients with hemostatic disorders, but is becoming important for the monitoring of anti platelet agents, mostly aspirin and clopidogrel. Beside platelet counting, morphological analysis and bleeding time, a number of dedicated platelet function instruments are now available, generally allowing a rapid evaluation of platelet function in whole blood. The other tests including aggregometry and ELISA measurement of activation markers are generally restricted to specialized laboratories. Although aggregometry is still considered as the "gold standard", the recently developed flow cytometric-based platelet function analysis provides a wide choice of tests that assess the number of surface receptors, the measure of secretion and aggregation, the quantification of microparticules and leukocyte-platelet aggregates. It also allows the measure of the function of the ADP receptor P2Y12 by the phosphorylation level of the VASP protein, method currently under evaluation to monitor the platelet response to clopidogrel treatment. PMID:17243268

  3. Binding of ATP to the progesterone receptor.

    PubMed Central

    Moudgil, V K; Toft, D O

    1975-01-01

    The possible interaction of progesterone--receptor complexes with nucleotides was tested by affinity chromatography. The cytosol progesterone receptor from hen oviduct was partially purified by ammonium sulfate precipitation before use. When progesterone was bound to the receptor, the resulting complex could be selectively adsorbed onto columns of ATP-Sepharose. This interaction was reversible and of an ionic nature since it could be disrupted by high-salt conditions. A competitive binding assay was used to test the specificity of receptor binding to several other nucleotides, including ADP, AMP, and cAMP. A clear specificity for binding ATP was evident from these studies. When ATP was added to receptor preparations, the nucleotide did not affect the sedimentation properties or hormone binding characteristics of the receptor. Although the function of ATP remains unknown, these studies indicate a role of this nucleotide in some aspect of hormone receptor activity. PMID:165493

  4. Structure-Based Design of 3-(4-Aryl-1H-1,2,3-triazol-1-yl)-Biphenyl Derivatives as P2Y14 Receptor Antagonists

    PubMed Central

    2016-01-01

    UDP and UDP-glucose activate the P2Y14 receptor (P2Y14R) to modulate processes related to inflammation, diabetes, and asthma. A computational pipeline suggested alternatives to naphthalene of a previously reported P2Y14R antagonist (3, PPTN) using docking and molecular dynamics simulations on a hP2Y14R homology model based on P2Y12R structures. By reevaluating the binding of 3 to P2Y14R computationally, two alternatives, i.e., alkynyl and triazolyl derivatives, were identified. Improved synthesis of fluorescent antagonist 4 enabled affinity quantification (IC50s, nM) using flow cytometry of P2Y14R-expressing CHO cells. p-F3C-phenyl-triazole 65 (32) was more potent than a corresponding alkyne 11. Thus, additional triazolyl derivatives were prepared, as guided by docking simulations, with nonpolar aryl substituents favored. Although triazoles were less potent than 3 (6), simpler synthesis facilitated further structural optimization. Additionally, relative P2Y14R affinities agreed with predicted binding of alkynyl and triazole analogues. These triazoles, designed through a structure-based approach, can be assessed in disease models. PMID:27331270

  5. 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.

  6. Botulinum ADP-ribosyltransferase activity as affected by detergents and phospholipids.

    PubMed

    Maehama, T; Ohoka, Y; Ohtsuka, T; Takahashi, K; Nagata, K; Nozawa, Y; Ueno, K; Ui, M; Katada, T

    1990-04-24

    GTP-binding proteins with Mr values of 22,000 and 25,000 in bovine brain cytosol were ADP-ribosylated by an exoenzyme (termed C3) purified from Clostridium botulinum type C. The rate of C3-catalyzed ADP-ribosylation of the partially purified substrates was extremely low by itself, but was increased enormously when a protein factor(s) obtained from the cytosol was simultaneously added. The rate of the C3-catalyzed reaction was also stimulated by the addition of certain types of detergents or phospholipids even in the absence of the protein factors. The ADP-ribosylation appeared to be enhanced to an extent more than the additive effect of either the protein factors or the detergents (and phospholipids). Thus, ADP-ribosylation catalyzed by botulinum C3 enzyme was affected not only by cytoplasmic protein factors but also by detergents or phospholipids in manners different from each other.

  7. Unidirectional growth of pure and L-lysine added ADP crystals from aqueous solution

    NASA Astrophysics Data System (ADS)

    Salarian, Samaneh; Dizaji, Hamid Rezagholipour

    2014-01-01

    Pure and L-lysine added ammonium dihydrogen phosphate (ADP) crystals were grown in the <001> direction by Sankaranarayanan-Ramasamy (S-R) method. The grown crystals were characterized by X-Ray diffractometry (XRD), UV-Vis spectroscopy, Fourier Transform Infrared (FT-IR) and Vicker's Microhardness analysis. XRD spectrum of each of the grown crystals proved its crystallinity. The crystals showed good transparency in the entire visible region. FT-IR spectra of the specimens revealed the presence of functional groups in them. The hardness of the pure and L-lysine added ADP crystals were measured and that of the added one was found higher. Meanwhile, it was found that the ADP crystals (pure and L-lysine added) grown by S-R method had higher hardness compared to ADP crystal grown by conventional method.

  8. Preliminary crystallographic analysis of ADP-glucose pyrophosphorylase from Agrobacterium tumefaciens

    SciTech Connect

    Cupp-Vickery, Jill R. Igarashi, Robert Y.; Meyer, Christopher R.

    2005-03-01

    Crystallization and X-ray diffraction methods for native A. tumefaciens ADP-glucose pyrophosphorylase and its selenomethionyl derivative are described. Two crystal forms are identified, both of which diffract to 2 Å.

  9. Use of genetically encoded sensors to monitor cytosolic ATP/ADP ratio in living cells.

    PubMed

    Tarasov, Andrei I; Rutter, Guy A

    2014-01-01

    ATP is not only recognized as the universal energy "currency" in most cells but also plays a less well-known role as an intracellular and extracellular messenger. Here, we review novel approaches for measuring free ATP (or ATP/ADP ratios) in living mammalian cells by using genetically encoded sensors. We also discuss the key technical aspects of routine real-time ATP/ADP monitoring using as a model one of the last-generation fluorescent probes, a fusion protein commonly known as "Perceval." Finally, we present detailed guidelines for the simultaneous measurement of cytosolic ATP/ADP ratios and Ca(2+) concentrations alongside electrical parameters in individual pancreatic β cells, in which energy metabolism is tightly linked to plasma membrane excitability to control the secretion of insulin. With appropriate variations, this approach can be adapted to the study of cytosolic ATP/ADP ratios and Ca(2+) concentrations in malignant cells, two important aspects of oncometabolism.

  10. Unscheduled synthesis of DNA and poly(ADP-ribose) in human fibroblasts following DNA damage

    SciTech Connect

    McCurry, L.S.; Jacobson, M.K.

    1981-01-01

    Unscheduled DNA synthesis has been measured in human fibroblasts under conditions of reduced rates of conversion of NAD to poly)ADP-ribose). Cells heterozygous for the xeroderma pigmentosum genotype showed normal rates of uv induced unscheduled DNA synthesis under conditions in which the rate of poly(ADP-ribose) synthesis was one-half the rate of normal cells. The addition of theophylline, a potent inhibitor of poly(ADP-ribose) polymerase, to the culture medium of normal cells blocked over 90% of the conversion of NAD to poly(ADP-ribose) following treatment with uv or N-methyl-N'-nitro-N-nitro-soguanidine but did not affect the rate of unscheduled DNA synthesis.

  11. The energetics of allosteric regulation of ADP release from myosin heads.

    PubMed

    Jackson, Del R; Baker, Josh E

    2009-06-28

    Myosin molecules are involved in a wide range of transport and contractile activities in cells. A single myosin head functions through its ATPase reaction as a force generator and as a mechanosensor, and when two or more myosin heads work together in moving along an actin filament, the interplay between these mechanisms contributes to collective myosin behaviors. For example, the interplay between force-generating and force-sensing mechanisms coordinates the two heads of a myosin V molecule in its hand-over-hand processive stepping along an actin filament. In muscle, it contributes to the Fenn effect and smooth muscle latch. In both examples, a key force-sensing mechanism is the regulation of ADP release via interhead forces that are generated upon actin-myosin binding. Here we present a model describing the mechanism of allosteric regulation of ADP release from myosin heads as a change, DeltaDeltaG(-D), in the standard free energy for ADP release that results from the work, Deltamicro(mech), performed by that myosin head upon ADP release, or DeltaDeltaG(-D) = Deltamicro(mech). We show that this model is consistent with previous measurements for strain-dependent kinetics of ADP release in both myosin V and muscle myosin II. The model makes explicit the energetic cost of accelerating ADP release, showing that acceleration of ADP release during myosin V processivity requires approximately 4 kT of energy whereas the energetic cost for accelerating ADP release in a myosin II-based actin motility assay is only approximately 0.4 kT. The model also predicts that the acceleration of ADP release involves a dissipation of interhead forces. To test this prediction, we use an in vitro motility assay to show that the acceleration of ADP release from both smooth and skeletal muscle myosin II correlates with a decrease in interhead force. Our analyses provide clear energetic constraints for models of the allosteric regulation of ADP release and provide novel, testable insights

  12. 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

  13. The energetics of allosteric regulation of ADP release from myosin heads.

    PubMed

    Jackson, Del R; Baker, Josh E

    2009-06-28

    Myosin molecules are involved in a wide range of transport and contractile activities in cells. A single myosin head functions through its ATPase reaction as a force generator and as a mechanosensor, and when two or more myosin heads work together in moving along an actin filament, the interplay between these mechanisms contributes to collective myosin behaviors. For example, the interplay between force-generating and force-sensing mechanisms coordinates the two heads of a myosin V molecule in its hand-over-hand processive stepping along an actin filament. In muscle, it contributes to the Fenn effect and smooth muscle latch. In both examples, a key force-sensing mechanism is the regulation of ADP release via interhead forces that are generated upon actin-myosin binding. Here we present a model describing the mechanism of allosteric regulation of ADP release from myosin heads as a change, DeltaDeltaG(-D), in the standard free energy for ADP release that results from the work, Deltamicro(mech), performed by that myosin head upon ADP release, or DeltaDeltaG(-D) = Deltamicro(mech). We show that this model is consistent with previous measurements for strain-dependent kinetics of ADP release in both myosin V and muscle myosin II. The model makes explicit the energetic cost of accelerating ADP release, showing that acceleration of ADP release during myosin V processivity requires approximately 4 kT of energy whereas the energetic cost for accelerating ADP release in a myosin II-based actin motility assay is only approximately 0.4 kT. The model also predicts that the acceleration of ADP release involves a dissipation of interhead forces. To test this prediction, we use an in vitro motility assay to show that the acceleration of ADP release from both smooth and skeletal muscle myosin II correlates with a decrease in interhead force. Our analyses provide clear energetic constraints for models of the allosteric regulation of ADP release and provide novel, testable insights

  14. Excitotoxicity in the Lung: N-Methyl-D-Aspartate-Induced, Nitric Oxide-Dependent, Pulmonary Edema is Attenuated by Vasoactive Intestinal Peptide and by Inhibitors of Poly(ADP-Ribose) Polymerase

    NASA Astrophysics Data System (ADS)

    Said, Sami I.; Berisha, Hasan I.; Pakbaz, Hedayatollah

    1996-05-01

    Excitatory amino acid toxicity, resulting from overactivation of N-methyl-D-aspartate (NMDA) glutamate receptors, is a major mechanism of neuronal cell death in acute and chronic neurological diseases. We have investigated whether excitotoxicity may occur in peripheral organs, causing tissue injury, and report that NMDA receptor activation in perfused, ventilated rat lungs triggered acute injury, marked by increased pressures needed to ventilate and perfuse the lung, and by high-permeability edema. The injury was prevented by competitive NMDA receptor antagonists or by channel-blocker MK-801, and was reduced in the presence of Mg2+. As with NMDA toxicity to central neurons, the lung injury was nitric oxide (NO) dependent: it required L-arginine, was associated with increased production of NO, and was attenuated by either of two NO synthase inhibitors. The neuropeptide vasoactive intestinal peptide and inhibitors of poly(ADP-ribose) polymerase also prevented this injury, but without inhibiting NO synthesis, both acting by inhibiting a toxic action of NO that is critical to tissue injury. The findings indicate that: (i) NMDA receptors exist in the lung (and probably elsewhere outside the central nervous system), (ii) excessive activation of these receptors may provoke acute edematous lung injury as seen in the ``adult respiratory distress syndrome,'' and (iii) this injury can be modulated by blockade of one of three critical steps: NMDA receptor binding, inhibition of NO synthesis, or activation of poly(ADP-ribose) polymerase.

  15. Multiple forms of ADP-glucose pyrophosphorylase from tomato fruit

    NASA Technical Reports Server (NTRS)

    Chen, B. Y.; Janes, H. W.

    1997-01-01

    ADP-glucose pyrophosphorylase (AGP) was purified from tomato (Lycopersicon esculentum Mill.) fruit to apparent homogeneity. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis the enzyme migrated as two close bands with molecular weights of 50,000 and 51,000. Two-dimensional polyacrylamide gel electrophoresis analysis of the purified enzyme, however, revealed at least five major protein spots that could be distinguished by their slight differences in net charge and molecular weight. Whereas all of the spots were recognized by the antiserum raised against tomato fruit AGP holoenzyme, only three of them reacted strongly with antiserum raised against the potato tuber AGP large subunit, and the other two spots (with lower molecular weights) reacted specifically with antisera raised against spinach leaf AGP holoenzyme and the potato tuber AGP small subunit. The results suggest the existence of at least three isoforms of the AGP large subunit and two isoforms of the small subunit in tomato fruit in vivo. The native molecular mass of the enzyme determined by gel filtration was 220 +/- 10 kD, indicating a tetrameric structure for AGP from tomato fruit. The purified enzyme is very sensitive to 3-phosphoglycerate/inorganic phosphate regulation.

  16. Poly (ADP-ribose) in the pathogenesis of Parkinson's disease

    PubMed Central

    Lee, Yunjong; Kang, Ho Chul; Lee, Byoung Dae; Lee, Yun-Il; Kim, Young Pil; Shin, Joo-Ho

    2014-01-01

    The defining feature of Parkinson’s disease is a progressive and selective demise of dopaminergic neurons. A recent report on Parkinson’s disease animal model demonstrates that poly (ADP-ribose) (PAR) dependent cell death, also named parthanatos, is accountable for selective dopaminergic neuronal loss. Parthanatos is a programmed necrotic cell death, characterized by PARP1 activation, apoptosis inducing factor (AIF) nuclear translocation, and large scale DNA fragmentation. Besides cell death regulation via interaction with AIF, PAR molecule mediates diverse cellular processes including genomic stability, cell division, transcription, epigenetic regulation, and stress granule formation. In this review, we will discuss the roles of PARP1 activation and PAR molecules in the pathological processes of Parkinson’s disease. Potential interaction between PAR molecule and Parkinson’s disease protein interactome are briefly introduced. Finally, we suggest promising points of therapeutic intervention in the pathological PAR signaling cascade to halt progression in Parkinson’s disease. [BMB Reports 2014; 47(8): 424-432] PMID:24874851

  17. ADP-stimulated contraction: A predictor of thin-filament activation in cardiac disease

    PubMed Central

    Sequeira, Vasco; Najafi, Aref; Wijnker, Paul J. M.; Michels, Michelle; Kuster, Diederik W. D.; van der Velden, Jolanda

    2015-01-01

    Diastolic dysfunction is general to all idiopathic dilated (IDCM) and hypertrophic cardiomyopathy (HCM) patients. Relaxation deficits may result from increased actin–myosin formation during diastole due to altered tropomyosin position, which blocks myosin binding to actin in the absence of Ca2+. We investigated whether ADP-stimulated force development (without Ca2+) can be used to reveal changes in actin–myosin blockade in human cardiomyopathy cardiomyocytes. Cardiac samples from HCM patients, harboring thick-filament (MYH7mut, MYBPC3mut) and thin-filament (TNNT2mut, TNNI3mut) mutations, and IDCM were compared with sarcomere mutation-negative HCM (HCMsmn) and nonfailing donors. Myofilament ADP sensitivity was higher in IDCM and HCM compared with donors, whereas it was lower for MYBPC3. Increased ADP sensitivity in IDCM, HCMsmn, and MYH7mut was caused by low phosphorylation of myofilament proteins, as it was normalized to donors by protein kinase A (PKA) treatment. Troponin exchange experiments in a TNNT2mut sample corrected the abnormal actin–myosin blockade. In MYBPC3trunc samples, ADP sensitivity highly correlated with cardiac myosin-binding protein-C (cMyBP-C) protein level. Incubation of cardiomyocytes with cMyBP-C antibody against the actin-binding N-terminal region reduced ADP sensitivity, indicative of cMyBP-C’s role in actin–myosin regulation. In the presence of Ca2+, ADP increased myofilament force development and sarcomere stiffness. Enhanced sarcomere stiffness in sarcomere mutation-positive HCM samples was irrespective of the phosphorylation background. In conclusion, ADP-stimulated contraction can be used as a tool to study how protein phosphorylation and mutant proteins alter accessibility of myosin binding on actin. In the presence of Ca2+, pathologic [ADP] and low PKA-phosphorylation, high actin–myosin formation could contribute to the impaired myocardial relaxation observed in cardiomyopathies. PMID:26621701

  18. Analytical Design Package (ADP2): A computer aided engineering tool for aircraft transparency design

    NASA Technical Reports Server (NTRS)

    Wuerer, J. E.; Gran, M.; Held, T. W.

    1994-01-01

    The Analytical Design Package (ADP2) is being developed as a part of the Air Force Frameless Transparency Program (FTP). ADP2 is an integrated design tool consisting of existing analysis codes and Computer Aided Engineering (CAE) software. The objective of the ADP2 is to develop and confirm an integrated design methodology for frameless transparencies, related aircraft interfaces, and their corresponding tooling. The application of this methodology will generate high confidence for achieving a qualified part prior to mold fabrication. ADP2 is a customized integration of analysis codes, CAE software, and material databases. The primary CAE integration tool for the ADP2 is P3/PATRAN, a commercial-off-the-shelf (COTS) software tool. The open architecture of P3/PATRAN allows customized installations with different applications modules for specific site requirements. Integration of material databases allows the engineer to select a material, and those material properties are automatically called into the relevant analysis code. The ADP2 materials database will be composed of four independent schemas: CAE Design, Processing, Testing, and Logistics Support. The design of ADP2 places major emphasis on the seamless integration of CAE and analysis modules with a single intuitive graphical interface. This tool is being designed to serve and be used by an entire project team, i.e., analysts, designers, materials experts, and managers. The final version of the software will be delivered to the Air Force in Jan. 1994. The Analytical Design Package (ADP2) will then be ready for transfer to industry. The package will be capable of a wide range of design and manufacturing applications.

  19. Biosynthesis Pathway of ADP-l-glycero-β-d-manno-Heptose in Escherichia coli

    PubMed Central

    Kneidinger, Bernd; Marolda, Cristina; Graninger, Michael; Zamyatina, Alla; McArthur, Fiona; Kosma, Paul; Valvano, Miguel A.; Messner, Paul

    2002-01-01

    The steps involved in the biosynthesis of the ADP-l-glycero-β-d-manno-heptose (ADP-l-β-d-heptose) precursor of the inner core lipopolysaccharide (LPS) have not been completely elucidated. In this work, we have purified the enzymes involved in catalyzing the intermediate steps leading to the synthesis of ADP-d-β-d-heptose and have biochemically characterized the reaction products by high-performance anion-exchange chromatography. We have also constructed a deletion in a novel gene, gmhB (formerly yaeD), which results in the formation of an altered LPS core. This mutation confirms that the GmhB protein is required for the formation of ADP-d-β-d-heptose. Our results demonstrate that the synthesis of ADP-d-β-d-heptose in Escherichia coli requires three proteins, GmhA (sedoheptulose 7-phosphate isomerase), HldE (bifunctional d-β-d-heptose 7-phosphate kinase/d-β-d-heptose 1-phosphate adenylyltransferase), and GmhB (d,d-heptose 1,7-bisphosphate phosphatase), as well as ATP and the ketose phosphate precursor sedoheptulose 7-phosphate. A previously characterized epimerase, formerly named WaaD (RfaD) and now renamed HldD, completes the pathway to form the ADP-l-β-d-heptose precursor utilized in the assembly of inner core LPS. PMID:11751812

  20. Vault-poly-ADP-ribose polymerase in the Octopus vulgaris brain: a regulatory factor of actin polymerization dynamic.

    PubMed

    De Maio, Anna; Natale, Emiliana; Rotondo, Sergio; Di Cosmo, Anna; Faraone-Mennella, Maria Rosaria

    2013-09-01

    Our previous behavioural, biochemical and immunohistochemical analyses conducted in selected regions (supra/sub oesophageal masses) of the Octopus vulgaris brain detected a cytoplasmic poly-ADP-ribose polymerase (more than 90% of total enzyme activity). The protein was identified as the vault-free form of vault-poly-ADP-ribose polymerase. The present research extends and integrates the biochemical characterization of poly-ADP-ribosylation system, namely, reaction product, i.e., poly-ADP-ribose, and acceptor proteins, in the O. vulgaris brain. Immunochemical analyses evidenced that the sole poly-ADP-ribose acceptor was the octopus cytoskeleton 50-kDa actin. It was present in both free, endogenously poly-ADP-ribosylated form (70kDa) and in complex with V-poly-ADP-ribose polymerase and poly-ADP-ribose (260kDa). The components of this complex, alkali and high salt sensitive, were purified and characterized. The kind and the length of poly-ADP-ribose corresponded to linear chains of 30-35 ADP-ribose units, in accordance with the features of the polymer synthesized by the known vault-poly-ADP-ribose polymerase. In vitro experiments showed that V-poly-ADP-ribose polymerase activity of brain cytoplasmic fraction containing endogenous actin increased upon the addition of commercial actin and was highly reduced by ATP. Anti-actin immunoblot of the mixture in the presence and absence of ATP showed that the poly-ADP-ribosylation of octopus actin is a dynamic process balanced by the ATP-dependent polymerization of the cytoskeleton protein, a fundamental mechanism for synaptic plasticity.

  1. ADP-dependent 6-Phosphofructokinase from Pyrococcus horikoshii OT3: STRUCTURE DETERMINATION AND BIOCHEMICAL CHARACTERIZATION OF PH1645

    SciTech Connect

    Currie, Mark A.; Merino, Felipe; Skarina, Tatiana; Wong, Andrew H.Y.; Singer, Alexander; Brown, Greg; Savchenko, Alexei; Caniuguir, Andrés; Guixé, Victoria; Yakunin, Alexander F.; Jia, Zongchao

    2009-12-01

    Some hyperthermophilic archaea use a modified glycolytic pathway that employs an ADP-dependent glucokinase (ADP-GK) and an ADP-dependent phosphofructokinase (ADP-PFK) or, in the case of Methanococcus jannaschii, a bifunctional ADP-dependent glucophosphofructokinase (ADP-GK/PFK). The crystal structures of three ADP-GKs have been determined. However, there is no structural information available for ADP-PFKs or the ADP-GK/PFK. Here, we present the first crystal structure of an ADP-PFK from Pyrococcus horikoshii OT3 (PhPFK) in both apo- and AMP-bound forms determined to 2.0-{angstrom} and 1.9-{angstrom} resolution, respectively, along with biochemical characterization of the enzyme. The overall structure of PhPFK maintains a similar large and small {alpha}/{beta} domain structure seen in the ADP-GK structures. A large conformational change accompanies binding of phosphoryl donor, acceptor, or both, in all members of the ribokinase superfamily characterized thus far, which is believed to be critical to enzyme function. Surprisingly, no such conformational change was observed in the AMP-bound PhPFK structure compared with the apo structure. Through comprehensive site-directed mutagenesis of the substrate binding pocket we identified residues that were critical for both substrate recognition and the phosphotransfer reaction. The catalytic residues and many of the substrate binding residues are conserved between PhPFK and ADP-GKs; however, four key residues differ in the sugar-binding pocket, which we have shown determine the sugar-binding specificity. Using these results we were able to engineer a mutant PhPFK that mimics the ADP-GK/PFK and is able to phosphorylate both fructose 6-phosphate and glucose.

  2. The use of a PC-ADP in Shallow Rivers: Potential and Limitations for Turbulence Measurements

    NASA Astrophysics Data System (ADS)

    Cassista, A.; Roy, A. G.

    2005-12-01

    Turbulence plays an important role for processes such as mixing, sediment transport, scour and energy dissipation in rivers. Information in both space and time is essential to characterize flow turbulence in rivers, which would lead to a better understanding of sediment transport processes and bedform dynamics. Characterization of turbulent structures and their dynamics from conventionnal single point velocity measurements is time- and labor-intensive. Acoustic Doppler Profilers (ADP) have the ability to measure instantaneous velocity profiles in three dimensions and have shown a potential to measure turbulence characteristics. New Pulse-Coherent Acoustic Doppler Profiler (PC-ADP) technology provides a higher spatial resolution than conventional ADP and allows their use in shallow rivers. However, the measurement noise, the spreading of the instrument beams and the temporal and spatial averaging from this device may limit its application in rivers. The aim of this work is to evaluate the potential and the limitations of turbulence measurement using PC-ADP in shallow rivers. Field measurements were undertaken on the Rouge River (Quebec, Canada) during summer 2005 using a 1.5 MHz PC-ADP. The downward-looking PC-ADP was used to measure velocities at fixed river locations in ten 8-cm cells at a frequency of 1 Hz for long sampling duration. PC-ADP data were compared to a reference instrument, a 10 MHz Acoustic Doppler Velocimeter (ADV), that can measure flow velocities in three dimensions in a small sampling volume. Comparison with single point measurements were conducted to evaluate the effects of temporal and spatial averaging, Doppler noise and beam spreading in PC-ADP data. An algorithm for correcting ambiguity errors was developed to measure velocities up to 70 cms-1. Autocorrelation, power spectra and profiles of turbulence characteristics such as Reynolds stresses and TKE estimated from PC-ADP velocities were compared to those obtained from ADV data. Results show

  3. Overview on poly(ADP-ribose) immuno-biomedicine and future prospects.

    PubMed

    Kanai, Yoshiyuki

    2016-01-01

    Poly(ADP-ribose), identified in 1966 independently by three groups Strassbourg, Kyoto and Tokyo, is synthesized by poly(ADP-ribose) polymerases (PARP) from NAD(+) as a substrate in the presence of Mg(2+). The structure was unique in that it has ribose-ribose linkage. In the early-1970s, however, its function in vivo/in vitro was still controversial and the antibody against it was desired to help clear its significance. Thereupon, the author tried to produce antibody against poly(ADP-ribose) in rabbits and succeeded in it for the first time in the world. Eventually, this success has led to the following two groundbreaking papers in Nature: "Naturally-occurring antibody against poly(ADP-ribose) in patients with autoimmune disease SLE", and "Induction of anti-poly(ADP-ribose) antibody by immunization with synthetic double-stranded RNA, poly(A)·poly(U)".On the way to the publication of the first paper, a reviewer gave me a friendly comment that there is "heteroclitic" fashion as a mechanism of the production of natural antibody. This comment was really a God-send for me, and became a train of power for publication of another paper, as described above. Accordingly, I thought this, I would say, episode is worth describing herein. Because of its importance in biomedical phenomena, a certain number of articles related to "heteroclitic" have become to be introduced in this review, although they were not always directly related to immuno-biological works on poly(ADP-ribose). Also, I tried to speculate on the future prospects of poly(ADP-ribose), product of PARP, as an immuno-regulatory molecule, including either induced or naturally-occurring antibodies, in view of "heteroclitic".

  4. Overview on poly(ADP-ribose) immuno-biomedicine and future prospects.

    PubMed

    Kanai, Yoshiyuki

    2016-01-01

    Poly(ADP-ribose), identified in 1966 independently by three groups Strassbourg, Kyoto and Tokyo, is synthesized by poly(ADP-ribose) polymerases (PARP) from NAD(+) as a substrate in the presence of Mg(2+). The structure was unique in that it has ribose-ribose linkage. In the early-1970s, however, its function in vivo/in vitro was still controversial and the antibody against it was desired to help clear its significance. Thereupon, the author tried to produce antibody against poly(ADP-ribose) in rabbits and succeeded in it for the first time in the world. Eventually, this success has led to the following two groundbreaking papers in Nature: "Naturally-occurring antibody against poly(ADP-ribose) in patients with autoimmune disease SLE", and "Induction of anti-poly(ADP-ribose) antibody by immunization with synthetic double-stranded RNA, poly(A)·poly(U)".On the way to the publication of the first paper, a reviewer gave me a friendly comment that there is "heteroclitic" fashion as a mechanism of the production of natural antibody. This comment was really a God-send for me, and became a train of power for publication of another paper, as described above. Accordingly, I thought this, I would say, episode is worth describing herein. Because of its importance in biomedical phenomena, a certain number of articles related to "heteroclitic" have become to be introduced in this review, although they were not always directly related to immuno-biological works on poly(ADP-ribose). Also, I tried to speculate on the future prospects of poly(ADP-ribose), product of PARP, as an immuno-regulatory molecule, including either induced or naturally-occurring antibodies, in view of "heteroclitic". PMID:27477457

  5. Negative feedback of extracellular ADP on ATP release in goldfish hepatocytes: a theoretical study.

    PubMed

    Chara, Osvaldo; Pafundo, Diego E; Schwarzbaum, Pablo J

    2010-06-21

    A mathematical model was built to account for the kinetic of extracellular ATP (ATPe) and extracellular ADP (ADPe) concentrations from goldfish hepatocytes exposed to hypotonicity. The model was based on previous experimental results on the time course of ATPe accumulation, ectoATPase activity, and cell viability [Pafundo et al., 2008]. The kinetic of ATPe is controlled by a lytic ATP flux, a non-lytic ATP flux, and ecto-ATPase activity, whereas ADPe kinetic is governed by a lytic ADP flux and both ecto-ATPase and ecto-ADPase activities. Non-lytic ATPe efflux was included as a diffusion equation modulated by ATPe activation (positive feedback) and ADPe inhibition (negative feedback). The model yielded physically meaningful and stable steady-state solutions, was able to fit the experimental time evolution of ATPe and simulated the concomitant kinetic of ADPe. According to the model during the first minute of hypotonicity the concentration of ATPe is mainly governed by both lytic and non-lytic ATP efflux, with almost no contribution from ecto-ATPase activity. Later on, ecto-ATPase activity becomes important in defining the time dependent decay of ATPe levels. ADPe inhibition of the non-lytic ATP efflux was strong, whereas ATPe activation was minimal. Finally, the model was able to predict the consequences of partial inhibition of ecto-ATPase activity on the ATPe kinetic, thus emulating the exposure of goldfish cells to hypotonic medium in the presence of the ATP analog AMP-PCP. The model predicts this analog to both inhibit ectoATPase activity and increase non-lytic ATP release.

  6. Deficiency of terminal ADP-ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease

    PubMed Central

    Sharifi, Reza; Morra, Rosa; Denise Appel, C; Tallis, Michael; Chioza, Barry; Jankevicius, Gytis; Simpson, Michael A; Matic, Ivan; Ozkan, Ege; Golia, Barbara; Schellenberg, Matthew J; Weston, Ria; Williams, Jason G; Rossi, Marianna N; Galehdari, Hamid; Krahn, Juno; Wan, Alexander; Trembath, Richard C; Crosby, Andrew H; Ahel, Dragana; Hay, Ron; Ladurner, Andreas G; Timinszky, Gyula; Williams, R Scott; Ahel, Ivan

    2013-01-01

    Adenosine diphosphate (ADP)-ribosylation is a post-translational protein modification implicated in the regulation of a range of cellular processes. A family of proteins that catalyse ADP-ribosylation reactions are the poly(ADP-ribose) (PAR) polymerases (PARPs). PARPs covalently attach an ADP-ribose nucleotide to target proteins and some PARP family members can subsequently add additional ADP-ribose units to generate a PAR chain. The hydrolysis of PAR chains is catalysed by PAR glycohydrolase (PARG). PARG is unable to cleave the mono(ADP-ribose) unit directly linked to the protein and although the enzymatic activity that catalyses this reaction has been detected in mammalian cell extracts, the protein(s) responsible remain unknown. Here, we report the homozygous mutation of the c6orf130 gene in patients with severe neurodegeneration, and identify C6orf130 as a PARP-interacting protein that removes mono(ADP-ribosyl)ation on glutamate amino acid residues in PARP-modified proteins. X-ray structures and biochemical analysis of C6orf130 suggest a mechanism of catalytic reversal involving a transient C6orf130 lysyl-(ADP-ribose) intermediate. Furthermore, depletion of C6orf130 protein in cells leads to proliferation and DNA repair defects. Collectively, our data suggest that C6orf130 enzymatic activity has a role in the turnover and recycling of protein ADP-ribosylation, and we have implicated the importance of this protein in supporting normal cellular function in humans. PMID:23481255

  7. 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

  8. Beryllium(II) binding to ATP and ADP: potentiometric determination of the thermodynamic constants and implications for in vivo toxicity.

    PubMed

    Boukhalfa, Hakim; Lewis, James G; Crumbliss, Alvin L

    2004-04-01

    Highly toxic beryllium(II) is divalent metal ion with a high charge density, making it a potential target for binding to bio-molecules rich in O donor groups. In aqueous solution Be2+ binds to ATP and ADP to form 1:1 Be2+:ATP and Be2+:ADP complexes in relatively acidic media. At neutral pH the complex formed undergoes hydrolysis. Be2+ binding to ATP and ADP is much stronger than Ca2+ and Mg2+ binding. The high affinity of Be2+ toward ATP and ADP binding suggests a mechanism relevant to understanding the in vivo chemical toxicity of this metal.

  9. Synergistic role of ADP and Ca2+ in diastolic myocardial stiffness

    PubMed Central

    Sequeira, Vasco; Najafi, Aref; McConnell, Mark; Fowler, Ewan D; Bollen, Ilse A E; Wüst, Rob C I; dos Remedios, Cris; Helmes, Michiel; White, Ed; Stienen, Ger J M; Tardiff, Jil; Kuster, Diederik W D; van der Velden, Jolanda

    2015-01-01

    Abstract Heart failure (HF) with diastolic dysfunction has been attributed to increased myocardial stiffness that limits proper filling of the ventricle. Altered cross-bridge interaction may significantly contribute to high diastolic stiffness, but this has not been shown thus far. Cross-bridge interactions are dependent on cytosolic [Ca2+] and the regeneration of ATP from ADP. Depletion of myocardial energy reserve is a hallmark of HF leading to ADP accumulation and disturbed Ca2+ handling. Here, we investigated if ADP elevation in concert with increased diastolic [Ca2+] promotes diastolic cross-bridge formation and force generation and thereby increases diastolic stiffness. ADP dose-dependently increased force production in the absence of Ca2+ in membrane-permeabilized cardiomyocytes from human hearts. Moreover, physiological levels of ADP increased actomyosin force generation in the presence of Ca2+ both in human and rat membrane-permeabilized cardiomyocytes. Diastolic stress measured at physiological lattice spacing and 37°C in the presence of pathological levels of ADP and diastolic [Ca2+] revealed a 76 ± 1% contribution of cross-bridge interaction to total diastolic stress in rat membrane-permeabilized cardiomyocytes. Inhibition of creatine kinase (CK), which increases cytosolic ADP, in enzyme-isolated intact rat cardiomyocytes impaired diastolic re-lengthening associated with diastolic Ca2+ overload. In isolated Langendorff-perfused rat hearts, CK inhibition increased ventricular stiffness only in the presence of diastolic [Ca2+]. We propose that elevations of intracellular ADP in specific types of cardiac disease, including those where myocardial energy reserve is limited, contribute to diastolic dysfunction by recruiting cross-bridges, even at low Ca2+, and thereby increase myocardial stiffness. Key points Diastolic dysfunction in heart failure patients is evident from stiffening of the passive properties of the ventricular wall. Increased actomyosin

  10. Synergistic role of ADP and Ca(2+) in diastolic myocardial stiffness.

    PubMed

    Sequeira, Vasco; Najafi, Aref; McConnell, Mark; Fowler, Ewan D; Bollen, Ilse A E; Wüst, Rob C I; dos Remedios, Cris; Helmes, Michiel; White, Ed; Stienen, Ger J M; Tardiff, Jil; Kuster, Diederik W D; van der Velden, Jolanda

    2015-09-01

    Diastolic dysfunction in heart failure patients is evident from stiffening of the passive properties of the ventricular wall. Increased actomyosin interactions may significantly limit diastolic capacity, however, direct evidence is absent. From experiments at the cellular and whole organ level, in humans and rats, we show that actomyosin-related force development contributes significantly to high diastolic stiffness in environments where high ADP and increased diastolic [Ca(2+) ] are present, such as the failing myocardium. Our basal study provides a mechanical mechanism which may partly underlie diastolic dysfunction. Heart failure (HF) with diastolic dysfunction has been attributed to increased myocardial stiffness that limits proper filling of the ventricle. Altered cross-bridge interaction may significantly contribute to high diastolic stiffness, but this has not been shown thus far. Cross-bridge interactions are dependent on cytosolic [Ca(2+) ] and the regeneration of ATP from ADP. Depletion of myocardial energy reserve is a hallmark of HF leading to ADP accumulation and disturbed Ca(2+) handling. Here, we investigated if ADP elevation in concert with increased diastolic [Ca(2+) ] promotes diastolic cross-bridge formation and force generation and thereby increases diastolic stiffness. ADP dose-dependently increased force production in the absence of Ca(2+) in membrane-permeabilized cardiomyocytes from human hearts. Moreover, physiological levels of ADP increased actomyosin force generation in the presence of Ca(2+) both in human and rat membrane-permeabilized cardiomyocytes. Diastolic stress measured at physiological lattice spacing and 37°C in the presence of pathological levels of ADP and diastolic [Ca(2+) ] revealed a 76 ± 1% contribution of cross-bridge interaction to total diastolic stress in rat membrane-permeabilized cardiomyocytes. Inhibition of creatine kinase (CK), which increases cytosolic ADP, in enzyme-isolated intact rat cardiomyocytes impaired

  11. A fluorometric assay for measurement of mono-ADP-ribosyltransferase activity.

    PubMed

    Klebl, B M; Pette, D

    1996-08-01

    Using 1,N6-etheno NAD, a fluorescent analog of NAD, we extended an existing assay for NAD glycohydrolase to the measurement of mono-ADP-ribosyltransferase (mADP-RT) activity using agmatine as acceptor for ADP-ribose. The reaction products were analyzed by reversed-phase chromatography. In the presence of agmatine two newly formed fluorescent products were tentatively identified as ADP-ribosylagmatine anomers. Fluorescence intensity increased upon splitting the N-glycoside bondage of 1,N6-etheno NAD. Therefore, 1, N6-etheno AMP could be used for calibration. The nonradioactive assay yielded values nearly identical to those obtained with the [carbonyl-14C]NAD method. It proved to be highly reproducible, rapid, and suitable for an improved purification protocol yielding a 76,000-fold enriched mADP-RT preparation from rabbit skeletal muscle. The identity and high purity of the enzyme were confirmed immunochemically. The assay served to determine the pH optimum of the enzyme (pH 9.0) and its KM for 1,N6-etheno NAD (287 microM). PMID:8811894

  12. Study on A.C. electrical properties of pure and L-serine doped ADP crystals

    NASA Astrophysics Data System (ADS)

    Joshi, J. H.; Dixit, K. P.; Joshi, M. J.; Parikh, K. D.

    2016-05-01

    Ammonium Dihydrogen Phosphate (ADP) crystals have a wide range of applications in integrated and nonlinear optics. Amino acids having significant properties like molecular chirality, zwitter ionic nature, etc. attracted many researchers to dope them in various NLO crystals. In the present study, pure and different weight percentage L-serine doped ADP crystals were grown by slow solvent evaporation technique at room temperature. The A.C. electrical study was carried out for palletized samples at room temperature. The Nyquist plot showed two semi circles for pure ADP indicated the effect of grain and grain boundary, whereas the doped ADP samples exhibited the single semi circle suggesting the effect of grain. The values resistance and capacitance for grain and grain boundary were calculated. The effect of doping was clearly seen in the grain capacitance and resistance values. The dielectric constant and dielectric loss decreased with increase in frequency for all samples. The Jonscher power law was applied for A.C. conductivity for pure and doped ADP samples. The imaginary part of modulus and impedance versus frequency were drawn and the value of stretch exponent (β) was calculated for all the samples.

  13. 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

  14. Diphosphosinositol polyphosphates and energy metabolism: assay for ATP/ADP ratio.

    PubMed

    Nagel, Andreas; Barker, Christopher J; Berggren, Per-Olof; Illies, Christopher

    2010-01-01

    Several inositol compounds undergo rapid cycles of phosphorylation and dephosphorylation. These cycles are dependent on ATP and energy metabolism. Therefore, interfering with the cellular energy metabolism can change the concentration of rapidly turning over inositols. Many pharmacological inhibitors, apart from their intended action, also affect the energy metabolism of the cells and lower ATP. This can unspecifically influence rapidly turning over inositol phosphates. Thus, the ATP concentration should be checked when reduced inositol phosphates are observed after application of pharmacological inhibitors. A luminescence-based assay for the measurement of ATP and ADP is described. ATP is measured luminometrically using firefly luciferase. Detection of ADP is performed in a two-step enzymatic procedure: (1) The sample ATP is degraded to AMP and (2) ADP is phosphorylated to ATP, which can then be measured luminometrically. This method gives a better signal-to-noise ratio than other methods that do not degrade the sample ATP, but convert ADP directly to ATP and then measure the sum of ATP plus ADP.

  15. Genetic evidence of a high-affinity cyanuric acid transport system in Pseudomonas sp. ADP.

    PubMed

    Platero, Ana I; Santero, Eduardo; Govantes, Fernando

    2014-03-01

    The Pseudomonas sp. ADP plasmid pADP-1 encodes the activities involved in the hydrolytic degradation of the s-triazine herbicide atrazine. Here, we explore the presence of a specific transport system for the central intermediate of the atrazine utilization pathway, cyanuric acid, in Pseudomonas sp. ADP. Growth in fed-batch cultures containing limiting cyanuric acid concentrations is consistent with high-affinity transport of this substrate. Acquisition of the ability to grow at low cyanuric acid concentrations upon conjugal transfer of pADP1 to the nondegrading host Pseudomonas putida KT2442 suggests that all activities required for this phenotype are encoded in this plasmid. Co-expression of the pADP1-borne atzDEF and atzTUVW genes, encoding the cyanuric acid utilization pathway and the subunits of an ABC-type solute transport system, in P. putida KT2442 was sufficient to promote growth at cyanuric acid concentrations as low as 50 μM in batch culture. Taken together, our results strongly suggest that the atzTUVW gene products are involved in high-affinity transport of cyanuric acid.

  16. Niacin, poly(ADP-ribose) polymerase-1 and genomic stability.

    PubMed

    Hageman, G J; Stierum, R H

    2001-04-18

    Nicotinic acid (NA) and nicotinamide (NAM), commonly called niacin, are the dietary precursors for NAD(+) (nicotinamide adenine dinucleotide), which is required for DNA synthesis, as well as for the activity of the enzyme poly(ADP-ribose) polymerase-1 (PARP-1; EC 2.4.2.30) for which NAD(+) is the sole substrate. The enzyme PARP-1 is highly activated by DNA strand breaks during the cellular genotoxic stress response, is involved in base excision repair, plays a role in p53 expression and activation, and hence, is thought to be important for genomic stability. In this review, first the absorption, metabolism of niacin to NAD(+), as well as the assessment of niacin status are discussed. Since NAD(+) is important for PARP-1 activity, various aspects of PARP-1 in relation to DNA synthesis and repair, and regulation of gene expression are addressed. This is followed by a discussion on interactions between dietary methyl donor deficiency, niacin status, PARP-1 activity and genomic stability. In vitro studies show that PARP-1 function is impaired and genomic stability decreased when cells are either depleted from NAD(+) or incubated with high concentrations of NAM which is a PARP-1 inhibitor. In vitro as well as animal studies indicate that niacin deficiency increases genomic instability especially in combination with genotoxic and oxidative stress. Niacin deficiency may also increase the risk for certain tumors. Preliminary data suggest that niacin supplementation may protect against UV-induced tumors of the skin in mice, but data on similar preventive effects in humans are not available. NAM has been shown in vitro to have an antioxidant activity comparable to that of ascorbic acid. Data on niacin status and genomic stability in vivo in humans are limited and yield ambiguous results. Therefore, no firm conclusions with respect to optimal niacin intake are possible. As a consequence of oral niacin supplementation, however, NAM levels in the body may increase, which may

  17. Inhibition of the hepatitis C virus helicase-associated ATPase activity by the combination of ADP, NaF, MgCl2, and poly(rU). Two ADP binding sites on the enzyme-nucleic acid complex.

    PubMed

    Porter, D J

    1998-03-27

    Hepatitis C virus (HCV) helicase has an intrinsic ATPase activity and a nucleic acid (poly(rU))-stimulated ATPase activity. The poly(rU)-stimulated ATPase activity was inhibited by F- in a time-dependent manner during ATP hydrolysis. Inhibition was the result of trapping an enzyme-bound ADP-poly(rU) ternary complex generated during the catalytic cycle and was not the result of generating enzyme-free ADP that subsequently inhibited the enzyme. However, catalysis was not required for efficient inhibition by F-. The stimulated and the intrinsic ATPase activities were also inhibited by treatment of the enzyme with F-, ADP, and poly(rU). The inhibited enzyme slowly recovered (t1/2 = 23 min) ATPase activity after a 2000-fold dilution into assay buffer. The onset of inhibition by 500 microM ADP and 15 mM F- in the absence of nucleic acid was very slow (t1/2 > 40 min). However, the sequence of addition of poly(rU) to a diluted solution of ADP/NaF-treated enzyme had a profound effect on the extent of inhibition. If the ADP/NaF-treated enzyme was diluted into an assay that lacked poly(rU) and the assay was subsequently initiated with poly(rU), the treated enzyme was not inhibited. Alternatively, if the treated enzyme was diluted into an assay containing poly(rU), the enzyme was inhibited. ATP protected the enzyme from inhibition by ADP/NaF. The stoichiometry between ADP and enzyme monomer in the inhibited enzyme complex was 2, as determined from titration of the ATPase activity ([ADP]/[E] = 2.2) and from the number of radiolabeled ADP bound to the inhibited enzyme ([ADP]/[E] = 1.7) in the presence of excess NaF, MgCl2, and poly(rU). The Hill coefficient for titration of ATPase activity with F- (n = 2.8) or MgCl2 (n = 2.1) in the presence of excess ADP and poly(rU) suggested that multiple F- and Mg2+ were involved in forming the inhibited enzyme complex. The stoichiometry between (dU)18, a defined oligomeric nucleic acid substituting for poly(rU), and enzyme monomer in the

  18. Office of Inspector General report on audit of controls over the ADP support services contract

    SciTech Connect

    1997-08-15

    In March 1995, the Department awarded a cost-plus-award-fee contract to DynCorp valued at approximately $246 million over 5 years for ADP support services at Headquarters. The performance period for the contract was a 3-year base period with two 1-year options. The contract statement of work identified 24 information management functional areas that required technical support services, including Automated Office Systems Support and Local Area Network support. The purpose of the audit was to evaluate the cost-plus-award-fee contract for ADP support services at Headquarters. The objective was to determine whether the Department`s program offices at Headquarters were managing their ADP support services contract costs.

  19. ADP-2Ho as a Phasing Tool for Nucleotide-Containing Proteins

    SciTech Connect

    Ku,S.; Smith, G.; Howell, P.

    2007-01-01

    Trivalent holmium ions were shown to isomorphously replace magnesium ions to form an ADP-2Ho complex in the nucleotide-binding domain of Bacillus subtilis 5-methylthioribose (MTR) kinase. This nucleotide-holmium complex provided sufficient phasing power to allow SAD and SIRAS phasing of this previously unknown structure using the L{sub III} absorption edge of holmium. The structure of ADP-2Ho reveals that the two Ho ions are approximately 4 {angstrom} apart and are likely to share their ligands: the phosphoryl O atoms of ADP and a water molecule. The structure determination of MTR kinase using data collected using Cu K X-radiation was also attempted. Although the heavy-atom substructure determination was successful, interpretation of the map was more challenging. The isomorphous substitution of holmium for magnesium in the MTR kinase-nucleotide complex suggests that this could be a useful phasing tool for other metal-dependent nucleotide-containing proteins.

  20. 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

  1. 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

  2. Effect of Co2+ doping on solubility, crystal growth and properties of ADP crystals

    NASA Astrophysics Data System (ADS)

    Ganesh, V.; Shkir, Mohd.; AlFaify, S.; Yahia, I. S.

    2016-09-01

    Bulk size crystal growth of ADP with different concentrations doping of cobalt (Co2+) has been done by low cost slow evaporation technique at ambient conditions. The solubility measurement was carried out on pure and doped crystals and found that the solubility is decreasing with doping concentrations. The presence of Co2+ ion in crystalline matrix of ADP has been confirmed by structural, vibrational and elemental analyses. Scanning electron microscopic study reveals that the doping has strong effect on the quality of the crystals. The optical absorbance and transmission confirms the enhancement of quality of ADP crystals due to Co2+ doping and so the optical band gap. Further the dislocation, photoluminescence, dielectric and mechanical studies confirms that the properties of grown crystals with Co2+ doping has been enriched and propose it as a better candidate for optoelectronic applications.

  3. Thiol reagents are substrates for the ADP-ribosyltransferase activity of pertussis toxin.

    PubMed

    Lobban, M D; van Heyningen, S

    1988-06-20

    Thiols such as cysteine and dithiothreitol are substrates for the ADP-ribosyltransferase activity of pertussis toxin. When cysteine was incubated with NAD+ and toxin at pH 7.5, a product containing ADP-ribose and cysteine (presumably ADP-ribosylcysteine) was isolated by high-performance liquid chromatography, and characterized by its composition and release of AMP with phosphodiesterase. Cysteine has a Km of 105 mM at saturating NAD+ concentration. The ability of thiols to act as a substrate is one explanation for the very high concentrations (250 mM or greater) that have been observed to enhance the apparent NAD glycohydrolase activity of the toxin. PMID:3133246

  4. ADP-ribose-derived nuclear ATP synthesis by NUDIX5 is required for chromatin remodeling.

    PubMed

    Wright, Roni H G; Lioutas, Antonios; Le Dily, Francois; Soronellas, Daniel; Pohl, Andy; Bonet, Jaume; Nacht, A S; Samino, Sara; Font-Mateu, Jofre; Vicent, Guillermo P; Wierer, Michael; Trabado, Miriam A; Schelhorn, Constanze; Carolis, Carlo; Macias, Maria J; Yanes, Oscar; Oliva, Baldo; Beato, Miguel

    2016-06-01

    Key nuclear processes in eukaryotes, including DNA replication, repair, and gene regulation, require extensive chromatin remodeling catalyzed by energy-consuming enzymes. It remains unclear how the ATP demands of such processes are met in response to rapid stimuli. We analyzed this question in the context of the massive gene regulation changes induced by progestins in breast cancer cells and found that ATP is generated in the cell nucleus via the hydrolysis of poly(ADP-ribose) to ADP-ribose. In the presence of pyrophosphate, ADP-ribose is used by the pyrophosphatase NUDIX5 to generate nuclear ATP. The nuclear source of ATP is essential for hormone-induced chromatin remodeling, transcriptional regulation, and cell proliferation. PMID:27257257

  5. Binding of ADP to beef-heart mitochondrial ATPase (F1).

    PubMed

    Wielders, J P; Slater, E C; Muller, J L

    1980-02-01

    1. ADP binding to beef-heart mitochondrial ATPase (F1), in the absence of Mg2+, has been determined by separating the free ligand by ultrafiltration and determining it in the filtrate by a specially modified isotachophoretic procedure. 2. Since during the binding experiments the 'tightly' bound ADP (but not the ATP) dissociates, it is necessary to take this into account in calculating the binding parameters. 3. The binding data show that only one tight binding site (Kd about 0.5 microM) for ADP is present. 4. It is not possible to calculate from the binding data alone the number of or the dissociation constants for the weak binding sites. It can be concluded, however, that the latter is not less than about 50 microM.

  6. 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.

  7. Enlightening energy parasitism by analysis of an ATP/ADP transporter from chlamydiae.

    PubMed

    Trentmann, Oliver; Horn, Matthias; van Scheltinga, Anke C Terwisscha; Neuhaus, H Ekkehard; Haferkamp, Ilka

    2007-09-01

    Energy parasitism by ATP/ADP transport proteins is an essential, common feature of intracellular bacteria such as chlamydiae and rickettsiae, which are major pathogens of humans. Although several ATP/ADP transport proteins have so far been characterized, some fundamental questions regarding their function remained unaddressed. In this study, we focused on the detailed biochemical analysis of a representative ATP/ADP transporter (PamNTT1), from the amoeba symbiont Protochlamydia amoebophila (UWE25) to further clarify the principle of energy exploitation. We succeeded in the purification of the first bacterial nucleotide transporter (NTT) and its functional reconstitution into artificial lipid vesicles. Reconstituted PamNTT1 revealed high import velocities for ATP and an unexpected and previously unobserved stimulating effect of the luminal ADP on nucleotide import affinities. Latter preference of the nucleotide hetero-exchange is independent of the membrane potential, and therefore, PamNTT1 not only structurally but also functionally differs from the well-characterized mitochondrial ADP/ATP carriers. Reconstituted PamNTT1 exhibits a bidirectional orientation in lipid vesicles, but interestingly, only carriers inserted with the N-terminus directed to the proteoliposomal interior are functional. The data presented here comprehensively explain the functional basis of how the intracellular P. amoebophila manages to exploit the energy pool of its host cell effectively by using the nucleotide transporter PamNTT1. This membrane protein mediates a preferred import of ATP, which is additionally stimulated by a high internal (bacterial) ADP/ATP ratio, and the orientation-dependent functionality of the transporter ensures that it is not working in a mode that is detrimental to P. amoebophila. Heterologous expression and purification of high amounts of PamNTT1 provides the basis for its crystallization and detailed structure/function analyses. Furthermore, functional

  8. 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

  9. 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

  10. Host Cell Poly(ADP-Ribose) Glycohydrolase Is Crucial for Trypanosoma cruzi Infection Cycle

    PubMed Central

    Vilchez Larrea, Salomé C.; Schlesinger, Mariana; Kevorkian, María L.; Flawiá, Mirtha M.; Alonso, Guillermo D.; Fernández Villamil, Silvia H.

    2013-01-01

    Trypanosoma cruzi, etiological agent of Chagas’ disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose) glycohydrolase in a trypanosomatid (TcPARG). In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose) glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl) pyrrolidinediol) or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate) to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose) glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose) glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas’ disease. PMID:23776710

  11. Host cell poly(ADP-ribose) glycohydrolase is crucial for Trypanosoma cruzi infection cycle.

    PubMed

    Vilchez Larrea, Salomé C; Schlesinger, Mariana; Kevorkian, María L; Flawiá, Mirtha M; Alonso, Guillermo D; Fernández Villamil, Silvia H

    2013-01-01

    Trypanosoma cruzi, etiological agent of Chagas' disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose) glycohydrolase in a trypanosomatid (TcPARG). In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose) glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl) pyrrolidinediol) or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate) to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose) glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose) glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas' disease. PMID:23776710

  12. Nuclear poly(ADP-ribose) polymerase-1 rapidly triggers mitochondrial dysfunction.

    PubMed

    Cipriani, Giulia; Rapizzi, Elena; Vannacci, Alfredo; Rizzuto, Rosario; Moroni, Flavio; Chiarugi, Alberto

    2005-04-29

    To obtain further information on time course and mechanisms of cell death after poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation, we used HeLa cells exposed for 1 h to the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. This treatment activated PARP-1 and caused a rapid drop of cellular NAD(H) and ATP contents, culminating 8-12 h later in cell death. PARP-1 antagonists fully prevented nucleotide depletion and death. Interestingly, in the early 60 min after challenge with N-methyl-N'-nitro-N-nitrosoguanidine, mitochondrial membrane potential and superoxide production significantly increased, whereas cellular ADP contents decreased. Again, these events were prevented by PARP-1 inhibitors, suggesting that PARP-1 hyperactivity leads to mitochondrial state 4 respiration. Mitochondrial membrane potential collapsed at later time points (3 h), when mitochondria released apoptosis-inducing factor and cytochrome c. Using immunocytochemistry and targeted luciferase transfection, we found that, despite an exclusive localization of PARP-1 and poly(ADP-ribose) in the nucleus, ATP levels first decreased in mitochondria and then in the cytoplasm of cells undergoing PARP-1 activation. PARP-1 inhibitors rescued ATP (but not NAD(H) levels) in cells undergoing hyper-poly(ADP-ribosyl)ation. Glycolysis played a central role in the energy recovery, whereas mitochondria consumed ATP in the early recovery phase and produced ATP in the late phase after PARP-1 inhibition, further indicating that nuclear poly(ADP-ribosyl)ation rapidly modulates mitochondrial functioning. Together, our data provide evidence for rapid nucleus-mitochondria cross-talk during hyper-poly(ADP-ribosyl)ation-dependent cell death.

  13. Design, Synthesis, and Chemical and Biological Properties of Cyclic ADP-4-Thioribose as a Stable Equivalent of Cyclic ADP-Ribose

    PubMed Central

    Tsuzuki, Takayoshi; Takano, Satoshi; Sakaguchi, Natsumi; Kudoh, Takashi; Murayama, Takashi; Sakurai, Takashi; Hashii, Minako; Higashida, Haruhiro; Weber, Karin; Guse, Andreas H.; Kameda, Tomoshi; Hirokawa, Takatsugu; Kumaki, Yasuhiro; Arisawa, Mitsuhiro; Potter, Barry V. L.; Shuto, Satoshi

    2016-01-01

    Here we describe the successful synthesis of cyclic ADP-4-thioribose (cADPtR, 3), designed as a stable mimic of cyclic ADP-ribose (cADPR, 1), a Ca2+-mobilizing second messenger, in which the key N1-β-thioribosyladenosine structure was stereoselectively constructed by condensation between the imidazole nucleoside derivative 8 and the 4-thioribosylamine 7 via equilibrium in 7 between the α-anomer (7α) and the β-anomer (7β) during the reaction course. cADPtR is, unlike cADPR, chemically and biologically stable, while it effectively mobilizes intracellular Ca2+ like cADPR in various biological systems, such as sea urchin homogenate, NG108-15 neuronal cells, and Jurkat T-lymphocytes. Thus, cADPtR is a stable equivalent of cADPR, which can be useful as a biological tool for investigating cADPR-mediated Ca2+-mobilizing pathways. PMID:27200225

  14. 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.

  15. Structure and Mechanism of an ADP-Glucose Phosphorylase from Arabidopsis thaliana†,‡

    PubMed Central

    McCoy, Jason G.; Arabshahi, Abolfazl; Bitto, Eduard; Bingman, Craig A.; Ruzicka, Frank J.; Frey, Perry A.; Phillips, George N.

    2008-01-01

    The X-ray crystal structure of the At5g18200.1 protein has been solved to a nominal resolution of 2.30 Å. The structure has a histidine triad (HIT)-like fold containing two distinct HIT-like motifs. The sequence of At5g18200.1 indicates a distant family relationship to the Escherichia coli galactose-1-P uridylyltransferase (GalT): the determined structure of the At5g18200.1 protein confirms this relationship. The At5g18200.1 protein does not demonstrate GalT activity but instead catalyzes adenylyltransfer in the reaction of ADP-glucose with various phosphates. The best acceptor among those evaluated is phosphate itself, thus the At5g18200.1 enzyme appears to be an ADP-glucose phosphorylase. The enzyme catalyzes the exchange of 14C between ADP-[14C]glucose and glucose-1-P in the absence of phosphate. The steady state kinetics of exchange follows the ping pong bi bi kinetic mechanism, with kcat = 4.1 s−1 and Km-values of 1.4 µM and 83 µM for ADP-[14C]glucose and glucose-1-P, respectively, at pH 8.5 and 25 °C. The overall reaction of ADP-glucose with phosphate to produce ADP and glucose-1-P follows ping pong bi bi steady state kinetics, with kcat = 2.7 s−1 and Km-values of 6.9 µM and 90 µM for ADP-glucose and phosphate, respectively, at pH 8.5 and 25 °C. The kinetics are consistent with a double displacement mechanism that involves a covalent adenylyl-enzyme intermediate. The X-ray crystal structure of this intermediate was solved to 1.83 Å resolution, and shows the AMP-group bonded to His186. The value of Keq in the direction of ADP and glucose-1-P formation is 5.0 at pH 7.0 and 25 °C in the absence of a divalent metal ion, and it is 40 in the presence of 1 mM MgCl2. PMID:16519510

  16. 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.

  17. 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

  18. 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

  19. Do heterotrimeric G proteins redistribute upon G protein-coupled receptor stimulation in platelets?

    PubMed

    Kahner, Bryan N; Quinton, Todd M; Langan, Sarah; Kunapuli, Satya P

    2006-09-01

    Previous studies have proposed that stimulation of G protein-coupled receptors can cause a redistribution of G proteins to other receptors. The redistribution would cause a greater functional sensitivity of unsensitized 'secondary' receptors toward their agonists. Using platelets as a model system, we utilized a proximal signaling event, intracellular calcium mobilization, to determine if agonist stimulation of particular Gq-coupled receptors would result in increased sensitivity for stimulation of other Gq-coupled receptors. Platelets express three Gq-coupled receptors for thrombin, thromboxane A2, and ADP with different potencies. Varying concentrations of a primary agonist (PAR-1 agonist SFLLRN, or the TXA2 agonist U46619) was followed by a constant submaximal concentration of a secondary agonist (U46619, or the P2Y1 agonist ADP). We observed that initial stimulation by SFLLRN was followed by a decrease in the extent of secondary U46619 or ADP-mediated calcium mobilization in comparison to control responses (i.e. without primary stimulation). To extend these studies we examined calcium mobilization in platelets from mice that were either wild-type or homozygous null for the PAR-4 or P2Y1 receptors, hypothesizing that the loss of PAR-4 or P2Y1 receptors would cause redistribution of its Galphaq proteins to other receptors, and elicit a greater response when stimulated with other agonists than in platelets from a wild-type mouse. However, our results showed almost identical levels of peak calcium between wild-type or PAR-4 null mice when stimulated with either ADP or U46619. Similar results were obtained for the P2Y1 null mice stimulated with AYPGKF or U46619. We conclude that stimulation of one Gq coupled receptor does not result in redistribution of Gq to other Gq-coupled receptors. PMID:16973501

  20. Increase in the ADP/ATP exchange in rat liver mitochondria irradiated in vitro by helium-neon laser

    SciTech Connect

    Passarella, S.; Ostuni, A.; Atlante, A.; Quagliariello, E.

    1988-10-31

    To gain some insight into the mechanism of cell photostimulation by laser light, measurements were made of the rate of ADP/ATP exchange in mitochondria irradiated with the low power continuous wave Helium Neon laser (energy dose 5 Joules/cm2). To do this a method has been developed to continuously monitor ATP efflux from phosphorylating mitochondria caused by externally added ADP, by photometrically following the NADP+ reduction which occurs in the presence of glucose, hexokinase, glucose-6-phosphate dehydrogenase and effluxed ATP. The NADP+ reduction rate shows hyperbolic dependence on ADP concentration (Km and Vmax values 8.5 +/- 0.87 microM and 20.7 +/- 0.49 nmoles NADP+ reduced/min x mg mitochondrial protein, respectively), and proves to measure the activity of the ADP/ATP translocator as shown by inhibition experiments using atracyloside, powerful inhibitor of this carrier. Irradiation was found to enhance the rate of ADP/ATP antiport, with externally added ADP ranging between 5 and 100 microM. As a result of experiments carried out with mitochondria loaded with either ATP or ADP, the increase in the activity of the ADP/ATP translocator is here proposed to depend on the increase in the electrochemical proton gradient which occurs owing to irradiation of mitochondria.

  1. The role of ADP in the modulation of the calcium-efflux pathway in rat brain mitochondria.

    PubMed Central

    Vitorica, J; Satrústegui, J

    1985-01-01

    The role of ADP in the regulation of Ca2+ efflux in rat brain mitochondria was investigated. ADP was shown to inhibit Ruthenium-Red-insensitive H+- and Na+-dependent Ca2+-efflux rates if Pi was present, but had no effect in the absence of Pi. The primary effect of ADP is an inhibition of Pi efflux, and therefore it allows the formation of a matrix Ca2+-Pi complex at concentrations above 0.2 mM-Pi and 25 nmol of Ca2+/mg of protein, which maintains a constant free matrix Ca2+ concentration. ADP inhibition of Pi and Ca2+ efflux is nucleotide-specific, since in the presence of oligomycin and an inhibitor of adenylate kinase ATP does not substitute for ADP, is dependent on the amount of ADP present, and requires ADP concentrations in excess of the concentrations of translocase binding sites. Brain mitochondria incubated with 0.2 mM-Pi and ADP showed Ca2+-efflux rates dependent on Ca2+ loads at Ca2+ concentrations below those required for the formation of a Pi-Ca2+ complex, and behaved as perfect cytosolic buffers exclusively at high Ca2+ loads. The possible role of brain mitochondrial Ca2+ in the regulation of the tricarboxylic acid-cycle enzymes and in buffering cytosolic Ca2+ is discussed. PMID:3977831

  2. Mitochondrial ADP/ATP exchange inhibition: a novel off-target mechanism underlying ibipinabant-induced myotoxicity.

    PubMed

    Schirris, Tom J J; Ritschel, Tina; Herma Renkema, G; Willems, Peter H G M; Smeitink, Jan A M; Russel, Frans G M

    2015-01-01

    Cannabinoid receptor 1 (CB1R) antagonists appear to be promising drugs for the treatment of obesity, however, serious side effects have hampered their clinical application. Rimonabant, the first in class CB1R antagonist, was withdrawn from the market because of psychiatric side effects. This has led to the search for more peripherally restricted CB1R antagonists, one of which is ibipinabant. However, this 3,4-diarylpyrazoline derivative showed muscle toxicity in a pre-clinical dog study with mitochondrial dysfunction. Here, we studied the molecular mechanism by which ibipinabant induces mitochondrial toxicity. We observed a strong cytotoxic potency of ibipinabant in C2C12 myoblasts. Functional characterization of mitochondria revealed increased cellular reactive oxygen species generation and a decreased ATP production capacity, without effects on the catalytic activities of mitochondrial enzyme complexes I-V or the complex specific-driven oxygen consumption. Using in silico off-target prediction modelling, combined with in vitro validation in isolated mitochondria and mitoplasts, we identified adenine nucleotide translocase (ANT)-dependent mitochondrial ADP/ATP exchange as a novel molecular mechanism underlying ibipinabant-induced toxicity. Minor structural modification of ibipinabant could abolish ANT inhibition leading to a decreased cytotoxic potency, as observed with the ibipinabant derivative CB23. Our results will be instrumental in the development of new types of safer CB1R antagonists.

  3. TRPM2: a calcium influx pathway regulated by oxidative stress and the novel second messenger ADP-ribose.

    PubMed

    Kühn, Frank J P; Heiner, Inka; Lückhoff, Andreas

    2005-10-01

    A unique functional property within the transient receptor potential (TRP) family of cation channels is the gating of TRP (melastatin) 2 (TRPM2) channels by ADP-ribose (ADPR). ADPR binds to the intracellular C-terminal tail of TRPM2, a domain that shows homology to enzymes with pyrophosphatase activity. Cytosolic Ca(2+) enhances TRPM2 gating by ADPR; ADPR and Ca(2+) in concert may be an important messenger system mediating Ca(2+) influx. Other stimuli of TRPM2 include NAD and H(2)O(2) and cyclic ADPR, which may act synergistically with ADPR. H(2)O(2), an experimental paradigm of oxidative stress, may also induce the formation of ADPR in the nucleus or mitochondria. In this review, we summarize the gating properties of TRPM2 and the proposed pathways of channel activation in vivo. TRPM2 is likely to be a key player in several signalling pathways, mediating cell death in response to oxidative stress or in reperfusion injury. Moreover, it plays a decisive role in experimentally induced diabetes mellitus and in the activation of leukocytes.

  4. Mitochondrial ADP/ATP exchange inhibition: a novel off-target mechanism underlying ibipinabant-induced myotoxicity

    PubMed Central

    Schirris, Tom J. J.; Ritschel, Tina; Herma Renkema, G.; Willems, Peter H. G. M.; Smeitink, Jan A. M.; Russel, Frans G. M.

    2015-01-01

    Cannabinoid receptor 1 (CB1R) antagonists appear to be promising drugs for the treatment of obesity, however, serious side effects have hampered their clinical application. Rimonabant, the first in class CB1R antagonist, was withdrawn from the market because of psychiatric side effects. This has led to the search for more peripherally restricted CB1R antagonists, one of which is ibipinabant. However, this 3,4-diarylpyrazoline derivative showed muscle toxicity in a pre-clinical dog study with mitochondrial dysfunction. Here, we studied the molecular mechanism by which ibipinabant induces mitochondrial toxicity. We observed a strong cytotoxic potency of ibipinabant in C2C12 myoblasts. Functional characterization of mitochondria revealed increased cellular reactive oxygen species generation and a decreased ATP production capacity, without effects on the catalytic activities of mitochondrial enzyme complexes I–V or the complex specific-driven oxygen consumption. Using in silico off-target prediction modelling, combined with in vitro validation in isolated mitochondria and mitoplasts, we identified adenine nucleotide translocase (ANT)-dependent mitochondrial ADP/ATP exchange as a novel molecular mechanism underlying ibipinabant-induced toxicity. Minor structural modification of ibipinabant could abolish ANT inhibition leading to a decreased cytotoxic potency, as observed with the ibipinabant derivative CB23. Our results will be instrumental in the development of new types of safer CB1R antagonists. PMID:26416158

  5. Discovery and mapping of ten novel G protein-coupled receptor genes.

    PubMed

    Lee, D K; Nguyen, T; Lynch, K R; Cheng, R; Vanti, W B; Arkhitko, O; Lewis, T; Evans, J F; George, S R; O'Dowd, B F

    2001-09-01

    We report the identification, cloning and tissue distributions of ten novel human genes encoding G protein-coupled receptors (GPCRs) GPR78, GPR80, GPR81, GPR82, GPR93, GPR94, GPR95, GPR101, GPR102, GPR103 and a pseudogene, psi GPR79. Each novel orphan GPCR (oGPCR) gene was discovered using customized searches of the GenBank high-throughput genomic sequences database with previously known GPCR-encoding sequences. The expressed genes can now be used in assays to determine endogenous and pharmacological ligands. GPR78 shared highest identity with the oGPCR gene GPR26 (56% identity in the transmembrane (TM) regions). psi GPR79 shared highest sequence identity with the P2Y(2) gene and contained a frame-shift truncating the encoded receptor in TM5, demonstrating a pseudogene. GPR80 shared highest identity with the P2Y(1) gene (45% in the TM regions), while GPR81, GPR82 and GPR93 shared TM identities with the oGPCR genes HM74 (70%), GPR17 (30%) and P2Y(5) (40%), respectively. Two other novel GPCR genes, GPR94 and GPR95, encoded a subfamily with the genes encoding the UDP-glucose and P2Y(12) receptors (sharing >50% identities in the TM regions). GPR101 demonstrated only distant identities with other GPCR genes and GPR102 shared identities with GPR57, GPR58 and PNR (35-42% in the TM regions). GPR103 shared identities with the neuropeptide FF 2, neuropeptide Y2 and galanin GalR1 receptors (34-38% in the TM regions). Northern analyses revealed GPR78 mRNA expression in the pituitary and placenta and GPR81 expression in the pituitary. A search of the GenBank databases with the GPR82 sequence retrieved an identical sequence in an expressed sequence tag (EST) partially encoding GPR82 from human colonic tissue. The GPR93 sequence retrieved an identical, human EST sequence from human primary tonsil B-cells and an EST partially encoding mouse GPR93 from small intestinal tissue. GPR94 was expressed in the frontal cortex, caudate putamen and thalamus of brain while GPR95 was expressed

  6. 41 CFR 109-45.309-54 - Automatic Data Processing Equipment (ADPE).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 41 Public Contracts and Property Management 3 2010-07-01 2010-07-01 false Automatic Data Processing Equipment (ADPE). 109-45.309-54 Section 109-45.309-54 Public Contracts and Property Management Federal Property Management Regulations System (Continued) DEPARTMENT OF ENERGY PROPERTY...

  7. 41 CFR 109-45.309-54 - Automatic Data Processing Equipment (ADPE).

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 41 Public Contracts and Property Management 3 2011-01-01 2011-01-01 false Automatic Data Processing Equipment (ADPE). 109-45.309-54 Section 109-45.309-54 Public Contracts and Property Management Federal Property Management Regulations System (Continued) DEPARTMENT OF ENERGY PROPERTY...

  8. 41 CFR 109-45.309-54 - Automatic Data Processing Equipment (ADPE).

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 41 Public Contracts and Property Management 3 2012-01-01 2012-01-01 false Automatic Data Processing Equipment (ADPE). 109-45.309-54 Section 109-45.309-54 Public Contracts and Property Management Federal Property Management Regulations System (Continued) DEPARTMENT OF ENERGY PROPERTY...

  9. 41 CFR 109-45.309-54 - Automatic Data Processing Equipment (ADPE).

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 41 Public Contracts and Property Management 3 2014-01-01 2014-01-01 false Automatic Data Processing Equipment (ADPE). 109-45.309-54 Section 109-45.309-54 Public Contracts and Property Management Federal Property Management Regulations System (Continued) DEPARTMENT OF ENERGY PROPERTY...

  10. 41 CFR 109-45.309-54 - Automatic Data Processing Equipment (ADPE).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 41 Public Contracts and Property Management 3 2013-07-01 2013-07-01 false Automatic Data Processing Equipment (ADPE). 109-45.309-54 Section 109-45.309-54 Public Contracts and Property Management Federal Property Management Regulations System (Continued) DEPARTMENT OF ENERGY PROPERTY...

  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. 32 CFR Appendix J to Part 154 - ADP Position Categories and Criteria for Designating Positions

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Designating Positions J Appendix J to Part 154 National Defense Department of Defense OFFICE OF THE SECRETARY OF DEFENSE SECURITY DEPARTMENT OF DEFENSE PERSONNEL SECURITY PROGRAM REGULATION Pt. 154, App. J Appendix J to Part 154—ADP Position Categories and Criteria for Designating Positions OMB Circular...

  13. The NASA/USRA ADP at the University of Central Florida

    NASA Technical Reports Server (NTRS)

    Anderson, L. A.; Armitage, P. K.

    1992-01-01

    An approach to learning engineering design is discussed with particular attention given to the impact of the NASA/Universities Space Research Association (USRA) Advanced Design Program (ADP) on that process. Attention is also given to a teaching method stressing science discipline and creativity and various selected space related designs.

  14. ATP/ADP Ratio, the Missed Connection between Mitochondria and the Warburg Effect

    PubMed Central

    Maldonado, Eduardo N.; Lemasters, John J.

    2014-01-01

    Non-proliferating cells generate the bulk of cellular ATP by fully oxidizing respiratory substrates in mitochondria. Respiratory substrates cross the mitochondrial outer membrane through only one channel, the voltage dependent anion channel (VDAC). Once in the matrix, respiratory substrates are oxidized in the tricarboxylic acid cycle to generate mostly NADH that is further oxidized in the respiratory chain to generate a proton motive force comprised mainly of membrane potential (ΔΨ) to synthesize ATP. Mitochondrial ΔΨ then drives release of ATP−4 from the matrix in exchange for ADP−3 in the cytosol via the adenine nucleotide translocator (ANT) located in the mitochondrial inner membrane. Thus, mitochondrial function in non-proliferating cells drives a high cytosolic ATP/ADP ratio, essential to inhibit glycolysis. By contrast, the bioenergetics of the Warburg phenotype of proliferating cells is characterized by enhanced aerobic glycolysis and suppression of mitochondrial metabolism. Suppressed mitochondrial function leads to lower production of mitochondrial ATP and hence lower cytosolic ATP/ADP ratios that favor enhanced glycolysis. Thus, cytosolic ATP/ADP ratio is a key feature that determines if cell metabolism is predominantly oxidative or glycolytic. Here, we describe two novel mechanisms to explain the suppression of mitochondrial metabolism in cancer cells: the relative closure of VDAC by free tubulin and inactivation of ANT. Both mechanisms contribute to low ATP/ADP ratios that activate glycolysis. PMID:25229666

  15. Impaired ADP channeling to mitochondria and elevated reactive oxygen species in hypertensive hearts.

    PubMed

    Power, Amelia S C; Pham, Toan; Loiselle, Denis S; Crossman, David H; Ward, Marie-Louise; Hickey, Anthony J

    2016-06-01

    Systemic hypertension initially promotes a compensatory cardiac hypertrophy, yet it progresses to heart failure (HF), and energetic deficits appear to be central to this failure. However, the transfer of energy between the mitochondria and the myofibrils is not often considered as part of the energetic equation. We compared hearts from old spontaneously hypertensive rats (SHRs) and normotensive Wistar controls. SHR hearts showed a 35% depression in mitochondrial function, yet produced at least double the amount of reactive oxygen species (ROS) in all respiration states in left ventricular (LV) homogenates. To test the connectivity between mitochondria and myofibrils, respiration was further tested in situ with LV permeabilized fibers by addition of multiple substrates and ATP, which requires hydrolysis to mediate oxidative phosphorylation. By trapping ADP using a pyruvate kinase enzyme system, we tested ADP channeling towards mitochondria, and this suppressed respiration and elevated ROS production more in the SHR fibers. The ADP-trapped state was also less relieved on creatine addition, likely reflecting the 30% depression in total CK activity in the SHR heart fibers. Confocal imaging identified a 34% longer distance between the centers of myofibril to mitochondria in the SHR hearts, which increases transverse metabolite diffusion distances (e.g., for ATP, ADP, and creatine phosphate). We propose that impaired connectivity between mitochondria and myofibrils may contribute to elevated ROS production. Impaired energy exchange could be the result of ultrastructural changes that occur with hypertrophy in this model of hypertension. PMID:27084386

  16. Selective Small Molecule Inhibition of Poly(ADP-Ribose) Glycohydrolase (PARG)

    PubMed Central

    Finch, Kristin E.; Knezevic, Claire E.; Nottbohm, Amanda C.; Partlow, Kathryn C.; Hergenrother, Paul J.

    2012-01-01

    The poly(ADP-ribose) (PAR) post-translational modification is essential for diverse cellular functions, including regulation of transcription, response to DNA damage, and mitosis. Cellular PAR is predominantly synthesized by the enzyme poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 is a critical node in the DNA damage response pathway, and multiple potent PARP-1 inhibitors have been described, some of which show considerable promise in the clinic for the treatment of certain cancers. Cellular PAR is efficiently degraded by poly(ADP-ribose) glycohydrolase (PARG), an enzyme for which no potent, readily accessible, and specific inhibitors exist. Herein we report the discovery of small molecules that effectively inhibit PARG in vitro and in cellular lysates. These potent PARG inhibitors can be produced in two chemical steps from commercial starting materials and have complete specificity for PARG over the other known PAR glycohydrolase (ADP-ribosylhydrolase 3, ARH3) and over PARP-1, and thus will be useful tools to study the biochemistry of PAR signaling. PMID:22220926

  17. Inhibition of poly(ADP-ribose) polymerase-1 attenuates the toxicity of carbon tetrachloride

    PubMed Central

    Banasik, Marek; Stedeford, Todd; Strosznajder, Robert P; Takehashi, Masanori; Tanaka, Seigo; Ueda, Kunihiro

    2011-01-01

    Carbon tetrachloride (CCl4) is routinely used as a model compound for eliciting centrilobular hepatotoxicity. It can be bioactivated to the trichloromethyl radical, which causes extensive lipid peroxidation and ultimately cell death by necrosis. Overactivation of poly(ADP-ribose) polymerase-1 (PARP-1) can rapidly reduce the levels of (β-nicotinamide adenine dinucleotide and adenosine triphosphate and ultimately promote necrosis. The aim of this study was to determine whether inhibition of PARP-1 could decrease CCl4-induced hepatotoxicity, as measured by degree of poly(ADP-ribosyl)ation, serum levels of lactate dehydrogenase (LDH), lipid peroxidation,and oxidative DNA damage. For this purpose, male ICR mice were administered intraperitoneally a hepatotoxic dose of CCl4 with or without 6(5H)-phenanthridinone, a potent inhibitor of PARP-1. Animals treated with CCl4 exhibited extensive poly(ADP-ribosyl)ation in centrilobular hepatocytes, elevated serum levels of LDH, and increased lipid peroxidation. In contrast, animals treated concomitantly with CCl4 and 6(5H)-phenanthridinone showed significantly lower levels of poly(ADP-ribosyl) ation, serum LDH, and lipid peroxidation. No changes were observed in the levels of oxidative DNA damage regardless of treatment. These results demonstrated that the hepatotoxicity of CCl4is dependent on the overactivation of PARP-1 and that inhibition of this enzyme attenuates the hepatotoxicity of CCl4. PMID:21395487

  18. VERO cells harbor a poly-ADP-ribose belt partnering their epithelial adhesion belt.

    PubMed

    Lafon-Hughes, Laura; Vilchez Larrea, Salomé C; Kun, Alejandra; Fernández Villamil, Silvia H

    2014-01-01

    Poly-ADP-ribose (PAR) is a polymer of up to 400 ADP-ribose units synthesized by poly-ADP-ribose-polymerases (PARPs) and degraded by poly-ADP-ribose-glycohydrolase (PARG). Nuclear PAR modulates chromatin compaction, affecting nuclear functions (gene expression, DNA repair). Diverse defined PARP cytoplasmic allocation patterns contrast with the yet still imprecise PAR distribution and still unclear functions. Based on previous evidence from other models, we hypothesized that PAR could be present in epithelial cells where cadherin-based adherens junctions are linked with the actin cytoskeleton (constituting the adhesion belt). In the present work, we have examined through immunofluorescence and confocal microscopy, the subcellular localization of PAR in an epithelial monkey kidney cell line (VERO). PAR was distinguished colocalizing with actin and vinculin in the epithelial belt, a location that has not been previously reported. Actin filaments disruption with cytochalasin D was paralleled by PAR belt disruption. Conversely, PARP inhibitors 3-aminobenzamide, PJ34 or XAV 939, affected PAR belt synthesis, actin distribution, cell shape and adhesion. Extracellular calcium chelation displayed similar effects. Our results demonstrate the existence of PAR in a novel subcellular localization. An initial interpretation of all the available evidence points towards TNKS-1 as the most probable PAR belt architect, although TNKS-2 involvement cannot be discarded. Forthcoming research will test this hypothesis as well as explore the existence of the PAR belt in other epithelial cells and deepen into its functional implications.

  19. 32 CFR Appendix J to Part 154 - ADP Position Categories and Criteria for Designating Positions

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Designating Positions J Appendix J to Part 154 National Defense Department of Defense OFFICE OF THE SECRETARY OF DEFENSE SECURITY DEPARTMENT OF DEFENSE PERSONNEL SECURITY PROGRAM REGULATION Pt. 154, App. J Appendix J to Part 154—ADP Position Categories and Criteria for Designating Positions OMB Circular...

  20. 32 CFR Appendix J to Part 154 - ADP Position Categories and Criteria for Designating Positions

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Designating Positions J Appendix J to Part 154 National Defense Department of Defense OFFICE OF THE SECRETARY OF DEFENSE SECURITY DEPARTMENT OF DEFENSE PERSONNEL SECURITY PROGRAM REGULATION Pt. 154, App. J Appendix J to Part 154—ADP Position Categories and Criteria for Designating Positions OMB Circular...

  1. 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

  2. VERO cells harbor a poly-ADP-ribose belt partnering their epithelial adhesion belt

    PubMed Central

    Vilchez Larrea, Salomé C.; Kun, Alejandra

    2014-01-01

    Poly-ADP-ribose (PAR) is a polymer of up to 400 ADP-ribose units synthesized by poly-ADP-ribose-polymerases (PARPs) and degraded by poly-ADP-ribose-glycohydrolase (PARG). Nuclear PAR modulates chromatin compaction, affecting nuclear functions (gene expression, DNA repair). Diverse defined PARP cytoplasmic allocation patterns contrast with the yet still imprecise PAR distribution and still unclear functions. Based on previous evidence from other models, we hypothesized that PAR could be present in epithelial cells where cadherin-based adherens junctions are linked with the actin cytoskeleton (constituting the adhesion belt). In the present work, we have examined through immunofluorescence and confocal microscopy, the subcellular localization of PAR in an epithelial monkey kidney cell line (VERO). PAR was distinguished colocalizing with actin and vinculin in the epithelial belt, a location that has not been previously reported. Actin filaments disruption with cytochalasin D was paralleled by PAR belt disruption. Conversely, PARP inhibitors 3-aminobenzamide, PJ34 or XAV 939, affected PAR belt synthesis, actin distribution, cell shape and adhesion. Extracellular calcium chelation displayed similar effects. Our results demonstrate the existence of PAR in a novel subcellular localization. An initial interpretation of all the available evidence points towards TNKS-1 as the most probable PAR belt architect, although TNKS-2 involvement cannot be discarded. Forthcoming research will test this hypothesis as well as explore the existence of the PAR belt in other epithelial cells and deepen into its functional implications. PMID:25332845

  3. 48 CFR 245.608-72 - Screening excess automatic data processing equipment (ADPE).

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 3 2010-10-01 2010-10-01 false Screening excess automatic... Reporting, Redistribution, and Disposal of Contractor Inventory 245.608-72 Screening excess automatic data... screening, including General Services Administration screening, for ADPE. (See the Defense...

  4. 45 CFR 95.625 - Increased FFP for certain ADP systems.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ...-D program are contained in 45 CFR Part 307. The applicable regulations for the Title IV-E program are contained in 45 CFR 1355.55. The applicable regulations for the Title XIX program are contained in 42 CFR Part 433, Subpart C. Federal Financial Participation in Costs of ADP Acquisitions...

  5. Guidelines for developing NASA (National Aeronautics and Space Administration) ADP security risk management plans

    NASA Technical Reports Server (NTRS)

    Tompkins, F. G.

    1983-01-01

    This report presents guidance to NASA Computer security officials for developing ADP security risk management plans. The six components of the risk management process are identified and discussed. Guidance is presented on how to manage security risks that have been identified during a risk analysis performed at a data processing facility or during the security evaluation of an application system.

  6. 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.

  7. 10 CFR 95.49 - Security of automatic data processing (ADP) systems.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false Security of automatic data processing (ADP) systems. 95.49 Section 95.49 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) FACILITY SECURITY CLEARANCE AND SAFEGUARDING OF NATIONAL SECURITY INFORMATION AND RESTRICTED DATA Control of Information § 95.49 Security...

  8. Does inhibition of poly(ADP-ribose) polymerase prevent energy overconsumption under microgravity?

    NASA Astrophysics Data System (ADS)

    Dobrota, C.; Piso, M. I.; Keul, A.

    When plants are exposed to a stress signal they expend a lot of energy and exhibit enhanced respiration rates This is partially due to a breakdown in the NAD pool caused by the enhanced activity PARP which uses NAD as a substrate to synthesize polymers of ADP-ribose Stress-induced depletion of NAD results in a similar depletion of energy since ATP molecules are required to resynthesize the depleted NAD It seems that plants with lowered poly ADP ribosyl ation activity appear tolerant to multiple stresses Inhibiting PARP activity prevents energy overconsumption under stress allowing normal mitochondrial respiration We intend to study if the microgravity is perceived by plants as a stress factor and if experimental inhibition of poly ADP-ribose polymerase may improve the energetic level of the cells References DeBlock M Verduyn C De Brouwer D and Cornelissen M 2005 Poly ADP-ribose polymerase in plants affects energy homeostasis cell death and stress tolerance The Plant Journal 41 95--106 Huang S Greenway H Colmerm T D and Millar A H 2005 Protein synthesis by rice coleoptiles during prolonged anoxia Implications for glycolysis growth and energy utilization Annals of Botany 96 703--715 Mittler R Vanderauwera S Gollery M and Van Breusegem F 2005 Reactive oxygen gene network of plants Trends in Plant Science 9 10 490-498

  9. 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

  10. Aero-Propulsion Technology (APT) Task V Low Noise ADP Engine Definition Study

    NASA Technical Reports Server (NTRS)

    Holcombe, V.

    2003-01-01

    A study was conducted to identify and evaluate noise reduction technologies for advanced ducted prop propulsion systems that would allow increased capacity operation and result in an economically competitive commercial transport. The study investigated the aero/acoustic/structural advancements in fan and nacelle technology required to match or exceed the fuel burned and economic benefits of a constrained diameter large Advanced Ducted Propeller (ADP) compared to an unconstrained ADP propulsion system with a noise goal of 5 to 10 EPNDB reduction relative to FAR 36 Stage 3 at each of the three measuring stations namely, takeoff (cutback), approach and sideline. A second generation ADP was selected to operate within the maximum nacelle diameter constrain of 160 deg to allow installation under the wing. The impact of fan and nacelle technologies of the second generation ADP on fuel burn and direct operating costs for a typical 3000 nm mission was evaluated through use of a large, twin engine commercial airplane simulation model. The major emphasis of this study focused on fan blade aero/acoustic and structural technology evaluations and advanced nacelle designs. Results of this study have identified the testing required to verify the interactive performance of these components, along with noise characteristics, by wind tunnel testing utilizing and advanced interaction rig.

  11. Three-Dimensional Structures Reveal Multiple ADP/ATP Binding Modes

    SciTech Connect

    C Simmons; C Magee; D Smith; L Lauman; J Chaput; J Allen

    2011-12-31

    The creation of synthetic enzymes with predefined functions represents a major challenge in future synthetic biology applications. Here, we describe six structures of de novo proteins that have been determined using protein crystallography to address how simple enzymes perform catalysis. Three structures are of a protein, DX, selected for its stability and ability to tightly bind ATP. Despite the addition of ATP to the crystallization conditions, the presence of a bound but distorted ATP was found only under excess ATP conditions, with ADP being present under equimolar conditions or when crystallized for a prolonged period of time. A bound ADP cofactor was evident when Asp was substituted for Val at residue 65, but ATP in a linear configuration is present when Phe was substituted for Tyr at residue 43. These new structures complement previously determined structures of DX and the protein with the Phe 43 to Tyr substitution [Simmons, C. R., et al. (2009) ACS Chem. Biol. 4, 649-658] and together demonstrate the multiple ADP/ATP binding modes from which a model emerges in which the DX protein binds ATP in a configuration that represents a transitional state for the catalysis of ATP to ADP through a slow, metal-free reaction capable of multiple turnovers. This unusual observation suggests that design-free methods can be used to generate novel protein scaffolds that are tailor-made for catalysis.

  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. The ADP/ATP Carrier and Its Relationship to Oxidative Phosphorylation in Ancestral Protist Trypanosoma brucei

    PubMed Central

    Gnipová, Anna; Šubrtová, Karolína; Panicucci, Brian; Horváth, Anton; Lukeš, Julius

    2015-01-01

    The highly conserved ADP/ATP carrier (AAC) is a key energetic link between the mitochondrial (mt) and cytosolic compartments of all aerobic eukaryotic cells, as it exchanges the ATP generated inside the organelle for the cytosolic ADP. Trypanosoma brucei, a parasitic protist of medical and veterinary importance, possesses a single functional AAC protein (TbAAC) that is related to the human and yeast ADP/ATP carriers. However, unlike previous studies performed with these model organisms, this study showed that TbAAC is most likely not a stable component of either the respiratory supercomplex III+IV or the ATP synthasome but rather functions as a physically separate entity in this highly diverged eukaryote. Therefore, TbAAC RNA interference (RNAi) ablation in the insect stage of T. brucei does not impair the activity or arrangement of the respiratory chain complexes. Nevertheless, RNAi silencing of TbAAC caused a severe growth defect that coincides with a significant reduction of mt ATP synthesis by both substrate and oxidative phosphorylation. Furthermore, TbAAC downregulation resulted in a decreased level of cytosolic ATP, a higher mt membrane potential, an elevated amount of reactive oxygen species, and a reduced consumption of oxygen in the mitochondria. Interestingly, while TbAAC has previously been demonstrated to serve as the sole ADP/ATP carrier for ADP influx into the mitochondria, our data suggest that a second carrier for ATP influx may be present and active in the T. brucei mitochondrion. Overall, this study provides more insight into the delicate balance of the functional relationship between TbAAC and the oxidative phosphorylation (OXPHOS) pathway in an early diverged eukaryote. PMID:25616281

  14. The substrate specificity of the human ADP/ATP carrier AAC1.

    PubMed

    Mifsud, John; Ravaud, Stéphanie; Krammer, Eva-Maria; Chipot, Chris; Kunji, Edmund R S; Pebay-Peyroula, Eva; Dehez, Francois

    2013-03-01

    The mitochondrial ADP/ATP carrier imports ADP from the cytosol into the mitochondrial matrix for its conversion to ATP by ATP synthase and exports ATP out of the mitochondrion to replenish the eukaryotic cell with chemical energy. Here the substrate specificity of the human mitochondrial ADP/ATP carrier AAC1 was determined by two different approaches. In the first the protein was functionally expressed in Escherichia coli membranes as a fusion protein with maltose binding protein and the effect of excess of unlabeled compounds on the uptake of [(32)P]-ATP was measured. In the second approach the protein was expressed in the cytoplasmic membrane of Lactococcus lactis. The uptake of [(14)C]-ADP in whole cells was measured in the presence of excess of unlabeled compounds and in fused membrane vesicles loaded with unlabeled compounds to demonstrate their transport. A large number of nucleotides were tested, but only ADP and ATP are suitable substrates for human AAC1, demonstrating a very narrow specificity. Next we tried to understand the molecular basis of this specificity by carrying out molecular-dynamics simulations with selected nucleotides, which were placed at the entrance of the central cavity. The binding of the phosphate groups of guanine and adenine nucleotides is similar, yet there is a low probability for the base moiety to be bound, likely to be rooted in the greater polarity of guanine compared to adenine. AMP is unlikely to engage fully with all contact points of the substrate binding site, suggesting that it cannot trigger translocation.

  15. 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

  16. 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

  17. 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

  18. 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.

  19. Altered poly(ADP-ribose) metabolism impairs cellular responses to genotoxic stress in a hypomorphic mutant of poly(ADP-ribose) glycohydrolase

    SciTech Connect

    Gao Hong; Coyle, Donna L.; Meyer-Ficca, Mirella L.; Meyer, Ralph G.; Jacobson, Elaine L.; Wang, Zhao-Qi; Jacobson, Myron K. . E-mail: mjacobson@pharmacy.arizona.edu

    2007-03-10

    Genotoxic stress activates nuclear poly(ADP-ribose) (PAR) metabolism leading to PAR synthesis catalyzed by DNA damage activated poly(ADP-ribose) polymerases (PARPs) and rapid PAR turnover by action of nuclear poly(ADP-ribose) glycohydrolase (PARG). The involvement of PARP-1 and PARP-2 in responses to DNA damage has been well studied but the involvement of nuclear PARG is less well understood. To gain insights into the function of nuclear PARG in DNA damage responses, we have quantitatively studied PAR metabolism in cells derived from a hypomorphic mutant mouse model in which exons 2 and 3 of the PARG gene have been deleted (PARG-{delta}2,3 cells), resulting in a nuclear PARG containing a catalytic domain but lacking the N-terminal region (A domain) of the protein. Following DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), we found that the activity of both PARG and PARPs in intact cells is increased in PARG-{delta}2,3 cells. The increased PARG activity leads to decreased PARP-1 automodification with resulting increased PARP activity. The degree of PARG activation is greater than PARP, resulting in decreased PAR accumulation. Following MNNG treatment, PARG-{delta}2,3 cells show reduced formation of XRCC1 foci, delayed H2AX phosphorylation, decreased DNA break intermediates during repair, and increased cell death. Our results show that a precise coordination of PARPs and PARG activities is important for normal cellular responses to DNA damage and that this coordination is defective in the absence of the PARG A domain.

  20. Purinergic Receptors in Ocular Inflammation

    PubMed Central

    Guzman-Aranguez, Ana; Gasull, Xavier; Diebold, Yolanda; Pintor, Jesús

    2014-01-01

    Inflammation is a complex process that implies the interaction between cells and molecular mediators, which, when not properly “tuned,” can lead to disease. When inflammation affects the eye, it can produce severe disorders affecting the superficial and internal parts of the visual organ. The nucleoside adenosine and nucleotides including adenine mononucleotides like ADP and ATP and dinucleotides such as P1,P4-diadenosine tetraphosphate (Ap4A), and P1,P5-diadenosine pentaphosphate (Ap5A) are present in different ocular locations and therefore they may contribute/modulate inflammatory processes. Adenosine receptors, in particular A2A adenosine receptors, present anti-inflammatory action in acute and chronic retinal inflammation. Regarding the A3 receptor, selective agonists like N6-(3-iodobenzyl)-5′-N-methylcarboxamidoadenosine (CF101) have been used for the treatment of inflammatory ophthalmic diseases such as dry eye and uveoretinitis. Sideways, diverse stimuli (sensory stimulation, large intraocular pressure increases) can produce a release of ATP from ocular sensory innervation or after injury to ocular tissues. Then, ATP will activate purinergic P2 receptors present in sensory nerve endings, the iris, the ciliary body, or other tissues surrounding the anterior chamber of the eye to produce uveitis/endophthalmitis. In summary, adenosine and nucleotides can activate receptors in ocular structures susceptible to suffer from inflammatory processes. This involvement suggests the possible use of purinergic agonists and antagonists as therapeutic targets for ocular inflammation. PMID:25132732

  1. Ticagrelor: Pharmacokinetic, Pharmacodynamic and Pharmacogenetic Profile: An Update.

    PubMed

    Teng, Renli

    2015-11-01

    Despite advancements in treatments for acute coronary syndromes over the last 10 years, they continue to be life-threatening disorders. Currently, the standard of treatment includes dual antiplatelet therapy consisting of aspirin plus a P2Y12 receptor antagonist. The thienopyridine class of P2Y12 receptor antagonists, clopidogrel and prasugrel, have demonstrated efficacy. However, their use is associated with several limitations, including the need for metabolic activation and irreversible P2Y12 receptor binding causing prolonged recovery of platelet function. In addition, response to clopidogrel is variable and efficacy is reduced in patients with certain genotypes. Although prasugrel is a more consistent inhibitor of platelet aggregation than clopidogrel, it is associated with an increased risk of life-threatening and fatal bleeding. Ticagrelor is an oral antiplatelet agent of the cyclopentyltriazolopyrimidine class and also acts through the P2Y12 receptor. In contrast to clopidogrel and prasugrel, ticagrelor does not require metabolic activation and binds rapidly and reversibly to the P2Y12 receptor. In light of new data, this review provides an update on the pharmacokinetic, pharmacodynamic and pharmacogenetic profiles of ticagrelor in different study populations. Recent studies report that no dose adjustment for ticagrelor is required on the basis of age, gender, ethnicity, severe renal impairment or mild hepatic impairment. The non-P2Y12 actions of ticagrelor are reviewed, showing indirect positive effects on cellular adenosine concentration and biological activity, by inhibition of equilibrative nucleoside transporter-1 independently of the P2Y12 receptor. CYP2C19 and ABCB1 genotypes do not appear to influence ticagrelor pharmacodynamics. A summary of drug interactions is also presented. PMID:26063049

  2. Cyclic ADP-ribose and hydrogen peroxide synergize with ADP-ribose in the activation of TRPM2 channels.

    PubMed

    Kolisek, Martin; Beck, Andreas; Fleig, Andrea; Penner, Reinhold

    2005-04-01

    The melastatin-related transient receptor potential channel TRPM2 is a plasma membrane Ca2+-permeable cation channel that is activated by intracellular adenosine diphosphoribose (ADPR) binding to the channel's enzymatic Nudix domain. Channel activity is also seen with nicotinamide dinucleotide (NAD+) and hydrogen peroxide (H2O2), but their mechanisms of action remain unknown. Here, we identify cyclic adenosine diphosphoribose (cADPR) as an agonist of TRPM2 with dual activity: at concentrations above 100 microM, cADPR can gate the channel by itself, whereas lower concentrations of 10 microM have a potentiating effect that enables ADPR to gate the channel at nanomolar concentrations. ADPR's breakdown product adenosine monophosphate (AMP) specifically inhibits ADPR, but not cADPR-mediated gating of TRPM2, whereas the cADPR antagonist 8-Br-cADPR exhibits the reverse block specificity. Our results establish TRPM2 as a coincidence detector for ADPR and cADPR signaling and provide a functional context for cADPR as a second messenger for Ca2+ influx.

  3. Differential and Concordant Roles for Poly(ADP-Ribose) Polymerase 1 and Poly(ADP-Ribose) in Regulating WRN and RECQL5 Activities

    PubMed Central

    Khadka, Prabhat; Hsu, Joseph K.; Veith, Sebastian; Tadokoro, Takashi; Shamanna, Raghavendra A.; Mangerich, Aswin; Croteau, Deborah L.

    2015-01-01

    Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) catalyzes the poly(ADP-ribosyl)ation (PARylation) of proteins, a posttranslational modification which forms the nucleic acid-like polymer PAR. PARP1 and PAR are integral players in the early DNA damage response, since PARylation orchestrates the recruitment of repair proteins to sites of damage. Human RecQ helicases are DNA unwinding proteins that are critical responders to DNA damage, but how their recruitment and activities are regulated by PARPs and PAR is poorly understood. Here we report that all human RecQ helicases interact with PAR noncovalently. Furthermore, we define the effects that PARP1, PARylated PARP1, and PAR have on RECQL5 and WRN, using both in vitro and in vivo assays. We show that PARylation is involved in the recruitment of RECQL5 and WRN to laser-induced DNA damage and that RECQL5 and WRN have differential responses to PARylated PARP1 and PAR. Furthermore, we show that the loss of RECQL5 or WRN resulted in increased sensitivity to PARP inhibition. In conclusion, our results demonstrate that PARP1 and PAR actively, and in some instances differentially, regulate the activities and cellular localization of RECQL5 and WRN, suggesting that PARylation acts as a fine-tuning mechanism to coordinate their functions in time and space during the genotoxic stress response. PMID:26391948

  4. Differential and Concordant Roles for Poly(ADP-Ribose) Polymerase 1 and Poly(ADP-Ribose) in Regulating WRN and RECQL5 Activities.

    PubMed

    Khadka, Prabhat; Hsu, Joseph K; Veith, Sebastian; Tadokoro, Takashi; Shamanna, Raghavendra A; Mangerich, Aswin; Croteau, Deborah L; Bohr, Vilhelm A

    2015-12-01

    Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) catalyzes the poly(ADP-ribosyl)ation (PARylation) of proteins, a posttranslational modification which forms the nucleic acid-like polymer PAR. PARP1 and PAR are integral players in the early DNA damage response, since PARylation orchestrates the recruitment of repair proteins to sites of damage. Human RecQ helicases are DNA unwinding proteins that are critical responders to DNA damage, but how their recruitment and activities are regulated by PARPs and PAR is poorly understood. Here we report that all human RecQ helicases interact with PAR noncovalently. Furthermore, we define the effects that PARP1, PARylated PARP1, and PAR have on RECQL5 and WRN, using both in vitro and in vivo assays. We show that PARylation is involved in the recruitment of RECQL5 and WRN to laser-induced DNA damage and that RECQL5 and WRN have differential responses to PARylated PARP1 and PAR. Furthermore, we show that the loss of RECQL5 or WRN resulted in increased sensitivity to PARP inhibition. In conclusion, our results demonstrate that PARP1 and PAR actively, and in some instances differentially, regulate the activities and cellular localization of RECQL5 and WRN, suggesting that PARylation acts as a fine-tuning mechanism to coordinate their functions in time and space during the genotoxic stress response.

  5. Differential and Concordant Roles for Poly(ADP-Ribose) Polymerase 1 and Poly(ADP-Ribose) in Regulating WRN and RECQL5 Activities.

    PubMed

    Khadka, Prabhat; Hsu, Joseph K; Veith, Sebastian; Tadokoro, Takashi; Shamanna, Raghavendra A; Mangerich, Aswin; Croteau, Deborah L; Bohr, Vilhelm A

    2015-12-01

    Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) catalyzes the poly(ADP-ribosyl)ation (PARylation) of proteins, a posttranslational modification which forms the nucleic acid-like polymer PAR. PARP1 and PAR are integral players in the early DNA damage response, since PARylation orchestrates the recruitment of repair proteins to sites of damage. Human RecQ helicases are DNA unwinding proteins that are critical responders to DNA damage, but how their recruitment and activities are regulated by PARPs and PAR is poorly understood. Here we report that all human RecQ helicases interact with PAR noncovalently. Furthermore, we define the effects that PARP1, PARylated PARP1, and PAR have on RECQL5 and WRN, using both in vitro and in vivo assays. We show that PARylation is involved in the recruitment of RECQL5 and WRN to laser-induced DNA damage and that RECQL5 and WRN have differential responses to PARylated PARP1 and PAR. Furthermore, we show that the loss of RECQL5 or WRN resulted in increased sensitivity to PARP inhibition. In conclusion, our results demonstrate that PARP1 and PAR actively, and in some instances differentially, regulate the activities and cellular localization of RECQL5 and WRN, suggesting that PARylation acts as a fine-tuning mechanism to coordinate their functions in time and space during the genotoxic stress response. PMID:26391948

  6. 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.

  7. Inhibition of Poly(ADP-Ribose) Polymerase by Nucleic Acid Metabolite 7-Methylguanine

    PubMed Central

    Nilov, D. K.; Tararov, V. I.; Kulikov, A. V.; Zakharenko, A. L.; Gushchina, I. V.; Mikhailov, S. N.; Lavrik, O. I.; Švedas, V. K.

    2016-01-01

    The ability of 7-methylguanine, a nucleic acid metabolite, to inhibit poly(ADP-ribose)polymerase-1 (PARP-1) and poly(ADP-ribose)polymerase-2 (PARP-2) has been identified in silico and studied experimentally. The amino group at position 2 and the methyl group at position 7 were shown to be important substituents for the efficient binding of purine derivatives to PARPs. The activity of both tested enzymes, PARP-1 and PARP-2, was suppressed by 7-methylguanine with IC50 values of 150 and 50 μM, respectively. At the PARP inhibitory concentration, 7-methylguanine itself was not cytotoxic, but it was able to accelerate apoptotic death of BRCA1-deficient breast cancer cells induced by cisplatin and doxorubicin, the widely used DNA-damaging chemotherapeutic agents. 7-Methylguanine possesses attractive predictable pharmacokinetics and an adverse-effect profile and may be considered as a new additive to chemotherapeutic treatment. PMID:27437145

  8. Poly(ADP-ribose) polymerase inhibitor induces accelerated senescence in irradiated breast cancer cells and tumors

    PubMed Central

    Efimova, Elena V.; Mauceri, Helena J.; Golden, Daniel W.; Labay, Edwardine; Bindokas, Vytautas P.; Darga, Thomas E.; Chakraborty, Chaitali; Andrade, Juan Camilo Barreto; Crawley, Clayton; Sutton, Harold G.; Kron, Stephen J.; Weichselbaum, Ralph R.

    2010-01-01

    Persistent DNA double strand breaks (DSBs) may determine the anti-tumor effects of ionizing radiation (IR) by inducing apoptosis, necrosis, mitotic catastrophe or permanent growth arrest. Ionizing radiation (IR) induces rapid modification of megabase chromatin domains surrounding double strand breaks (DSBs) via poly-ADP-ribosylation, phosphorylation, acetylation, and protein assembly. The dynamics of these ionizing radiation-induced foci (IRIF) have been implicated in DNA damage signaling and DNA repair. As an IRIF reporter, we tracked relocalization of GFP fused to a chromatin binding domain of the checkpoint adapter protein 53BP1 after IR of breast cancer cells and tumors. To block DSB repair in breast cancer cells and tumors, we targeted poly(ADP-ribose) polymerase with ABT-888 (veliparib), one of several PARP inhibitors currently in clinical trials. PARP inhibition markedly enhanced IRIF persistence and increased breast cancer cell senescence both in vitro and in vivo, arguing for targeting IRIF resolution as a novel therapeutic strategy. PMID:20610628

  9. 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.

  10. The Neisseria meningitidis ADP-Ribosyltransferase NarE Enters Human Epithelial Cells and Disrupts Epithelial Monolayer Integrity.

    PubMed

    Valeri, Maria; Zurli, Vanessa; Ayala, Inmaculada; Colanzi, Antonino; Lapazio, Lucia; Corda, Daniela; Soriani, Marco; Pizza, Mariagrazia; Rossi Paccani, Silvia

    2015-01-01

    Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and impair essential functions of eukaryotic cells. It has been previously reported that Neisseria meningitidis possesses an ADP-ribosyltransferase enzyme, NarE, retaining the capacity to hydrolyse NAD and to transfer ADP-ribose moiety to arginine residues in target acceptor proteins. Here we show that upon internalization into human epithelial cells, NarE gains access to the cytoplasm and, through its ADP-ribosylating activity, targets host cell proteins. Notably, we observed that these events trigger the disruption of the epithelial monolayer integrity and the activation of the apoptotic pathway. Overall, our findings provide, for the first time, evidence for a biological activity of NarE on host cells, suggesting its possible involvement in Neisseria pathogenesis.

  11. Effect of L-cysteine on optical, thermal and mechanical properties of ADP crystal for NLO application

    NASA Astrophysics Data System (ADS)

    Shaikh, R. N.; Shirsat, M. D.; Koinkar, P. M.; Hussaini, S. S.

    2015-06-01

    The ammonium dihydrogen phosphate (ADP) crystal doped with amino acid L-cysteine (LC) was grown by a slow evaporation technique. The grown crystal was transparent in the entire visible region, which is an essential requirement for a nonlinear crystal. The LC doping enhances the optical band gap of ADP (5.35 eV). The TG/DTA analysis of LC doped ADP crystal confirms the optimum thermal stability of grown crystal. The enhancement in the mechanical stability after LC doping was confirmed by Vicker's microhardness test. The LC doping showed significant impact on dielectric properties (dielectric constant and dielectric loss) of grown crystal. The third order nonlinear behavior of LC doped ADP crystal was investigated using a Z-scan technique at 632.8 nm and effective nonlinear optical parameters were evaluated.

  12. The Molecular Mechanism of P2Y1 Receptor Activation

    PubMed Central

    Chan, H. C. Stephen; Vogel, Horst; Filipek, Slawomir

    2016-01-01

    Human purinergic G protein-coupled receptor P2Y1 (P2Y1R) is activated by adenosine 5’-diphosphate (ADP) to induce platelet activation and thereby serves as an important antithrombotic drug target. Crystal structures of P2Y1R revealed that one ligand (MRS2500) binds to the extracellular vestibule of this GPCR, whereas another (BPTU) occupies the surface between transmembrane (TM) helices TM2 and TM3. We introduced a total of 20 µs all-atom long-timescale molecular dynamic (MD) simulations to inquire why two molecules in completely different locations both serve as antagonists while ADP activates the receptor. Our results indicate that BPTU acts as an antagonist by stabilizing extracellular helix bundles leading to an increase of the lipid order, whereas MRS2500 blocks signaling by occupying the ligand binding site. Both antagonists stabilize an ionic lock within the receptor. However, binding of ADP breaks this ionic lock, forming a continuous water channel that leads to P2Y1R activation. PMID:27460867

  13. Guidelines for contingency planning NASA (National Aeronautics and Space Administration) ADP security risk reduction decision studies

    NASA Technical Reports Server (NTRS)

    Tompkins, F. G.

    1984-01-01

    Guidance is presented to NASA Computer Security Officials for determining the acceptability or unacceptability of ADP security risks based on the technical, operational and economic feasibility of potential safeguards. The risk management process is reviewed as a specialized application of the systems approach to problem solving and information systems analysis and design. Reporting the results of the risk reduction analysis to management is considered. Report formats for the risk reduction study are provided.

  14. The switching mechanism of the mitochondrial ADP/ATP carrier explored by free-energy landscapes.

    PubMed

    Pietropaolo, Adriana; Pierri, Ciro Leonardo; Palmieri, Ferdinando; Klingenberg, Martin

    2016-06-01

    The ADP/ATP carrier (AAC) of mitochondria has been an early example for elucidating the transport mechanism alternating between the external (c-) and internal (m-) states (M. Klingenberg, Biochim. Biophys. Acta 1778 (2008) 1978-2021). An atomic resolution crystal structure of AAC is available only for the c-state featuring a three repeat transmembrane domain structure. Modeling of transport mechanism remained hypothetical for want of an atomic structure of the m-state. Previous molecular dynamics studies simulated the binding of ADP or ATP to the AAC remaining in the c-state. Here, a full description of the AAC switching from the c- to the m-state is reported using well-tempered metadynamics simulations. Free-energy landscapes of the entire translocation from the c- to the m-state, based on the gyration radii of the c- and m-gates and of the center of mass, were generated. The simulations revealed three free-energy basins attributed to the c-, intermediate- and m-states separated by activation barriers. These simulations were performed with the empty and with the ADP- and ATP-loaded AAC as well as with the poorly transported AMP and guanine nucleotides, showing in the free energy landscapes that ADP and ATP lowered the activation free-energy barriers more than the other substrates. Upon binding AMP and guanine nucleotides a deeper free-energy level stabilized the intermediate-state of the AAC2 hampering the transition to the m-state. The structures of the substrate binding sites in the different states are described producing a full picture of the translocation events in the AAC.

  15. Sodium pump-mediated ATP:ADP exchange. The sided effects of sodium and potassium ions

    PubMed Central

    1982-01-01

    Resealed human red cell ghosts containing caged ATP (Kaplan et al., 1978) and [3H]ADP were irradiated at 340 nm. The photochemical release of free ATP initiated a rapid transphosphorylation reaction (ATP:ADP exchange), a component of which is inhibited by ouabain. The reaction rate was measured by following the rate of appearance of [3H]ATP. The sodium pump-mediated ATP:ADP exchange reaction showed high-affinity stimulation by Mg ions (less than 10 microM) and was inhibited at higher levels. At optimal [Mg], extracellular Na (Nao) had a biphasic effect. Nao progressively inhibited the reaction rate between 0 and 10 mM and stimulated at higher levels. Intracellular Na (Nai) activated the reaction; the rate was maximal when Nai was 1 mM and remained unaltered up to 115 mM Nai at constant Nao. Extracellular K ions (Ko) inhibited the reaction; at high Nao, half-maximal inhibition was observed with 0.9 mM Ko. Lio inhibited the exchange rate with a lower affinity than Ko; half-maximal inhibition was produced by approximately 50 mM Lio. Intracellular K ions were without dramatic effect on the reaction rate in the concentration range where Ko inhibited completely. The relationship between these observations and previous studies on porous preparations is discussed, as well as the extent to which these observations support the hypothesis that the sodium pump-mediated ATP:ADP exchange reaction accompanies the Na:Na exchange transport mode of the sodium pump. PMID:6294224

  16. G-protein-mediated activation of turkey erythrocyte phospholipase C by beta-adrenergic and P2y-purinergic receptors.

    PubMed Central

    Vaziri, C; Downes, C P

    1992-01-01

    Isoprenaline, previously known only to stimulate adenylate cyclase via the stimulatory G-protein, Gs, activates turkey erythrocyte ghost phospholipase C (PLC) in a dose-dependent manner when GTP or guanosine 5'-[gamma-thio]triphosphate (GTP[S]) is present. The effect is specific in that it is abolished by beta-adrenergic-receptor antagonists. Stimulation of adenosine receptors, which also couple to adenylate cyclase via Gs in turkey erythrocytes, does not activate PLC, indicating that the stimulation observed in the presence of isoprenaline is not due to Gs activation. Furthermore, the stimulation seen is independent of cyclic AMP production. Purified turkey erythrocyte PLC is activated in an adenosine 5'-[beta-thio]diphosphate (ADP[S]; a P2y-purinergic-receptor agonist)- or isoprenaline-regulated manner when reconstituted with turkey erythrocyte ghosts, demonstrating that a single species of PLC effector enzyme can be regulated by both the purinergic and the beta-adrenergic receptor populations present in turkey erythrocyte membranes. Pretreatment of intact turkey erythrocytes with the P2y agonist ADP[S] causes decreased PLC responsiveness of subsequent ghost preparations to ADP[S] stimulation, although responses to isoprenaline are unaffected (homologous desensitization). In contrast, pretreatment of intact erythrocytes with isoprenaline results in heterologous desensitization of both the P2y and the beta-adrenergic receptors. These effects occur at the level of receptor-G-protein coupling, since PLC stimulation by GTP[S] (which directly activates G-proteins) in the absence of agonists is unaffected. PMID:1352448

  17. Pyruvate dehydrogenase kinase isoform 2 activity limited and further inhibited by slowing down the rate of dissociation of ADP.

    PubMed

    Bao, Haiying; Kasten, Shane A; Yan, Xiaohua; Roche, Thomas E

    2004-10-26

    Pyruvate dehydrogenase kinase 2 (PDK2) activity is enhanced by the dihydrolipoyl acetyltransferase core (E2 60mer) that binds PDK2 and a large number of its pyruvate dehydrogenase (E1) substrate. With E2-activated PDK2, K(+) at approximately 90 mM and Cl(-) at approximately 60 mM decreased the K(m) of PDK2 for ATP and competitive K(i) for ADP by approximately 3-fold and enhanced pyruvate inhibition. Comparing PDK2 catalysis +/- E2, E2 increased the K(m) of PDK2 for ATP by nearly 8-fold (from 5 to 39 microM), increased k(cat) by approximately 4-fold, and decreased the requirement for E1 by at least 400-fold. ATP binding, measured by a cold-trapping technique, occurred at two active sites with a K(d) of 5 microM, which equals the K(m) and K(d) of PDK2 for ATP measured in the absence of E2. During E2-aided catalysis, PDK2 had approximately 3 times more ADP than ATP bound at its active site, and the pyruvate analogue, dichloroacetate, led to 16-fold more ADP than ATP being bound (no added ADP). Pyruvate functioned as an uncompetitive inhibitor versus ATP, and inclusion of ADP transformed pyruvate inhibition to noncompetitive. At high pyruvate levels, pyruvate was a partial inhibitor but also induced substrate inhibition at high ATP levels. Our results indicate that, at physiological salt levels, ADP dissociation is a limiting step in E2-activated PDK2 catalysis, that PDK2.[ADP or ATP].pyruvate complexes form, and that PDK2.ATP.pyruvate.E1 reacts with PDK2.ADP.pyruvate accumulating. PMID:15491150

  18. 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

  19. PARP1 is a TRF2-associated poly(ADP-ribose) polymerase and protects eroded telomeres

    SciTech Connect

    Gomez, Marla V; Wu, Jun; Wang, Yisong; Liu, Yie

    2006-01-01

    Poly(ADP-ribose)polymerase 1 (PARP1) is well characterized for its role in base excision repair (BER), where it is activated by and binds to DNA breaks and catalyzes the poly(ADP-ribosyl)ation of several substrates involved in DNA damage repair. Here we demonstrate that PARP1 associates with telomere repeat binding factor 2 (TRF2) and is capable of poly(ADP-ribosyl)ation of TRF2, which affects binding of TRF2 to telomeric DNA. Immunostaining of interphase cells or metaphase spreads shows that PARP1 is detected sporadically at normal telomeres, but it appears preferentially at eroded telomeres caused by telomerase deficiency or damaged telomeres induced by DNA-damaging reagents. Although PARP1 is dispensable in the capping of normal telomeres, Parp1 deficiency leads to an increase in chromosome end-to-end fusions or chromosome ends without detectable telomeric DNA in primary murine cells after induction of DNA damage. Our results suggest that upon DNA damage, PARP1 is recruited to damaged telomeres, where it can help protect telomeres against chromosome end-to-end fusions and genomic instability.

  20. 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.

  1. PARP1 Is a TRF2-associated Poly(ADP-Ribose)Polymerase and Protects Eroded Telomeres

    SciTech Connect

    Liu, Yie; Wu, Jun; Schreiber, Valerie; Dunlap, John; Dantzer, Francoise; Wang, Yisong

    2006-01-01

    Poly(ADP-ribose)polymerase 1 (PARP1) is well characterized for its role in base excision repair (BER), where it is activated by and binds to DNA breaks and catalyzes the poly(ADP-ribosyl)ation of several substrates involved in DNA damage repair. Here we demonstrate that PARP1 associates with telomere repeat binding factor 2 (TRF2) and is capable of poly(ADP-ribosyl)ation of TRF2, which affects binding of TRF2 to telomeric DNA. Immunostaining of interphase cells or metaphase spreads shows that PARP1 is detected sporadically at normal telomeres, but it appears preferentially at eroded telomeres caused by telomerase deficiency or damaged telomeres induced by DNA-damaging reagents. Although PARP1 is dispensable in the capping of normal telomeres, Parp1 deficiency leads to an increase in chromosome end-to-end fusions or chromosome ends without detectable telomeric DNA in primary murine cells after induction of DNA damage. Our results suggest that upon DNA damage, PARP1 is recruited to damaged telomeres, where it can help protect telomeres against chromosome end-to-end fusions and genomic instability.

  2. Deciphering the kinetic mechanisms controlling selected plant ADP-glucose pyrophosphorylases.

    PubMed

    Boehlein, Susan K; Shaw, Janine R; Hwang, Seon K; Stewart, Jon D; Curtis Hannah, L

    2013-07-15

    ADP-Glc pyrophosphorylase (AGPase), a rate-limiting enzyme in starch biosynthesis, is controlled by thermostability and allosteric regulation. Previous studies suggested that redox affects turnover number and heat stability of AGPases. Here, we investigated how allostery and redox state affect kinetic mechanisms of the reduced, heat labile and the oxidized, heat stable potato tuber enzymes; the heat labile maize endosperm enzyme and a chimeric maize/potato heat stable enzyme that lacks the cysteine responsible for redox changes. With 3-PGA, all AGPases followed a Theorell-Chance Bi Bi mechanism with ATP binding first and ADP-Glc releasing last. 3-PGA increases the binding affinity for both substrates with little effect on velocity for the maize and MP isoforms. By contrast, 3-PGA increases the velocity and the affinity for G-1-P for the potato enzymes. Redox state does not affect kcat of the two potato isoforms. Without 3-PGA the oxidized potato enzyme exhibits a rapid equilibrium random Bi Bi mechanism with a dead end ternary complex. This fundamental change from rapid, ordered binding with little buildup of intermediates to a mechanism featuring relatively slow, random binding is unique to the oxidized potato tuber enzyme. Finally, ADP-Glc the physiologically relevant product of this enzyme has complex, isoform-specific effects on catalysis. PMID:23603314

  3. Photox, a novel actin-targeting mono-ADP-ribosyltransferase from Photorhabdus luminescens.

    PubMed

    Visschedyk, Danielle D; Perieteanu, Alexandru A; Turgeon, Zachari J; Fieldhouse, Robert J; Dawson, John F; Merrill, A Rod

    2010-04-30

    Photorhabdus luminescens is a pathogenic bacterium that produces many toxic proteins. The mono-ADP-ribosyltransferases (mARTs) are an enzyme class produced by numerous pathogenic bacteria and participate in disease in plants and animals, including humans. Herein we report a novel mART from P. luminescens called Photox. This 46-kDa toxin shows high homology to other actin-targeting mARTs in hallmark catalytic regions and a similar core catalytic fold. Furthermore, Photox shows in vivo cytotoxic activity against yeast, with protection occurring when catalytic residues are substituted with alanine. In vitro, enzymatic activity (k(cat), 1680 +/- 75 min(-1)) is higher than that of the related iota toxin, and diminishes by nearly 14,000-fold following substitution of the catalytic Glu (E355A). This toxin specifically ADP-ribosylates monomeric alpha-skeletal actin and nonmuscle beta- and gamma-actin at Arg(177), inhibiting regular polymerization of actin filaments. These results indicate that Photox is indeed an ADP-ribosyltransferase, making it the newest member of the actin-targeting mART family. PMID:20181945

  4. Novel metabolic features in Acinetobacter baylyi ADP1 revealed by a multiomics approach.

    PubMed

    Stuani, Lucille; Lechaplais, Christophe; Salminen, Aaro V; Ségurens, Béatrice; Durot, Maxime; Castelli, Vanina; Pinet, Agnès; Labadie, Karine; Cruveiller, Stéphane; Weissenbach, Jean; de Berardinis, Véronique; Salanoubat, Marcel; Perret, Alain

    2014-01-01

    Expansive knowledge of bacterial metabolism has been gained from genome sequencing output, but the high proportion of genes lacking a proper functional annotation in a given genome still impedes the accurate prediction of the metabolism of a cell. To access to a more global view of the functioning of the soil bacterium Acinetobacter baylyi ADP1, we adopted a multi 'omics' approach. Application of RNA-seq transcriptomics and LC/MS-based metabolomics, along with the systematic phenotyping of the complete collection of single-gene deletion mutants of A. baylyi ADP1 made possible to interrogate on the metabolic perturbations encountered by the bacterium upon a biotic change. Shifting the sole carbon source from succinate to quinate elicited in the cell not only a specific transcriptional response, necessary to catabolize the new carbon source, but also a major reorganization of the transcription pattern. Here, the expression of more than 12 % of the total number of genes was affected, most of them being of unknown function. These perturbations were ultimately reflected in the metabolome, in which the concentration of about 50 % of the LC/MS-detected metabolites was impacted. And the differential regulation of many genes of unknown function is probably related to the synthesis of the numerous unidentified compounds that were present exclusively in quinate-grown cells. Together, these data suggest that A. baylyi ADP1 metabolism involves unsuspected enzymatic reactions that await discovery. PMID:25374488

  5. An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells.

    PubMed

    James, Dominic I; Durant, Stephen; Eckersley, Kay; Fairweather, Emma; Griffiths, Louise A; Hamilton, Nicola; Kelly, Paul; O'Connor, Mark; Shea, Kerry; Waddell, Ian D; Ogilvie, Donald J

    2016-01-01

    After a DNA damage signal multiple polymers of ADP ribose attached to poly(ADP) ribose (PAR) polymerases (PARPs) are broken down by the enzyme poly(ADP) ribose glycohydrolase (PARG). Inhibition of PARG leads to a failure of DNA repair and small molecule inhibition of PARG has been a goal for many years. To determine whether biochemical inhibitors of PARG are active in cells we have designed an immunofluorescence assay to detect nuclear PAR after DNA damage. This 384-well assay is suitable for medium throughput high-content screening and can detect cell-permeable inhibitors of PARG from nM to µM potency. In addition, the assay has been shown to work in murine cells and in a variety of human cancer cells. Furthermore, the assay is suitable for detecting the DNA damage response induced by treatment with temozolomide and methylmethane sulfonate (MMS). Lastly, the assay has been shown to be robust over a period of several years. PMID:27610220

  6. An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells

    PubMed Central

    James, Dominic I.; Durant, Stephen; Eckersley, Kay; Fairweather, Emma; Griffiths, Louise A.; Hamilton, Nicola; Kelly, Paul; O'Connor, Mark; Shea, Kerry; Waddell, Ian D.; Ogilvie, Donald J.

    2016-01-01

    After a DNA damage signal multiple polymers of ADP ribose attached to poly(ADP) ribose (PAR) polymerases (PARPs) are broken down by the enzyme poly(ADP) ribose glycohydrolase (PARG). Inhibition of PARG leads to a failure of DNA repair and small molecule inhibition of PARG has been a goal for many years. To determine whether biochemical inhibitors of PARG are active in cells we have designed an immunofluorescence assay to detect nuclear PAR after DNA damage. This 384-well assay is suitable for medium throughput high-content screening and can detect cell-permeable inhibitors of PARG from nM to µM potency. In addition, the assay has been shown to work in murine cells and in a variety of human cancer cells. Furthermore, the assay is suitable for detecting the DNA damage response induced by treatment with temozolomide and methylmethane sulfonate (MMS). Lastly, the assay has been shown to be robust over a period of several years. PMID:27610220

  7. 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

  8. Residual force depression in single sarcomeres is abolished by MgADP-induced activation

    PubMed Central

    Trecarten, Neal; Minozzo, Fabio C.; Leite, Felipe S.; Rassier, Dilson E.

    2015-01-01

    The mechanisms behind the shortening-induced force depression commonly observed in skeletal muscles remain unclear, but have been associated with sarcomere length non-uniformity and/or crossbridge inhibition. The purpose of this study was twofold: (i) to evaluate if force depression is present in isolated single sarcomeres, a preparation that eliminates sarcomere length non-uniformities and (ii) to evaluate if force depression is inhibited when single sarcomeres are activated with MgADP, which biases crossbridges into a strongly-bound state. Single sarcomeres (n = 16) were isolated from rabbit psoas myofibrils using two micro-needles (one compliant, one rigid), piercing the sarcomere externally adjacent to the Z-lines. The sarcomeres were contracted isometrically and subsequently shortened, in both Ca2+- and MgADP-activating solutions. Shortening in Ca2+-activated samples resulted in a 27.44 ± 9.04% force depression when compared to isometric contractions produced at similar final sarcomere lengths (P < 0.001). There was no force depression in MgADP-activated sarcomeres (force depression = −1.79 ± 9.69%, P =  0.435). These results suggest that force depression is a sarcomeric property, and that is associated with an inhibition of myosin-actin interactions. PMID:26037312

  9. An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells

    PubMed Central

    James, Dominic I.; Durant, Stephen; Eckersley, Kay; Fairweather, Emma; Griffiths, Louise A.; Hamilton, Nicola; Kelly, Paul; O'Connor, Mark; Shea, Kerry; Waddell, Ian D.; Ogilvie, Donald J.

    2016-01-01

    After a DNA damage signal multiple polymers of ADP ribose attached to poly(ADP) ribose (PAR) polymerases (PARPs) are broken down by the enzyme poly(ADP) ribose glycohydrolase (PARG). Inhibition of PARG leads to a failure of DNA repair and small molecule inhibition of PARG has been a goal for many years. To determine whether biochemical inhibitors of PARG are active in cells we have designed an immunofluorescence assay to detect nuclear PAR after DNA damage. This 384-well assay is suitable for medium throughput high-content screening and can detect cell-permeable inhibitors of PARG from nM to µM potency. In addition, the assay has been shown to work in murine cells and in a variety of human cancer cells. Furthermore, the assay is suitable for detecting the DNA damage response induced by treatment with temozolomide and methylmethane sulfonate (MMS). Lastly, the assay has been shown to be robust over a period of several years.

  10. Impact of non-inhibited platelet supplementation on platelet reactivity in patients treated with prasugrel or ticagrelor for an acute coronary syndrome: An ex vivo study.

    PubMed

    Bonhomme, Fanny; Bonvini, Robert; Reny, Jean-Luc; Poncet, Antoine; Fontana, Pierre

    2015-01-01

    Managing bleeding in patients receiving P2Y12 inhibitors is challenging. Few data are available regarding the efficacy of platelet transfusion in patients treated with prasugrel or ticagrelor. The aim of this study was to evaluate the minimal amount of platelet supplementation (in terms of ratio of non-inhibited platelets to inhibited platelets) necessary to restore platelet reactivity in platelet-rich plasma (PRP) of patients treated with aspirin and a prasugrel or ticagrelor loading dose for an acute coronary syndrome. PRP samples from patients were mixed ex vivo with increasing proportions of pooled PRP from healthy volunteers. Platelet reactivity was challenged with adenosine diphosphate (ADP), arachidonic acid, collagen or thrombin receptor activating peptide using light transmission aggregometry. The primary endpoint was the proportion of patient samples recovering an ADP-induced maximal aggregation (ADP-Aggmax) value above 40%. In patients treated with prasugrel (n = 32), ADP-Aggmax increased progressively with supplements of pooled PRP, with an average increase of 7.9% (95% CI [7.1; 8.8], p < 0.001) per each 20% increase in the ratio of non-inhibited platelets to inhibited platelets. A ratio of 60% was associated with 90% of patients reaching the primary endpoint. In patients treated with ticagrelor (n = 15), ADP-Aggmax did not significantly increase with any level of supplements. In conclusions, ex vivo addition of non-inhibited platelets significantly improved ADP-Aggmax in patients treated with prasugrel with a dose-dependent effect. There was no evidence of such a reversal in patients treated with ticagrelor. These results suggest that platelet transfusion may be more effective in blunting bleeding in patients treated with prasugrel, than those treated with ticagrelor. PMID:25905916

  11. Structure and properties of Al-MIL-53-ADP, a breathing MOF based on the aliphatic linker molecule adipic acid.

    PubMed

    Reinsch, Helge; Pillai, Renjith S; Siegel, Renée; Senker, Jürgen; Lieb, Alexandra; Maurin, Guillaume; Stock, Norbert

    2016-03-14

    The new aluminium based metal-organic framework [Al(OH)(O2C-C4H8-CO2)]·H2O denoted as Al-MIL-53-ADP-lp (lp stands for large pore) was synthesised under solvothermal conditions. This solid is an analogue of the archetypical aluminium terephthalate Al-MIL-53 based on the aliphatic single-chain linker molecule adipic acid (H2ADP, hexanedioic acid). In contrast to its aromatic counterparts, Al-MIL-53-ADP exhibits a structural breathing behaviour solely upon dehydration/rehydration. The crystal structure of the anhydrous compound denoted as Al-MIL-53-ADP-np (np stands for narrow pore) was determined by a combination of forcefield-based computations and Rietveld refinement of the powder X-ray diffraction data while the structure of the hydrated form Al-MIL-53-ADP-lp was derived computationally by a combination of force field based methods and Density Functional Theory calculations. Both structures were further supported by (1)H, (13)C and (27)Al high-resolution NMR MAS 1D data coupled again with simulations. Al-MIL-53-ADP was further characterised by means of vibrational spectroscopy, elemental analysis, thermogravimetry and water vapour sorption. PMID:26498663

  12. The XRCC1 phosphate-binding pocket binds poly (ADP-ribose) and is required for XRCC1 function

    PubMed Central

    Breslin, Claire; Hornyak, Peter; Ridley, Andrew; Rulten, Stuart L.; Hanzlikova, Hana; Oliver, Antony W.; Caldecott, Keith W.

    2015-01-01

    Poly (ADP-ribose) is synthesized at DNA single-strand breaks and can promote the recruitment of the scaffold protein, XRCC1. However, the mechanism and importance of this process has been challenged. To address this issue, we have characterized the mechanism of poly (ADP-ribose) binding by XRCC1 and examined its importance for XRCC1 function. We show that the phosphate-binding pocket in the central BRCT1 domain of XRCC1 is required for selective binding to poly (ADP-ribose) at low levels of ADP-ribosylation, and promotes interaction with cellular PARP1. We also show that the phosphate-binding pocket is required for EGFP-XRCC1 accumulation at DNA damage induced by UVA laser, H2O2, and at sites of sub-nuclear PCNA foci, suggesting that poly (ADP-ribose) promotes XRCC1 recruitment both at single-strand breaks globally across the genome and at sites of DNA replication stress. Finally, we show that the phosphate-binding pocket is required following DNA damage for XRCC1-dependent acceleration of DNA single-strand break repair, DNA base excision repair, and cell survival. These data support the hypothesis that poly (ADP-ribose) synthesis promotes XRCC1 recruitment at DNA damage sites and is important for XRCC1 function. PMID:26130715

  13. The XRCC1 phosphate-binding pocket binds poly (ADP-ribose) and is required for XRCC1 function.

    PubMed

    Breslin, Claire; Hornyak, Peter; Ridley, Andrew; Rulten, Stuart L; Hanzlikova, Hana; Oliver, Antony W; Caldecott, Keith W

    2015-08-18

    Poly (ADP-ribose) is synthesized at DNA single-strand breaks and can promote the recruitment of the scaffold protein, XRCC1. However, the mechanism and importance of this process has been challenged. To address this issue, we have characterized the mechanism of poly (ADP-ribose) binding by XRCC1 and examined its importance for XRCC1 function. We show that the phosphate-binding pocket in the central BRCT1 domain of XRCC1 is required for selective binding to poly (ADP-ribose) at low levels of ADP-ribosylation, and promotes interaction with cellular PARP1. We also show that the phosphate-binding pocket is required for EGFP-XRCC1 accumulation at DNA damage induced by UVA laser, H2O2, and at sites of sub-nuclear PCNA foci, suggesting that poly (ADP-ribose) promotes XRCC1 recruitment both at single-strand breaks globally across the genome and at sites of DNA replication stress. Finally, we show that the phosphate-binding pocket is required following DNA damage for XRCC1-dependent acceleration of DNA single-strand break repair, DNA base excision repair, and cell survival. These data support the hypothesis that poly (ADP-ribose) synthesis promotes XRCC1 recruitment at DNA damage sites and is important for XRCC1 function. PMID:26130715

  14. Imaging energy status in live cells with a fluorescent biosensor of the intracellular ATP-to-ADP ratio.

    PubMed

    Tantama, Mathew; Martínez-François, Juan Ramón; Mongeon, Rebecca; Yellen, Gary

    2013-01-01

    The ATP:ADP ratio is a critical parameter of cellular energy status that regulates many metabolic activities. Here we report an optimized genetically encoded fluorescent biosensor, PercevalHR, that senses the ATP:ADP ratio. PercevalHR is tuned to the range of intracellular ATP:ADP expected in mammalian cells, and it can be used with one- or two-photon microscopy in live samples. We use PercevalHR to visualize activity-dependent changes in ATP:ADP when neurons are exposed to multiple stimuli, demonstrating that it is a sensitive reporter of physiological changes in energy consumption and production. We also use PercevalHR to visualize intracellular ATP:ADP while simultaneously recording currents from ATP-sensitive potassium (KATP) channels in single cells, showing that PercevalHR enables the study of coordinated variation in ATP:ADP and KATP channel open probability in intact cells. With its ability to monitor changes in cellular energetics within seconds, PercevalHR should be a versatile tool for metabolic research.

  15. Comparison of the luminescent ADP-Glo assay to a standard radiometric assay for measurement of protein kinase activity.

    PubMed

    Sanghera, Jasbinder; Li, Rick; Yan, Jun

    2009-12-01

    Many assay technologies have been developed and utilized to efficiently assay and screen against protein kinase targets. The radiometric assay format for assaying the protein kinase targets has been considered the "Gold Standard" format since it allows the direct readout of kinase functional activity and is a universal assay that is highly sensitive. However, the hazardous nature of the radiometric assay together with the regulatory hurdles has led to the development of alternative assay formats for assessing protein kinase activity measurements. The luminescent ADP-Glo assay has been developed as an alternative to radiometric format for assaying protein kinase targets. This assay allows the measurement of the ADP product formed during the kinase reaction. Therefore, the luminescent ADP-Glo assay is similar to the radiometric format in that it measures the direct product of the protein kinase reaction. Furthermore, since the ADP product is generated by all protein kinase reactions, this is a universal format that can be used for assaying any given protein kinase target. Analysis of data generated with multiple protein kinase targets and the luminescent ADP-Glo technology shows comparable results to the radiometric assay format. Therefore, the luminescent ADP-Glo assay is a robust new technology for evaluating catalytic function of protein kinases as well as other ATPases.

  16. 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

  17. Mechanical study of rat soleus muscle using caged ATP and X-ray diffraction: high ADP affinity of slow cross-bridges.

    PubMed Central

    Horiuti, K; Yagi, N; Takemori, S

    1997-01-01

    1. The cross-bridges in slow- and fast-twitch fibres (taken, respectively, from soleus and psoas muscles of rats) were examined in mechanical experiments using caged ATP and X-ray diffraction, to compare their binding of ATP and ADP. 2. Caged ATP was photolysed in rigor fibres. When ADP was removed from pre-photolysis fibres, the initial relaxation (+/- Ca2+) in soleus was as fast as that in psoas fibres, whereas the subsequent contraction (+Ca2+) was slower in soleus than in psoas. The ATPase rate during the steady-state contraction was also slower in soleus fibres. 3. When ADP was added to pre-photolysis fibres (+/- Ca2+), tension developed even in the initial phase, the overall tension development being biphasic. Both initial and late components of the Ca(2+)-free contraction were enhanced when ADP was added before photolysis, although pre-photolysis ADP was not a prerequisite for the late component. The effect of ADP was greater in soleus than in psoas fibres. Static experiments on rigor fibres revealed a higher ADP affinity in soleus fibres. 4. The intensity of the actin layer-line from ADP rigor soleus fibres decreased rapidly on photorelease of ATP. We conclude that, despite the tight ADP binding of the soleus cross-bridge, its isometric reaction is not rate limited by the 'off' rate of ADP. PMID:9263922

  18. Metabotropic glutamate receptor agonists potentiate a slow afterdepolarization in CNS neurons

    NASA Technical Reports Server (NTRS)

    Zheng, F.; Gallagher, J. P.

    1992-01-01

    We have previously reported that, in the rat dorsolateral septal nucleus (DLSN), metabotropic glutamate receptor (met-GluR) agonists evoked a slow depolarization accompanied by an increase in membrane conductance and burst firing. We have speculated that the burst firing elicited by met-GluR agonists may be due to activation or enhancement of a non-specific cation current, which exists in some DLSN neurons. Now we report that a slow afterdepolarization (sADP) mediated by a non-specific cation current was potentiated by both 1S,3R-ACPD and quisqualate. In addition, met-GluR agonists unmask a sADP in DLSN neurons which did not show a sADP under control conditions. Our data suggest that a non-specific cation current can be potentiated by activation of the met-GluR.

  19. Peroxynitrite-induced thymocyte apoptosis: the role of caspases and poly (ADP-ribose) synthetase (PARS) activation.

    PubMed Central

    Virág, L; Scott, G S; Cuzzocrea, S; Marmer, D; Salzman, A L; Szabó, C

    1998-01-01

    The mechanisms by which immature thymocyte apoptosis is induced during negative selection are poorly defined. Reports demonstrated that cross-linking of T-cell receptor leads to stromal cell activation, expression of inducible nitric oxide synthase (iNOS) and, subsequently, to thymocyte apoptosis. Therefore we examined, whether NO directly or indirectly, through peroxynitrite formation, causes thymocyte apoptosis. Immuno-histochemical detection of nitrotyrosine revealed in vivo peroxynitrite formation in the thymi of naive mice. Nitrotyrosine, the footprint of peroxynitrite, was predominantly found in the corticomedullary junction and the medulla of naive mice. In the thymi of mice deficient in the inducible isoform of nitric oxide synthase, considerably less nitrotyrosine was found. Exposure of thymocytes in vitro to low concentrations (10 microM) of peroxynitrite led to apoptosis, whereas higher concentrations (50 microM) resulted in intense cell death with the characteristics of necrosis. We also investigated the effect of poly (ADP-ribose) synthetase (PARS) inhibition on thymocyte apoptosis. Using the PARS inhibitor 3-aminobenzamide (3-AB), or thymocytes from PARS-deficient animals, we established that PARS determines the fate of thymocyte death. Suppression of cellular ATP levels, and the cellular necrosis in response to peroxynitrite were prevented by PARS inhibition. Therefore, in the absence of PARS, cells are diverted towards the pathway of apoptotic cell death. Similar results were obtained with H2O2 treatment, while apoptosis induced by non-oxidative stimuli such as dexamethasone or anti-FAS antibody was unaffected by PARS inhibition. In conclusion, we propose that peroxynitrite-induced apoptosis may play a role in the process of thymocyte negative selection. Furthermore, we propose that the physiological role of PARS cleavage by apopain during apoptosis may serve as an energy-conserving step, enabling the cell to complete the process of apoptosis

  20. The allosteric transition of GroEL induced by metal fluoride-ADP complexes.

    PubMed

    Inobe, Tomonao; Kikushima, Kenji; Makio, Tadashi; Arai, Munehito; Kuwajima, Kunihiro

    2003-05-23

    To understand the mechanism of a functionally important ATP-induced allosteric transition of GroEL, we have studied the effect of a series of metal fluoride-ADP complexes and vanadate-ADP on GroEL by kinetic fluorescence measurement of pyrene-labeled GroEL and by small-angle X-ray scattering measurement of wild-type GroEL. The metal fluorides and vanadate, complexed with ADP, are known to mimic the gamma-phosphate group of ATP, but they differ in geometry and size; it is expected that these compounds will be useful for investigating the strikingly high specificity of GroEL for ATP that enables the induction of the allosteric transition. The kinetic fluorescence measurement revealed that aluminium, beryllium, and gallium ions, when complexed with the fluoride ion and ADP, induced a biphasic fluorescence change of pyrenyl GroEL, while scandium and vanadate ions did not induce any kinetically observed change in fluorescence. The burst phase and the first phase of the fluorescence kinetics were reversible, while the second phase and subsequent changes were irreversible. The dependence of the burst-phase and the first-phase fluorescence changes on the ADP concentration indicated that the burst phase represents non-cooperative nucleotide binding to GroEL, and that the first phase represents the allosteric transition of GroEL. Both the amplitude and the rate constant of the first phase of the fluorescence kinetics were well understood in terms of a kinetic allosteric model, which is a combination of transition state theory and the Monod-Wyman-Changeux allosteric model. From the kinetic allosteric model analysis, the relative free energy of the transition state in the metal fluoride-ADP-induced allosteric transition of GroEL was found to be larger than the corresponding free energy of the ATP-induced allosteric transition by more than 5.5kcal/mol. However, the X-ray scattering measurements indicated that the allosteric state induced by these metal fluoride-ADP complexes is

  1. 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

  2. The molecular basis of the antiplatelet action of ajoene: direct interaction with the fibrinogen receptor.

    PubMed

    Apitz-Castro, R; Ledezma, E; Escalante, J; Jain, M K

    1986-11-26

    Ajoene, the major antiplatelet compound derived from garlic inhibits the fibrinogen-supported aggregation of washed human platelets (ID50 = 13 microM) and, inhibits binding of 125I-fibrinogen to ADP-stimulated platelets (ID50 = 0.8 microM). In both cases, the inhibition is of the mixed non-competitive type. Furthermore, fibrinogen-induced aggregation of chymotrypsin-treated platelets is also inhibited by ajoene in a dose-dependent manner (ID50 = 2.3 microM). Other membrane receptors such as ADP or epinephrine receptors are not affected by ajoene. Ajoene strongly quenches the intrinsic fluorescence emission of purified glycoproteins IIb-IIIa (ID50 = 10 microM). These results indicate that the antiaggregatory effect of ajoene is causally related to its direct interaction with the putative fibrinogen receptor.

  3. PARP2 Is the Predominant Poly(ADP-Ribose) Polymerase in Arabidopsis DNA Damage and Immune Responses

    PubMed Central

    Song, Junqi; Keppler, Brian D.; Wise, Robert R.; Bent, Andrew F.

    2015-01-01

    Poly (ADP-ribose) polymerases (PARPs) catalyze the transfer of multiple poly(ADP-ribose) units onto target proteins. Poly(ADP-ribosyl)ation plays a crucial role in a variety of cellular processes including, most prominently, auto-activation of PARP at sites of DNA breaks to activate DNA repair processes. In humans, PARP1 (the founding and most characterized member of the PARP family) accounts for more than 90% of overall cellular PARP activity in response to DNA damage. We have found that, in contrast with animals, in Arabidopsis thaliana PARP2 (At4g02390), rather than PARP1 (At2g31320), makes the greatest contribution to PARP activity and organismal viability in response to genotoxic stresses caused by bleomycin, mitomycin C or gamma-radiation. Plant PARP2 proteins carry SAP DNA binding motifs rather than the zinc finger domains common in plant and animal PARP1 proteins. PARP2 also makes stronger contributions than PARP1 to plant immune responses including restriction of pathogenic Pseudomonas syringae pv. tomato growth and reduction of infection-associated DNA double-strand break abundance. For poly(ADP-ribose) glycohydrolase (PARG) enzymes, we find that Arabidopsis PARG1 and not PARG2 is the major contributor to poly(ADP-ribose) removal from acceptor proteins. The activity or abundance of PARP2 is influenced by PARP1 and PARG1. PARP2 and PARP1 physically interact with each other, and with PARG1 and PARG2, suggesting relatively direct regulatory interactions among these mediators of the balance of poly(ADP-ribosyl)ation. As with plant PARP2, plant PARG proteins are also structurally distinct from their animal counterparts. Hence core aspects of plant poly(ADP-ribosyl)ation are mediated by substantially different enzymes than in animals, suggesting the likelihood of substantial differences in regulation. PMID:25950582

  4. Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: unravelling the role of Mg2+ in cell respiration.

    PubMed

    Gout, Elisabeth; Rébeillé, Fabrice; Douce, Roland; Bligny, Richard

    2014-10-28

    In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg(2+) concentrations must be considered as well. Here we developed in vivo/in vitro techniques using (31)P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg(2+) concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg(2+) in the mitochondrial matrix, where [Mg(2+)] is tenfold higher. In contrast, owing to a much higher affinity for Mg(2+), ATP is mostly complexed by Mg(2+) in both compartments. Mg(2+) starvation used to alter cytosolic and mitochondrial [Mg(2+)] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg(2+) concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis. PMID:25313036

  5. Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: Unravelling the role of Mg2+ in cell respiration

    PubMed Central

    Gout, Elisabeth; Rébeillé, Fabrice; Douce, Roland; Bligny, Richard

    2014-01-01

    In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg2+ concentrations must be considered as well. Here we developed in vivo/in vitro techniques using 31P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg2+ concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg2+ in the mitochondrial matrix, where [Mg2+] is tenfold higher. In contrast, owing to a much higher affinity for Mg2+, ATP is mostly complexed by Mg2+ in both compartments. Mg2+ starvation used to alter cytosolic and mitochondrial [Mg2+] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg2+ concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis. PMID:25313036

  6. Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: unravelling the role of Mg2+ in cell respiration.

    PubMed

    Gout, Elisabeth; Rébeillé, Fabrice; Douce, Roland; Bligny, Richard

    2014-10-28

    In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg(2+) concentrations must be considered as well. Here we developed in vivo/in vitro techniques using (31)P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg(2+) concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg(2+) in the mitochondrial matrix, where [Mg(2+)] is tenfold higher. In contrast, owing to a much higher affinity for Mg(2+), ATP is mostly complexed by Mg(2+) in both compartments. Mg(2+) starvation used to alter cytosolic and mitochondrial [Mg(2+)] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg(2+) concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis.

  7. 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

  8. Expression of ADP-ribosyltransferase 1 Is Associated with Poor Prognosis of Glioma Patients.

    PubMed

    Li, Zhen; Yan, Xinling; Sun, Yuyan; Yang, Xiaoqing

    2016-01-01

    Glioma has a poor prognosis due to its rapid overgrowth, diffuse invasion, and chemotherapy resistance. The improvements in clinical outcome are still limited and the identification of novel biomarkers involved in the progression of gliomas is still under critical demands. Amino acid ADP-ribosyltransferase 1 (ART1) is an enzyme that catalyzes the mono-ADP-ribosylation, a reversible post-translational modification. For example, the mono-ADP-ribosylation of transcription factors can affect their binding to target gene promoters. However, the functional significance of ART1 in glioma has not been reported. We collected 107 glioma cases from Qianfoshan Hospital and Yidu Central Hospital of Weifang between April 2008 and September 2015 to analyze the prognosis value of ART1 in gliomas. RT-qPCR analysis showed that the expression level of ART1 mRNA in glioma tissues was 4-fold higher than that in normal brain tissues. According to the immunohistochemical staining results, 44 patients (41.1%) were categorized as ART1 positive (≥ 20% of stained glioma cells), while the other 63 patients (58.9%) categorized as ART1 negative (< 20% of stained glioma cells). Moreover, the mean percentage of ART1-positive cells was 43.7%, 53.6% and 64.2% in WHO grade II, III and IV specimens, respectively. Through univariate and multivariate analyses, we identified ART1 as an independent prognostic factor. We also found that ART1 overexpression in U251 glioblastoma cells could significantly decrease the susceptibility to vincristine, one of tubulin-targeted drugs, which is widely used in clinical treatment for glioma. Taken together, we propose that up-regulation of ART1 expression is associated with the aggressiveness of glioma. PMID:27466078

  9. Poly(ADP-ribose) polymerase regulates glycolytic activity in kidney proximal tubule epithelial cells

    PubMed Central

    Song, Hana; Yoon, Sang Pil

    2016-01-01

    After renal injury, selective damage occurs in the proximal tubules as a result of inhibition of glycolysis. The molecular mechanism of damage is not known. Poly(ADP-ribose) polymerase (PARP) activation plays a critical role of proximal tubular cell death in several renal disorders. Here, we studied the role of PARP on glycolytic flux in pig kidney proximal tubule epithelial LLC-PK1 cells using XFp extracellular flux analysis. Poly(ADP-ribosyl)ation by PARP activation was increased approximately 2-fold by incubation of the cells in 10 mM glucose for 30 minutes, but treatment with the PARP inhibitor 3-aminobenzamide (3-AB) does-dependently prevented the glucose-induced PARP activation (approximately 14.4% decrease in 0.1 mM 3-AB–treated group and 36.7% decrease in 1 mM 3-AB–treated group). Treatment with 1 mM 3-AB significantly enhanced the glucose-mediated increase in the extracellular acidification rate (61.1±4.3 mpH/min vs. 126.8±6.2 mpH/min or approximately 2-fold) compared with treatment with vehicle, indicating that PARP inhibition increases only glycolytic activity during glycolytic flux including basal glycolysis, glycolytic activity, and glycolytic capacity in kidney proximal tubule epithelial cells. Glucose increased the activities of glycolytic enzymes including hexokinase, phosphoglucose isomerase, phosphofructokinase-1, glyceraldehyde-3-phosphate dehydrogenase, enolase, and pyruvate kinase in LLC-PK1 cells. Furthermore, PARP inhibition selectively augmented the activities of hexokinase (approximately 1.4-fold over vehicle group), phosphofructokinase-1 (approximately 1.6-fold over vehicle group), and glyceraldehyde-3-phosphate dehydrogenase (approximately 2.2-fold over vehicle group). In conclusion, these data suggest that PARP activation may regulate glycolytic activity via poly(ADP-ribosyl)ation of hexokinase, phosphofructokinase-1, and glyceraldehyde-3-phosphate dehydrogenase in kidney proximal tubule epithelial cells. PMID:27382509

  10. Multiscale prediction of patient-specific platelet function under flow.

    PubMed

    Flamm, Matthew H; Colace, Thomas V; Chatterjee, Manash S; Jing, Huiyan; Zhou, Songtao; Jaeger, Daniel; Brass, Lawrence F; Sinno, Talid; Diamond, Scott L

    2012-07-01

    During thrombotic or hemostatic episodes, platelets bind collagen and release ADP and thromboxane A(2), recruiting additional platelets to a growing deposit that distorts the flow field. Prediction of clotting function under hemodynamic conditions for a patient's platelet phenotype remains a challenge. A platelet signaling phenotype was obtained for 3 healthy donors using pairwise agonist scanning, in which calcium dye-loaded platelets were exposed to pairwise combinations of ADP, U46619, and convulxin to activate the P2Y(1)/P2Y(12), TP, and GPVI receptors, respectively, with and without the prostacyclin receptor agonist iloprost. A neural network model was trained on each donor's pairwise agonist scanning experiment and then embedded into a multiscale Monte Carlo simulation of donor-specific platelet deposition under flow. The simulations were compared directly with microfluidic experiments of whole blood flowing over collagen at 200 and 1000/s wall shear rate. The simulations predicted the ranked order of drug sensitivity for indomethacin, aspirin, MRS-2179 (a P2Y(1) inhibitor), and iloprost. Consistent with measurement and simulation, one donor displayed larger clots and another presented with indomethacin resistance (revealing a novel heterozygote TP-V241G mutation). In silico representations of a subject's platelet phenotype allowed prediction of blood function under flow, essential for identifying patient-specific risks, drug responses, and novel genotypes.

  11. 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

  12. Dynamics and Control of Orbiting Space Structures NASA Advanced Design Program (ADP)

    NASA Technical Reports Server (NTRS)

    Cruse, T. A.

    1996-01-01

    The report summarizes the advanced design program in the mechanical engineering department at Vanderbilt University for the academic years 1994-1995 and 1995-1996. Approximately 100 students participated in the two years of the subject grant funding. The NASA-oriented design projects that were selected included lightweight hydrogen propellant tank for the reusable launch vehicle, a thermal barrier coating test facility, a piezoelectric motor for space antenna control, and a lightweight satellite for automated materials processing. The NASA supported advanced design program (ADP) has been a success and a number of graduates are working in aerospace and are doing design.

  13. Growth and characterization of ADP single crystals doped with alkali and alkaline metal ions

    NASA Astrophysics Data System (ADS)

    Kavya, H.; Bhavyashree, M.; Kumari, R. Ananda

    2016-05-01

    Pure and KBr, KI, MgCl2 & LiCl added ammonium dihydrogen orthophosphate (ADP) single crystals have been grown at room temperature by the slow evaporation method. The grown crystals have been subjected to powder XRD, FTIR, UV-Vis, and SHG studies. The crystallinity and the functional groups are confirmed by powder XRD and FTIR spectroscopy. Good transparency in the entire visible region which is an essential requirement for a nonlinear optical crystal is observed for the grown crystals. Results of the non-linear optical measurements indicate the enhancement of second harmonic generation efficiency due to the dopants and show the suitability of the ingot for nonlinear optical application

  14. Bookmarking promoters in mitotic chromatin: poly(ADP-ribose)polymerase-1 as an epigenetic mark

    PubMed Central

    Lodhi, Niraj; Kossenkov, Andrew V.; Tulin, Alexei V.

    2014-01-01

    Epigenetics are the heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence. After mitosis, it is thought that bookmarking transcription factors remain at promoters, regulating which genes become active and which remain silent. Herein, we demonstrate that poly(ADP-ribose)polymerase-1 (PARP-1) is a genome-wide epigenetic memory mark in mitotic chromatin, and we further show that the presence of PARP-1 is absolutely crucial for reactivation of transcription after mitosis. Based on these findings, a novel molecular model of epigenetic memory transmission through the cell cycle is proposed. PMID:24861619

  15. Modeling regulation of cardiac KATP and L-type Ca2+ currents by ATP, ADP, and Mg2+

    NASA Technical Reports Server (NTRS)

    Michailova, Anushka; Saucerman, Jeffrey; Belik, Mary Ellen; McCulloch, Andrew D.

    2005-01-01

    Changes in cytosolic free Mg(2+) and adenosine nucleotide phosphates affect cardiac excitability and contractility. To investigate how modulation by Mg(2+), ATP, and ADP of K(ATP) and L-type Ca(2+) channels influences excitation-contraction coupling, we incorporated equations for intracellular ATP and MgADP regulation of the K(ATP) current and MgATP regulation of the L-type Ca(2+) current in an ionic-metabolic model of the canine ventricular myocyte. The new model: 1), quantitatively reproduces a dose-response relationship for the effects of changes in ATP on K(ATP) current, 2), simulates effects of ADP in modulating ATP sensitivity of K(ATP) channel, 3), predicts activation of Ca(2+) current during rapid increase in MgATP, and 4), demonstrates that decreased ATP/ADP ratio with normal total Mg(2+) or increased free Mg(2+) with normal ATP and ADP activate K(ATP) current, shorten action potential, and alter ionic currents and intracellular Ca(2+) signals. The model predictions are in agreement with experimental data measured under normal and a variety of pathological conditions.

  16. 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

  17. Stronger control of ATP/ADP by proton leak in pancreatic β-cells than skeletal muscle mitochondria

    PubMed Central

    Affourtit, Charles; Brand, Martin D.

    2005-01-01

    Pancreatic beta cells respond to rising blood glucose concentrations by increasing their oxidative metabolism, which leads to an increased ATP/ADP ratio, closure of KATP channels, depolarization of the plasma membrane potential, influx of calcium and the eventual secretion of insulin. Such a signalling mechanism implies that the ATP/ADP ratio is flexible in beta cells (β-cells), which is in contrast with other cell types (e.g. muscle and liver) that maintain a stable ATP/ADP poise while respiring at widely varying rates. To determine whether this difference in flexibility is accounted for by mitochondrial peculiarities, we performed a top-down metabolic control analysis to quantitatively assess how ATP/ADP is controlled in mitochondria isolated from rat skeletal muscle and cultured beta cells. We show that the ATP/ADP ratio is more strongly controlled (approx. 7.5-fold) by proton leak in beta cells than in muscle. The comparatively high importance of proton leak in beta cell mitochondria (relative to phosphorylation) is evidenced furthermore by its relatively high level of control over membrane potential and overall respiratory activity. Modular-kinetic analysis of oxidative phosphorylation reveals that these control differences can be fully explained by a higher relative leak activity in beta cell mitochondria, which results in a comparatively high contribution of proton leak to the overall respiratory activity in this system. PMID:16137248

  18. Overexpression, purification, and partial characterization of ADP-ribosyltransferases modA and modB of bacteriophage T4.

    PubMed

    Tiemann, B; Depping, R; Rüger, W

    1999-01-01

    There is increasing experimental evidence that ADP-ribosylation of host proteins is an important means to regulate gene expression of bacteriophage T4. Surprisingly, this phage codes for three different ADP-ribosyltransferases, gene products Alt, ModA, and ModB, modifying partially overlapping sets of host proteins. While gene product Alt already has been isolated as a recombinant protein and its action on host RNA polymerases and transcription regulation have been studied, the nucleotide sequences of the two mod genes was published only recently. Their mode of action in the course of the infection cycle and the consequences of the ADP-ribosylations catalyzed by these enzymes remain to be investigated. Here we describe the cloning of the genes, the overexpression, purification, and partial characterization of ADP-ribosyltransferases ModA and ModB. Both proteins seem to act independently, and the ADP-ribosyl moieties are transferred to different sets of host proteins. While gene product ModA, similarly to the Alt protein, acts also on the alpha-subunit of host RNA polymerase, the ModB activity serves another set of proteins, one of which was identified as the S1 protein associated with the 30S subunit of the E. coli ribosomes.

  19. Inhibitors of poly(ADP-ribose) synthesis enhance X-ray killing of log-phase Chinese hamster cells

    SciTech Connect

    Ben-Hur, E.; Utsumi, H.; Elkind, M.M.

    1984-03-01

    Postirradiation incubation of V79 Chinese hamster cells with inhibitors of poly(ADP-ribose) synthesis was found to potentiate the killing of cells by X rays. Potentiation increased with incubation time and with concentration of the inhibitor. Preirradiation incubation had only a small effect. The enhanced response correlated well with the known extent of the inhibition of poly(ADP-ribose) synthesis. A radiation-sensitive line, V79-AL162/S-10, was affected to a lesser extent than the normal cells. Cells repaired the radiation damage with which the inhibitors interacted within 1 hr, a process that has similar kinetics to what is observed when a postirradiation treatment with hypertonic buffer is used. However, the sectors of damage affected by inhibitors of poly(ADP-ribose) synthesis and hypertonic buffer do not entirely overlap. The inhibitor nicotinamide enhanced the killing mainly of late S-phase cells and did not affect cells at the G/sub 1//S border. It is concluded that the repair process(es) involving poly(ADP-ribose) synthesis is important for cell survival in repair-competent cells and that the radiation-sensitive cells that were examined are partially deficient in a repair pathway in which poly(ADP-ribose) participates.

  20. Molybdenum nitrogenase of Azotobacter chroococcum. Tight binding of MgADP to the MoFe protein.

    PubMed

    Miller, R W; Eady, R R

    1989-11-01

    The dye-oxidized or dithionite-reduced forms of the MoFe protein of molybdenum nitrogenase of Azotobacter chroococcum were shown to bind 2 mol of MgADP/mol of protein, as determined by column equilibrium techniques. The gel-filtration elution profile of unbound Mg[14C]ADP was not symmetrical, consistent with a low rate of dissociation from the protein. Symmetrical elution profiles were observed for the oxidized Fe protein of nitrogenase, which bound 2 mol of MgADP/mol of protein. The low rate of dissociation of MgADP from MoFe protein was shown by non-equilibrium column techniques, where 1 mol of MgADP/mol of MoFe protein remained tightly bound during chromatography. Very weak binding of MgATP (less than 0.01 mol of MgATP/mol of MoFe protein) to dye-oxidized but not to dithionite-reduced MoFe protein was observed. These results are discussed in terms of their relevance to the catalytic cycle of nitrogenase catalysis.

  1. Inhibition of poly(ADP-ribose) polymerase interferes with Trypanosoma cruzi infection and proliferation of the parasite.

    PubMed

    Vilchez Larrea, Salomé C; Haikarainen, Teemu; Narwal, Mohit; Schlesinger, Mariana; Venkannagari, Harikanth; Flawiá, Mirtha M; Villamil, Silvia H Fernández; Lehtiö, Lari

    2012-01-01

    Poly(ADP-ribosylation) is a post-translational covalent modification of proteins catalyzed by a family of enzymes termed poly(ADP-ribose) polymerases (PARPs). In the human genome, 17 different genes have been identified that encode members of the PARP superfamily. Poly (ADP-ribose) metabolism plays a role in a wide range of biological processes. In Trypanosoma cruzi, PARP enzyme appears to play a role in DNA repair mechanisms and may also be involved in controlling the different phases of cell growth. Here we describe the identification of potent inhibitors for T. cruzi PARP with a fluorescence-based activity assay. The inhibitors were also tested on T. cruzi epimastigotes, showing that they reduced ADP-ribose polymer formation in vivo. Notably, the identified inhibitors are able to reduce the growth rate of T. cruzi epimastigotes. The best inhibitor, Olaparib, is effective at nanomolar concentrations, making it an efficient chemical tool for chacterization of ADP-ribose metabolism in T. cruzi. PARP inhibition also decreases drastically the amount of amastigotes but interestingly has no effect on the amount of trypomastigotes in the cell culture. Knocking down human PARP-1 decreases both the amount of amastigotes and trypomastigotes in cell culture, indicating that the effect would be mainly due to inhibition of human PARP-1. The result suggests that the inhibition of PARP could be a potential way to interfere with T. cruzi infection. PMID:23049934

  2. Inhibition of poly(ADP-ribose) Polymerase Interferes with Trypanosoma cruzi Infection and Proliferation of the Parasite

    PubMed Central

    Vilchez Larrea, Salomé C.; Haikarainen, Teemu; Narwal, Mohit; Schlesinger, Mariana; Venkannagari, Harikanth; Flawiá, Mirtha M.; Villamil, Silvia H. Fernández; Lehtiö, Lari

    2012-01-01

    Poly(ADP-ribosylation) is a post-translational covalent modification of proteins catalyzed by a family of enzymes termed poly(ADP-ribose) polymerases (PARPs). In the human genome, 17 different genes have been identified that encode members of the PARP superfamily. Poly (ADP-ribose) metabolism plays a role in a wide range of biological processes. In Trypanosoma cruzi, PARP enzyme appears to play a role in DNA repair mechanisms and may also be involved in controlling the different phases of cell growth. Here we describe the identification of potent inhibitors for T. cruzi PARP with a fluorescence-based activity assay. The inhibitors were also tested on T. cruzi epimastigotes, showing that they reduced ADP-ribose polymer formation in vivo. Notably, the identified inhibitors are able to reduce the growth rate of T. cruzi epimastigotes. The best inhibitor, Olaparib, is effective at nanomolar concentrations, making it an efficient chemical tool for chacterization of ADP-ribose metabolism in T. cruzi. PARP inhibition also decreases drastically the amount of amastigotes but interestingly has no effect on the amount of trypomastigotes in the cell culture. Knocking down human PARP-1 decreases both the amount of amastigotes and trypomastigotes in cell culture, indicating that the effect would be mainly due to inhibition of human PARP-1. The result suggests that the inhibition of PARP could be a potential way to interfere with T. cruzi infection. PMID:23049934

  3. Modeling regulation of cardiac KATP and L-type Ca2+ currents by ATP, ADP, and Mg2+.

    PubMed

    Michailova, Anushka; Saucerman, Jeffrey; Belik, Mary Ellen; McCulloch, Andrew D

    2005-03-01

    Changes in cytosolic free Mg(2+) and adenosine nucleotide phosphates affect cardiac excitability and contractility. To investigate how modulation by Mg(2+), ATP, and ADP of K(ATP) and L-type Ca(2+) channels influences excitation-contraction coupling, we incorporated equations for intracellular ATP and MgADP regulation of the K(ATP) current and MgATP regulation of the L-type Ca(2+) current in an ionic-metabolic model of the canine ventricular myocyte. The new model: 1), quantitatively reproduces a dose-response relationship for the effects of changes in ATP on K(ATP) current, 2), simulates effects of ADP in modulating ATP sensitivity of K(ATP) channel, 3), predicts activation of Ca(2+) current during rapid increase in MgATP, and 4), demonstrates that decreased ATP/ADP ratio with normal total Mg(2+) or increased free Mg(2+) with normal ATP and ADP activate K(ATP) current, shorten action potential, and alter ionic currents and intracellular Ca(2+) signals. The model predictions are in agreement with experimental data measured under normal and a variety of pathological conditions. PMID:15738467

  4. 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

  5. Coexistence of ferroelectric and antiferroelectric microregions in the paraelectric phase of NH 4H 2PO 4 (ADP)

    NASA Astrophysics Data System (ADS)

    Lasave, J.; Koval, S.; Migoni, R. L.

    2009-10-01

    Ammonium dihydrogen phosphate NH 4H 2PO 4 (ADP) is an antiferroelectric (AFE) compound belonging to the KDP-type family of hydrogen-bonded ferroelectrics. Recent ab initio results have shown that the optimization of the N-H-O bridges in ADP leads to the stabilization of the AFE state over a FE one. However, electron spin probe measurements have suggested that microregions of both phases may coexist above the critical antiferroelectric-paraelectric transition temperature. We have performed first principles studies of the energetics and relative stability of different AFE and FE defects embedded in a paraelectric (PE) matrix of ADP. Our analysis indicates that FE and AFE clusters are stable and may coexist in the PE phase, thus confirming the above suggestion.

  6. By Releasing ADP, Acanthamoeba castellanii Causes an Increase in the Cytosolic Free Calcium Concentration and Apoptosis in Wish Cells

    PubMed Central

    Mattana, A.; Tozzi, M. G.; Costa, M.; Delogu, G.; Fiori, P. L.; Cappuccinelli, P.

    2001-01-01

    The role played by soluble molecules that may participate in acanthamoebal cytopathogenicity has yet to be fully characterized. We demonstrate here that Acanthamoeba castellanii trophozoites constitutively release ADP in the medium. Cell-free supernatants prepared from A. castellanii, by interaction with specific P2y2 purinoceptors expressed on the Wish cell membrane, caused a biphasic rise in [Ca2+]i, extensive cell membrane blebbing, cytoskeletal disorganization, and the breakdown of nuclei. Cell damage induced by amoebic supernatants was blocked by the P2y2 inhibitor Suramin. The same results were found in Wish cells exposed to purified ADP. These findings suggest that pathogenic free-living A. castellanii may have a cytopathic effect on human epithelial cells through ADP release, by a process that begins with a rise of cytosolic free-calcium concentration, and culminates in apoptosis. PMID:11349088

  7. Exchange of glutamine-217 to glutamate of Clostridium limosum exoenzyme C3 turns the asparagine-specific ADP-ribosyltransferase into an arginine-modifying enzyme.

    PubMed

    Vogelsgesang, Martin; Aktories, Klaus

    2006-01-24

    C3-like ADP-ribosyltransferaseses are produced by Clostridium species, Bacillus cereus, and various Staphylococcus aureus strains. The exoenzymes modify the low-molecular-mass GTPases RhoA, B, and C. In structural studies of C3-like exoenzymes, an ARTT-motif (ADP-ribosylating turn-turn motif) was identified that appears to be involved in substrate specificity and recognition (Han, S., Arvai, A. S., Clancy, S. B., Tainer, J. A. (2001) J. Mol. Biol. 305, 95-107). Exchange of Gln217, which is a key residue of the ARTT-motif, to Glu in C3 from Clostridium limosum results in inhibition of ADP-ribosyltransferase activity toward RhoA. The mutant protein is still capable of NAD-binding and possesses NAD+ glycohydrolase activity. Whereas recombinant wild-type C3 modifies Rho proteins specifically at an asparagine residue (Asn41), Gln217Glu-C3 is capable of ADP-ribosylation of poly-arginine but not poly-asparagine. Soybean trypsin inhibitor, a model substrate for many arginine-specific ADP-ribosyltransferases, is modified by the Gln217Glu-C3 transferase. Also in C3 ADP-ribosyltransferases from Clostridium botulinum and B. cereus, the exchange of the equivalent Gln residue to Glu blocked asparagine modification of RhoA but elicited arginine-specific ADP-ribosylation. Moreover, the Gln217Glu-C3lim transferase was able to ADP-ribosylate recombinant wild-type C3lim at Arg86, resulting in decrease in ADP-ribosyltransferase activity of the wild-type enzyme. The data indicate that the exchange of one amino acid residue in the ARTT-motif turns the asparagine-modifying ADP-ribosyltransferases of the C3 family into arginine-ADP-ribosylating transferases.

  8. Two small enzyme isoforms mediate mammalian mitochondrial poly(ADP-ribose) glycohydrolase (PARG) activity

    SciTech Connect

    Meyer, Ralph G. . E-mail: meyerg@vet.upenn.edu; Meyer-Ficca, Mirella L.; Whatcott, Clifford J.; Jacobson, Elaine L.; Jacobson, Myron K.

    2007-08-01

    Poly(ADP-ribose)glycohydrolase (PARG) is the major enzyme capable of rapidly hydrolyzing poly(ADP-ribose) (PAR) formed by the diverse members of the PARP enzyme family. This study presents an alternative splice mechanism by which two novel PARG protein isoforms of 60 kDa and 55 kDa are expressed from the human PARG gene, termed hPARG60 and hPARG55, respectively. Homologous forms were found in the mouse (mPARG63 and mPARG58) supporting the hypothesis that expression of small PARG isoforms is conserved among mammals. A PARG protein of {approx} 60 kDa has been described for decades but with its genetic basis unknown, it was hypothesized to be a product of posttranslational cleavage of larger PARG isoforms. While this is not excluded entirely, isolation and expression of cDNA clones from different sources of RNA indicate that alternative splicing leads to expression of a catalytically active hPARG60 in multiple cell compartments. A second enzyme, hPARG55, that can be expressed through alternative translation initiation from hPARG60 transcripts is strictly targeted to the mitochondria. Functional studies of a mitochondrial targeting signal (MTS) in PARG exon IV suggest that hPARG60 may be capable of shuttling between nucleus and mitochondria, which would be in line with a proposed function of PAR in genotoxic stress-dependent, nuclear-mitochondrial crosstalk.

  9. Readers of poly(ADP-ribose): designed to be fit for purpose

    PubMed Central

    Teloni, Federico; Altmeyer, Matthias

    2016-01-01

    Post-translational modifications (PTMs) regulate many aspects of protein function and are indispensable for the spatio-temporal regulation of cellular processes. The proteome-wide identification of PTM targets has made significant progress in recent years, as has the characterization of their writers, readers, modifiers and erasers. One of the most elusive PTMs is poly(ADP-ribosyl)ation (PARylation), a nucleic acid-like PTM involved in chromatin dynamics, genome stability maintenance, transcription, cell metabolism and development. In this article, we provide an overview on our current understanding of the writers of this modification and their targets, as well as the enzymes that degrade and thereby modify and erase poly(ADP-ribose) (PAR). Since many cellular functions of PARylation are exerted through dynamic interactions of PAR-binding proteins with PAR, we discuss the readers of this modification and provide a synthesis of recent findings, which suggest that multiple structurally highly diverse reader modules, ranging from completely folded PAR-binding domains to intrinsically disordered sequence stretches, evolved as PAR effectors to carry out specific cellular functions. PMID:26673700

  10. 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.

  11. A conserved loop in polynucleotide phosphorylase (PNPase) essential for both RNA and ADP/phosphate binding.

    PubMed

    Carzaniga, Thomas; Mazzantini, Elisa; Nardini, Marco; Regonesi, Maria Elena; Greco, Claudio; Briani, Federica; De Gioia, Luca; Dehò, Gianni; Tortora, Paolo

    2014-02-01

    Polynucleotide phosphorylase (PNPase) reversibly catalyzes RNA phosphorolysis and polymerization of nucleoside diphosphates. Its homotrimeric structure forms a central channel where RNA is accommodated. Each protomer core is formed by two paralogous RNase PH domains: PNPase1, whose function is largely unknown, hosts a conserved FFRR loop interacting with RNA, whereas PNPase2 bears the putative catalytic site, ∼20 Å away from the FFRR loop. To date, little is known regarding PNPase catalytic mechanism. We analyzed the kinetic properties of two Escherichia coli PNPase mutants in the FFRR loop (R79A and R80A), which exhibited a dramatic increase in Km for ADP/Pi binding, but not for poly(A), suggesting that the two residues may be essential for binding ADP and Pi. However, both mutants were severely impaired in shifting RNA electrophoretic mobility, implying that the two arginines contribute also to RNA binding. Additional interactions between RNA and other PNPase domains (such as KH and S1) may preserve the enzymatic activity in R79A and R80A mutants. Inspection of enzyme structure showed that PNPase has evolved a long-range acting hydrogen bonding network that connects the FFRR loop with the catalytic site via the F380 residue. This hypothesis was supported by mutation analysis. Phylogenetic analysis of PNPase domains and RNase PH suggests that such network is a unique feature of PNPase1 domain, which coevolved with the paralogous PNPase2 domain.

  12. On PAR with PARP: cellular stress signaling through poly(ADP-ribose) and PARP-1

    PubMed Central

    Luo, Xin; Kraus, W. Lee

    2012-01-01

    Cellular stress responses are mediated through a series of regulatory processes that occur at the genomic, transcriptional, post-transcriptional, translational, and post-translational levels. These responses require a complex network of sensors and effectors from multiple signaling pathways, including the abundant and ubiquitous nuclear enzyme poly(ADP-ribose) (PAR) polymerase-1 (PARP-1). PARP-1 functions at the center of cellular stress responses, where it processes diverse signals and, in response, directs cells to specific fates (e.g., DNA repair vs. cell death) based on the type and strength of the stress stimulus. Many of PARP-1's functions in stress response pathways are mediated by its regulated synthesis of PAR, a negatively charged polymer, using NAD+ as a donor of ADP-ribose units. Thus, PARP-1's functions are intimately tied to nuclear NAD+ metabolism and the broader metabolic profile of the cell. Recent studies in cell and animal models have highlighted the roles of PARP-1 and PAR in the response to a wide variety of extrinsic and intrinsic stress signals, including those initiated by oxidative, nitrosative, genotoxic, oncogenic, thermal, inflammatory, and metabolic stresses. These responses underlie pathological conditions, including cancer, inflammation-related diseases, and metabolic dysregulation. The development of PARP inhibitors is being pursued as a therapeutic approach to these conditions. In this review, we highlight the newest findings about PARP-1's role in stress responses in the context of the historical data. PMID:22391446

  13. Abnormalities of ADP/ATP carrier protein in J-2-N cardiomyopathic hamsters.

    PubMed

    Kato, M; Yang, J; Iwai, T; Tanamura, A; Arino, T; Kawashima, O; Takeda, N

    1993-02-17

    ADP/ATP carrier protein (AAC) is located in the mitochondrial inner membrane and has an important function in mitochondrial energy supply. This protein transports ATP to the cytoplasm and counter transports ADP into the mitochondria. J-2-N cardiomyopathic hamsters were investigated to determine the AAC content in cardiac mitochondria. After recording an electrocardiogram and collecting blood, the cardiac mitochondria were isolated. The mitochondrial membranes were labelled with eosin-5-maleimide (EMA) and separated on SDS polyacrylamide gels. The position of the AAC component was identified by exposing the gel under UV light, and the AAC content was determined by densitometry after staining with Coomassie blue. The AAC content ratio was significantly decreased in both 10-week-old and 1-year survived J-2-N hamsters when compared to control Golden hamster. Among 10-week-old J-2-N hamsters, the decrease in the AAC content ratio was more marked for the animals with more severe myocardial damage. The H(+)-ATPase activities of mitochondrial membrane were higher in 10-week-old J-2-N hamsters than in control hamsters. These results suggest that the decrease of AAC in J-2-N hamster plays an important role in the pathogenesis of cardiomyopathy in J-2-N hamsters. PMID:8455591

  14. 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

  15. Poly(ADP-Ribose) Glycohydrolase (PARG) Silencing Suppresses Benzo(a)pyrene Induced Cell Transformation

    PubMed Central

    Huang, Peiwu; Zhuang, Zhixiong; Liu, Jianjun; Gao, Wei; Liu, Yinpin; Huang, Haiyan

    2016-01-01

    Benzo(a)pyrene (BaP) is a ubiquitously distributed environmental pollutant and known carcinogen, which can induce malignant transformation in rodent and human cells. Poly(ADP-ribose) glycohydrolase (PARG), the primary enzyme that catalyzes the degradation of poly(ADP-ribose) (PAR), has been known to play an important role in regulating DNA damage repair and maintaining genomic stability. Although PARG has been shown to be a downstream effector of BaP, the role of PARG in BaP induced carcinogenesis remains unclear. In this study, we used the PARG-deficient human bronchial epithelial cell line (shPARG) as a model to examine how PARG contributed to the carcinogenesis induced by chronic BaP exposure under various concentrations (0, 10, 20 and 40 μM). Our results showed that PARG silencing dramatically reduced DNA damages, chromosome abnormalities, and micronuclei formations in the PARG-deficient human bronchial epithelial cells compared to the control cells (16HBE cells). Meanwhile, the wound healing assay showed that PARG silencing significantly inhibited BaP-induced cell migration. Furthermore, silencing of PARG significantly reduced the volume and weight of tumors in Balb/c nude mice injected with BaP induced transformed human bronchial epithelial cells. This was the first study that reported evidences to support an oncogenic role of PARG in BaP induced carcinogenesis, which provided a new perspective for our understanding in BaP exposure induced cancer. PMID:27003318

  16. Activation of the Poly(ADP-Ribose) Polymerase Pathway in Human Heart Failure

    PubMed Central

    Molnár, Andrea; Tóth, Attila; Bagi, Zsolt; Papp, Zoltán; Édes, István; Vaszily, Miklós; Galajda, Zoltán; Papp, Julius Gy.; Varró, András; Szüts, Viktória; Lacza, Zsombor; Gerö, Domokos; Szabó, Csaba

    2006-01-01

    Poly(ADP-ribose) polymerase (PARP) activation has been implicated in the pathogenesis of acute and chronic myocardial dysfunction and heart failure. The goal of the present study was to investigate PARP activation in human heart failure, and to correlate PARP activation with various indices of apoptosis and oxidative and nitrosative stress in healthy (donor) and failing (NYHA class III–IV) human heart tissue samples. Higher levels of oxidized protein end-products were found in failing hearts compared with donor heart samples. On the other hand, no differences in tyrosine nitration (a marker of peroxynitrite generation) were detected. Activation of PARP was demonstrated in the failing hearts by an increased abundance of poly-ADP ribosylated proteins. Immunohistochemical analysis revealed that PARP activation was localized to the nucleus of the cardiomyocytes from the failing hearts. The expression of full-length PARP-1 was not significantly different in donor and failing hearts. The expression of caspase-9, in contrast, was significantly higher in the failing than in the donor hearts. Immunohistochemical analysis was used to detect the activation of mitochondrial apoptotic pathways. We found no significant translocation of apoptosis-inducing factor (AIF) into the nucleus. Overall, the current data provide evidence of oxidative stress and PARP activation in human heart failure. Interventional studies with antioxidants or PARP inhibitors are required to define the specific roles of these factors in the pathogenesis of human heart failure. PMID:17088946

  17. 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

  18. Elucidation of human choline kinase crystal structures in complex with the products ADP or phosphocholine.

    PubMed

    Malito, Enrico; Sekulic, Nikolina; Too, Wei Cun See; Konrad, Manfred; Lavie, Arnon

    2006-11-24

    Choline kinase, responsible for the phosphorylation of choline to phosphocholine as the first step of the CDP-choline pathway for the biosynthesis of phosphatidylcholine, has been recognized as a new target for anticancer therapy. Crystal structures of human choline kinase in its apo, ADP and phosphocholine-bound complexes, respectively, reveal the molecular details of the substrate binding sites. ATP binds in a cavity where residues from both the N and C-terminal lobes contribute to form a cleft, while the choline-binding site constitutes a deep hydrophobic groove in the C-terminal domain with a rim composed of negatively charged residues. Upon binding of choline, the enzyme undergoes conformational changes independently affecting the N-terminal domain and the ATP-binding loop. From this structural analysis and comparison with other kinases, and from mutagenesis data on the homologous Caenorhabditis elegans choline kinase, a model of the ternary ADP.phosphocholine complex was built that reveals the molecular basis for the phosphoryl transfer activity of this enzyme.

  19. Ageing of atrazine in manure amended soils assessed by bioavailability to Pseudomonas sp. strain ADP.

    PubMed

    Glæsner, Nadia; Bælum, Jacob; Strobel, Bjarne W; Jacobsen, Carsten S

    2014-04-01

    Animal manure is applied to agricultural land in areas of high livestock production. In the present study, we evaluated ageing of atrazine in two topsoils with and without addition of manure and in one subsoil. Ageing was assessed as the bioavailability of atrazine to the atrazine mineralizing bacteria Pseudomonas sp. strain ADP. Throughout an ageing period of 90 days bioavailability was investigated at days 1, 10, 32, 60 and 90, where ~10(8) cells g(-1) of the ADP strain was inoculated to the (14)C-atrazine exposed soil and (14)CO2 was collected over 7 days as a measure of mineralized atrazine. Even though the bioavailable residue decreased in all of the three soils as time proceeded, we found that ageing occurred faster in the topsoils rich in organic carbon than in subsoil. For one topsoil rich in organic carbon content, Simmelkær, we observed a higher degree of ageing when treated with manure. Contrarily, sorption experiments showed less sorption to Simmelkær treated with manure than the untreated soil indicating that sorption processes are not the only mechanisms of ageing. The other topsoil low in organic carbon content, Ringe, showed no significant difference in ageing between the manure-treated and untreated soil. The present study illustrates that not simply the organic carbon content influences adsorption and ageing of atrazine in soil but the origin and composition of organic matter plays an important role.

  20. Cloning and characterization of ADP-glucose pyrophosphorylase small subunit gene in Cyperus esculentus (yellow nutsedge).

    PubMed

    Cheng, C; Hu, J; Zhi, Y; Su, J J; Zhang, X K; Huang, B Q

    2015-01-01

    ADP-glucose pyrophosphorylase (ADPGlcPPase) controls the first committed step of starch synthesis by catalyzing the biosynthesis of ADP-glucose from glucose-phosphate and ATP. It is a tetrameric protein consisting of two small and two large subunits. The small subunits have a catalytic function, while the large subunits regulate the enzyme activity. Cyperus esculentus (yellow nutsedge) is a perennial C4 plant grown from rhizomes and tubers. Previous studies on yellow nutsedge have mostly focused on the morphology and cultivation of tubers, their application in food, and biochemical analyses of the tubers. In this study, the gene encoding the ADPGlcPPase small subunit (CeAGPS) in yellow nutsedge was cloned and characterized. The full-length CeAGPS cDNA sequence contained an 81-bp 5'-untranslated region (UTR), a 188-bp 3'-UTR, and a 1539-bp open reading frame encoding 512-amino acid residues. The genomic sequence of CeAGPS comprises a nine exon-eight intron structure similar to the previously reported cotton and Arabidopsis thaliana AGPS genes. The deduced translation product of the CeAGPS gene contained a well-conserved catalytic domain and regulatory elements typical of plant AGPS. Reverse transcriptase polymerase chain reaction amplification of the target gene in various plant parts using gene-specific primers indicated that the expression of CeAGPS was most abundant in the tuber, and relatively lower in nutsedge roots. PMID:26782478

  1. Hydrogen ADPs with Cu Kα data? Invariom and Hirshfeld atom modelling of fluconazole.

    PubMed

    Orben, Claudia M; Dittrich, Birger

    2014-06-01

    For the structure of fluconazole [systematic name: 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-ol] monohydrate, C13H12F2N6O·H2O, a case study on different model refinements is reported, based on single-crystal X-ray diffraction data measured at 100 K with Cu Kα radiation to a resolution of sin θ/λ of 0.6 Å(-1). The structure, anisotropic displacement parameters (ADPs) and figures of merit from the independent atom model are compared to `invariom' and `Hirshfeld atom' refinements. Changing from a spherical to an aspherical atom model lowers the figures of merit and improves both the accuracy and the precision of the geometrical parameters. Differences between results from the two aspherical-atom refinements are small. However, a refinement of ADPs for H atoms is only possible with the Hirshfeld atom density model. It gives meaningful results even at a resolution of 0.6 Å(-1), but requires good low-order data.

  2. Sumoylation of poly(ADP-ribose) polymerase 1 inhibits its acetylation and restrains transcriptional coactivator function.

    PubMed

    Messner, Simon; Schuermann, David; Altmeyer, Matthias; Kassner, Ingrid; Schmidt, Darja; Schär, Primo; Müller, Stefan; Hottiger, Michael O

    2009-11-01

    Poly(ADP-ribose) polymerase 1 (PARP1) is a chromatin-associated nuclear protein and functions as a molecular stress sensor. At the cellular level, PARP1 has been implicated in a wide range of processes, such as maintenance of genome stability, cell death, and transcription. PARP1 functions as a transcriptional coactivator of nuclear factor kappaB (NF-kappaB) and hypoxia inducible factor 1 (HIF1). In proteomic studies, PARP1 was found to be modified by small ubiquitin-like modifiers (SUMOs). Here, we characterize PARP1 as a substrate for modification by SUMO1 and SUMO3, both in vitro and in vivo. PARP1 is sumoylated at the single lysine residue K486 within its automodification domain. Interestingly, modification of PARP1 with SUMO does not affect its ADP-ribosylation activity but completely abrogates p300-mediated acetylation of PARP1, revealing an intriguing crosstalk of sumoylation and acetylation on PARP1. Genetic complementation of PARP1-depleted cells with wild-type and sumoylation-deficient PARP1 revealed that SUMO modification of PARP1 restrains its transcriptional coactivator function and subsequently reduces gene expression of distinct PARP1-regulated target genes.

  3. Poly(ADP-Ribose) Glycohydrolase (PARG) Silencing Suppresses Benzo(a)pyrene Induced Cell Transformation.

    PubMed

    Li, Xuan; Li, Xiyi; Zhu, Zhiliang; Huang, Peiwu; Zhuang, Zhixiong; Liu, Jianjun; Gao, Wei; Liu, Yinpin; Huang, Haiyan

    2016-01-01

    Benzo(a)pyrene (BaP) is a ubiquitously distributed environmental pollutant and known carcinogen, which can induce malignant transformation in rodent and human cells. Poly(ADP-ribose) glycohydrolase (PARG), the primary enzyme that catalyzes the degradation of poly(ADP-ribose) (PAR), has been known to play an important role in regulating DNA damage repair and maintaining genomic stability. Although PARG has been shown to be a downstream effector of BaP, the role of PARG in BaP induced carcinogenesis remains unclear. In this study, we used the PARG-deficient human bronchial epithelial cell line (shPARG) as a model to examine how PARG contributed to the carcinogenesis induced by chronic BaP exposure under various concentrations (0, 10, 20 and 40 μM). Our results showed that PARG silencing dramatically reduced DNA damages, chromosome abnormalities, and micronuclei formations in the PARG-deficient human bronchial epithelial cells compared to the control cells (16HBE cells). Meanwhile, the wound healing assay showed that PARG silencing significantly inhibited BaP-induced cell migration. Furthermore, silencing of PARG significantly reduced the volume and weight of tumors in Balb/c nude mice injected with BaP induced transformed human bronchial epithelial cells. This was the first study that reported evidences to support an oncogenic role of PARG in BaP induced carcinogenesis, which provided a new perspective for our understanding in BaP exposure induced cancer. PMID:27003318

  4. Unique features revealed by the genome sequence of Acinetobacter sp. ADP1, a versatile and naturally transformation competent bacterium.

    PubMed

    Barbe, Valérie; Vallenet, David; Fonknechten, Nuria; Kreimeyer, Annett; Oztas, Sophie; Labarre, Laurent; Cruveiller, Stéphane; Robert, Catherine; Duprat, Simone; Wincker, Patrick; Ornston, L Nicholas; Weissenbach, Jean; Marlière, Philippe; Cohen, Georges N; Médigue, Claudine

    2004-01-01

    Acinetobacter sp. strain ADP1 is a nutritionally versatile soil bacterium closely related to representatives of the well-characterized Pseudomonas aeruginosa and Pseudomonas putida. Unlike these bacteria, the Acinetobacter ADP1 is highly competent for natural transformation which affords extraordinary convenience for genetic manipulation. The circular chromosome of the Acinetobacter ADP1, presented here, encodes 3325 predicted coding sequences, of which 60% have been classified based on sequence similarity to other documented proteins. The close evolutionary proximity of Acinetobacter and Pseudomonas species, as judged by the sequences of their 16S RNA genes and by the highest level of bidirectional best hits, contrasts with the extensive divergence in the GC content of their DNA (40 versus 62%). The chromosomes also differ significantly in size, with the Acinetobacter ADP1 chromosome <60% of the length of the Pseudomonas counterparts. Genome analysis of the Acinetobacter ADP1 revealed genes for metabolic pathways involved in utilization of a large variety of compounds. Almost all of these genes, with orthologs that are scattered in other species, are located in five major 'islands of catabolic diversity', now an apparent 'archipelago of catabolic diversity', within one-quarter of the overall genome. Acinetobacter ADP1 displays many features of other aerobic soil bacteria with metabolism oriented toward the degradation of organic compounds found in their natural habitat. A distinguishing feature of this genome is the absence of a gene corresponding to pyruvate kinase, the enzyme that generally catalyzes the terminal step in conversion of carbohydrates to pyruvate for respiration by the citric acid cycle. This finding supports the view that the cycle itself is centrally geared to the catabolic capabilities of this exceptionally versatile organism. PMID:15514110

  5. Full activation of mouse platelets requires ADP secretion regulated by SERCA3 ATPase-dependent calcium stores.

    PubMed

    Elaïb, Ziane; Adam, Frédéric; Berrou, Eliane; Bordet, Jean-Claude; Prévost, Nicolas; Bobe, Régis; Bryckaert, Marijke; Rosa, Jean-Philippe

    2016-08-25

    The role of the sarco-endoplasmic reticulum calcium (Ca(2+)) adenosine triphosphatase (ATPase) 3 (SERCA3) in platelet physiology remains poorly understood. Here, we show that SERCA3 knockout (SERCA3(-/-)) mice exhibit prolonged tail bleeding time and rebleeding. Thrombus formation was delayed both in arteries and venules in an in vivo ferric chloride-induced thrombosis model. Defective platelet adhesion and thrombus growth over collagen was confirmed in vitro. Adenosine 5'-diphosphate (ADP) removal by apyrase diminished adhesion and thrombus growth of control platelets to the level of SERCA3(-/-) platelets. Aggregation, dense granule secretion, and Ca(2+) mobilization of SERCA3(-/-) platelets induced by low collagen or low thrombin concentration were weaker than controls. Accordingly, SERCA3(-/-) platelets exhibited a partial defect in total stored Ca(2+) and in Ca(2+) store reuptake following thrombin stimulation. Importantly ADP, but not serotonin, rescued aggregation, secretion, and Ca(2+) mobilization in SERCA3(-/-) platelets, suggesting specificity. Dense granules appeared normal upon electron microscopy, mepacrine staining, and total serotonin content, ruling out a dense granule defect. ADP induced normal platelet aggregation, excluding a defect in ADP activation pathways. The SERCA3-specific inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroquinone diminished both Ca(2+) mobilization and secretion of control platelets, as opposed to the SERCA2b inhibitor thapsigargin. This confirmed the specific role of catalytically active SERCA3 in ADP secretion. Accordingly, SERCA3-dependent Ca(2+) stores appeared depleted in SERCA3(-/-) platelets. Finally, αIIbβ3 integrin blockade did not affect SERCA3-dependent secretion, therefore proving independent of αIIbβ3 engagement. Altogether, these results show that SERCA3-dependent Ca(2+) stores control a specific ADP secretion pathway required for full platelet secretion induced by agonists at low concentration and independent

  6. Crystallization and preliminary X-ray diffraction analysis of the amidase domain of allophanate hydrolase from Pseudomonas sp. strain ADP

    SciTech Connect

    Balotra, Sahil; Newman, Janet; French, Nigel G.; Briggs, Lyndall J.; Peat, Thomas S.; Scott, Colin

    2014-02-19

    The amidase domain of the allophanate hydrolase AtzF from Pseudomonas sp. strain ADP has been crystallized and preliminary X-ray diffraction data have been collected. The allophanate hydrolase from Pseudomonas sp. strain ADP was expressed and purified, and a tryptic digest fragment was subsequently identified, expressed and purified. This 50 kDa construct retained amidase activity and was crystallized. The crystals diffracted to 2.5 Å resolution and adopted space group P2{sub 1}, with unit-cell parameters a = 82.4, b = 179.2, c = 112.6 Å, β = 106.6°.

  7. Poly(ADP-ribose)--a unique natural polymer structural features, biological role and approaches to the chemical synthesis.

    PubMed

    Drenichev, Mikhail S; Mikhailov, Sergey N

    2015-01-01

    Poly(ADP-ribose) (PAR) is a natural polymer, taking part in numerous important cellular processes. Several enzymes are involved in biosynthesis and degradation of PAR. One of them, poly(ADP-ribose)polymerase-1 (PARP-1) is considered to be a perspective target for the design of new drugs, affecting PAR metabolism. The structure of PAR was established by enzymatic hydrolysis and further analysis of the products, but total chemical synthesis of PAR hasn't been described yet. Several approaches have been developed on the way to chemical synthesis of this unique biopolymer.

  8. Regulation of the clpP1clpP2 operon by the pleiotropic regulator AdpA in Streptomyces lividans.

    PubMed

    Guyet, Aurélie; Gominet, Myriam; Benaroudj, Nadia; Mazodier, Philippe

    2013-12-01

    Insertion of an apramycin resistance cassette in the clpP1clpP2 operon (encoding the ClpP1 and ClpP2 peptidase subunits) affects morphological and physiological differentiation of Streptomyces lividans. Another key factor controlling Streptomyces differentiation is the pleiotropic transcriptional regulator AdpA. We have identified a spontaneous missense mutation (-1 frameshift) in the adpA (bldH) open reading frame in a clpP1clpP2 mutant that led to the synthesis of a non-functional AdpA protein. Electrophoretic mobility shift assays showed that AdpA bound directly to clpP1clpP2 promoter region. Quantitative real-time PCR analysis showed that AdpA regulated the clpP1clpP2 operon expression at specific growth times. In vitro, AdpA and ClgR, a transcriptional activator of clpP1clpP2 operon and other genes, were able to bind simultaneously to clpP1 promoter, which suggests that AdpA binding to clpP1 promoter did not affect that of ClgR. This study allowed to uncover an interplay between the ClpP peptidases and AdpA in S. lividans.

  9. Burn and smoke injury activates poly(ADP-ribose)polymerase in circulating leukocytes

    PubMed Central

    Bartha, Eva; Asmussen, Sven; Olah, Gabor; Rehberg, Sebastian W.; Yamamoto, Yusuke; Traber, Daniel L.; Szabo, Csaba

    2011-01-01

    The nuclear enzyme poly(ADP-ribose)polymerase (PARP) plays a significant role in the pathogenesis of various forms of critical illness. DNA strand breaks induced by oxidative and nitrative stress trigger the activation of PARP, and PARP, in turn, mediates cell death and promotes pro-inflammatory responses. Until recently, most studies focused on the role of PARP in solid organs such as heart, liver, kidney. Here we investigated the effect of burn and smoke inhalation on the levels of poly(ADP-ribosylated) proteins (PAR) in circulating sheep leukocytes ex vivo. Adult female merino sheep were subjected to burn injury (2×20% each flank, 3 degree) and smoke inhalation injury (insufflated with a total of 48 breaths of cotton smoke) under deep anesthesia. Arterial and venous blood were collected at baseline, immediately after the injury and 1-24 hours after the injury. Leukocytes were isolated with the Histopaque method. The levels of poly(ADP-ribosyl)ated proteins were determined by Western blotting. The amount of reactive oxygen species (ROS) were quantified by the Oxyblot method. To examine whether PARP activation continues to increasing ex vivo in the leukocytes, blood samples were incubated at room temperature or at 37°C for 3h with or without the PARP inhibitor PJ34. To investigate whether the plasma of burn/smoke animals may trigger PARP activation, burn/smoke plasma was incubated with control leukocytes in vitro. The results show that burn and smoke injury induced a marked PARP activation in circulating leukocytes. The activity was the highest immediately after injury and at 1 hour, and decreased gradually over time. Incubation of whole blood at 37°C for 3 hours significantly increased PAR levels, indicative of the presence of an on-going cell activation process. In conclusion, PARP activity is elevated in leukocytes after burn and smoke inhalation injury and the response parallels the time-course of reactive oxygen species generation in these cells. PMID

  10. 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

  11. Ruling out pyridine dinucleotides as true TRPM2 channel activators reveals novel direct agonist ADP-ribose-2′-phosphate

    PubMed Central

    Tóth, Balázs; Iordanov, Iordan

    2015-01-01

    Transient receptor potential melastatin 2 (TRPM2), a Ca2+-permeable cation channel implicated in postischemic neuronal cell death, leukocyte activation, and insulin secretion, is activated by intracellular ADP ribose (ADPR). In addition, the pyridine dinucleotides nicotinamide-adenine-dinucleotide (NAD), nicotinic acid–adenine-dinucleotide (NAAD), and NAAD-2′-phosphate (NAADP) have been shown to activate TRPM2, or to enhance its activation by ADPR, when dialyzed into cells. The precise subset of nucleotides that act directly on the TRPM2 protein, however, is unknown. Here, we use a heterologously expressed, affinity-purified–specific ADPR hydrolase to purify commercial preparations of pyridine dinucleotides from substantial contaminations by ADPR or ADPR-2′-phosphate (ADPRP). Direct application of purified NAD, NAAD, or NAADP to the cytosolic face of TRPM2 channels in inside-out patches demonstrated that none of them stimulates gating, or affects channel activation by ADPR, indicating that none of these dinucleotides directly binds to TRPM2. Instead, our experiments identify for the first time ADPRP as a true direct TRPM2 agonist of potential biological interest. PMID:25918360

  12. Differential regulation of nicotinic acid-adenine dinucleotide phosphate and cADP-ribose production by cAMP and cGMP.

    PubMed Central

    Wilson, H L; Galione, A

    1998-01-01

    The sea urchin egg has been used as a system to study calcium-release mechanisms induced by inositol 1,4,5-trisphosphate (IP3), cADP-ribose (cADPR), and more recently, nicotinic acid-adenine dinucleotide phosphate (NAADP). In order that cADPR and NAADP may be established as endogenous messengers for calcium release, the existence of intracellular enzymes capable of metabolizing these molecules must be demonstrated. In addition, intracellular levels of cADPR and NAADP should be under the control of extracellular stimuli. It has been shown that cGMP stimulates the synthesis of cADPR in the sea urchin egg. The present study shows that the sea urchin egg is capable of synthesizing and degrading NAADP. cADPR and NAADP synthetic activities appear to be separate, with different cellular localizations, pH and temperature optima. We suggest that in the sea urchin egg, cADPR and NAADP production may be differentially regulated by receptor-coupled second messengers, with cADPR production being regulated by cGMP and NAADP production modulated by cAMP. PMID:9560312

  13. 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.

  14. Dysfunction of the PI3 kinase/Rap1/integrin α(IIb)β(3) pathway underlies ex vivo platelet hypoactivity in essential thrombocythemia.

    PubMed

    Moore, Samantha F; Hunter, Roger W; Harper, Matthew T; Savage, Joshua S; Siddiq, Samreen; Westbury, Sarah K; Poole, Alastair W; Mumford, Andrew D; Hers, Ingeborg

    2013-02-14

    Patients with myeloproliferative disorders (MPDs), such as essential thrombocythemia (ET) have increased risk of thrombosis and bleeding, which are major sources of morbidity and mortality. Most MPD patients have a gain of function mutation in Janus kinase 2 (JAK2V617F), but little is known how JAK2V617F affects platelet function. Here, we demonstrate that platelets from ET patients have impaired SFLLRN-mediated fibrinogen binding and have lost the potentiating effect of thrombopoietin (which couples to JAK2) on this pathway. In contrast, SFLLRN-mediated P-selectin expression, ATP secretion, phosphorylation of the PKC substrate pleckstrin, and Ca(2+) mobilization were unaffected in JAK2V617F positive platelets. In addition, thrombopoietin-mediated JAK2 phosphorylation was unchanged, suggesting that signaling pathways activated downstream of JAK2 are impaired. Indeed, we found that platelets from JAK2V617F positive ET patients have significantly reduced phosphorylation of the PI3 kinase substrate Akt, and have reduced activation of Rap1 in response to thrombopoietin, IGF-1,ADP, SFLLRN, and thrombin. This effect was independent of Giα P2Y12 purinergic receptor function as ADP-mediated inhibition of VASP phosphorylation was unchanged. These results demonstrate that the PI3 kinase/Rap1 pathway is intrinsically impaired in platelets from JAK2V617F-positive ET patients, resulting in diminished thrombin and thrombopoietin-mediated integrin α(IIb)β(3) activation. PMID:23243278

  15. Discovering Anti-platelet Drug Combinations with an Integrated Model of Activator-Inhibitor Relationships, Activator-Activator Synergies and Inhibitor-Inhibitor Synergies

    PubMed Central

    Lombardi, Federica; Golla, Kalyan; Fitzpatrick, Darren J.; Casey, Fergal P.; Moran, Niamh; Shields, Denis C.

    2015-01-01

    Identifying effective therapeutic drug combinations that modulate complex signaling pathways in platelets is central to the advancement of effective anti-thrombotic therapies. However, there is no systems model of the platelet that predicts responses to different inhibitor combinations. We developed an approach which goes beyond current inhibitor-inhibitor combination screening to efficiently consider other signaling aspects that may give insights into the behaviour of the platelet as a system. We investigated combinations of platelet inhibitors and activators. We evaluated three distinct strands of information, namely: activator-inhibitor combination screens (testing a panel of inhibitors against a panel of activators); inhibitor-inhibitor synergy screens; and activator-activator synergy screens. We demonstrated how these analyses may be efficiently performed, both experimentally and computationally, to identify particular combinations of most interest. Robust tests of activator-activator synergy and of inhibitor-inhibitor synergy required combinations to show significant excesses over the double doses of each component. Modeling identified multiple effects of an inhibitor of the P2Y12 ADP receptor, and complementarity between inhibitor-inhibitor synergy effects and activator-inhibitor combination effects. This approach accelerates the mapping of combination effects of compounds to develop combinations that may be therapeutically beneficial. We integrated the three information sources into a unified model that predicted the benefits of a triple drug combination targeting ADP, thromboxane and thrombin signaling. PMID:25875950

  16. Label-free detection of Alzheimer's disease through the ADP3 peptoid recognizing the serum amyloid-beta42 peptide.

    PubMed

    Zhao, Zijian; Zhu, Ling; Bu, Xiangli; Ma, Huailei; Yang, Shu; Yang, Yanlian; Hu, Zhiyuan

    2015-01-14

    The early diagnosis of Alzheimer's disease (AD) is challenging due to the lack of reliable methods for detecting its biomarkers in the noninvasive biopsies. We used surface plasmon resonance imaging to identify AD based on the detection of amyloid-beta42 in the serum by the ADP3 peptoid. PMID:25418132

  17. 3'-O-(5-fluoro-2,4-dinitrophenyl)ADP ether and ATP ether. Affinity reagents for labeling ATPases.

    PubMed

    Chuan, H; Wang, J H

    1988-09-15

    The affinity reagents 3'-O-(5-fluoro-2,4-dinitrophenyl)ADP ether (FDNP-ADP) and 3'-O-(5-fluoro-2,4-dinitrophenyl)ATP ether (FDNP-ATP) were synthesized and characterized. FDNP[14C]ADP was found to label the active site of mitochondrial F1-ATPase slowly at room temperature but with high specificity. F1 was effectively protected from the labeling reagent by ATP or ADP. An average number of 1.3 covalent label per F1 is sufficient for 100% inhibition of the ATPase. About 73% of the radioactive label was found covalently attached to beta subunits, 9% on alpha, practically none on gamma, delta, and epsilon. Cleavage of the labeled enzyme by pepsin and sequencing of the major radioactive peptide showed that the labeled amino acid residue in beta subunit was Lys beta 162. These results show that Lys beta 162 is indeed at the active site of F1 as assumed in the recently proposed models (Fry, D. C., Kuby, S. A., and Mildvan, A. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 907-911; Duncan, I. M., Parsonage, D., and Senior, A. E. (1986) FEBS Lett. 208, 1-6).

  18. Spermatid Head Elongation with Normal Nuclear Shaping Requires ADP-Ribosyltransferase PARP11 (ARTD11) in Mice1

    PubMed Central

    Meyer-Ficca, Mirella L.; Ihara, Motomasa; Bader, Jessica J.; Leu, N. Adrian; Beneke, Sascha; Meyer, Ralph G.

    2015-01-01

    ABSTRACT Sperm are highly differentiated cells characterized by their species-specific nuclear shapes and extremely condensed chromatin. Abnormal head shapes represent a form of teratozoospermia that can impair fertilization capacity. This study shows that poly(ADP-ribose) polymerase-11 (ARTD11/PARP11), a member of the ADP-ribosyltransferase (ARTD) family, is expressed preferentially in spermatids undergoing nuclear condensation and differentiation. Deletion of the Parp11 gene results in teratozoospermia and male infertility in mice due to the formation of abnormally shaped fertilization-incompetent sperm, despite normal testis weights and sperm counts. At the subcellular level, PARP11-deficient elongating spermatids reveal structural defects in the nuclear envelope and chromatin detachment associated with abnormal nuclear shaping, suggesting functional relevance of PARP11 for nuclear envelope stability and nuclear reorganization during spermiogenesis. In vitro, PARP11 exhibits mono(ADP-ribosyl)ation activity with the ability to ADP-ribosylate itself. In transfected somatic cells, PARP11 colocalizes with nuclear pore components, such as NUP153. Amino acids Y77, Q86, and R95 in the N-terminal WWE domain, as well as presence of the catalytic domain, are essential for colocalization of PARP11 with the nuclear envelope, but catalytic activity of the protein is not required for colocalization with NUP153. This study demonstrates that PARP11 is a novel enzyme important for proper sperm head shaping and identifies it as a potential factor involved in idiopathic mammalian teratozoospermia. PMID:25673562

  19. Effect of inhibitors of poly(ADP-ribose)polymerase on the radiation response of HeLa S3 cells

    SciTech Connect

    Burgman, P.; Konings, A.W. )

    1989-08-01

    The purpose of this study was to investigate possible involvement of poly(ADP-ribosyl)ation reactions in X-ray-induced cell killing, repair of potentially lethal damage (PLD), and formation and repair of radiation-induced DNA damage. As tools we used the inhibitors of poly(ADP-ribose)polymerase, 3-aminobenzamide (3AB), and 4-aminobenzamide (4AB). Both drugs inhibited PLD repair equally well but did not increase radiation-induced cell killing when cells were plated immediately after irradiation. 3AB affected repair of radiation-induced DNA damage, while 4AB had no effect. When 3AB was combined with aphidicolin (APC), it was found that the amount of DNA damage increased during the postirradiation incubation period. This means that the presence of 3AB stimulates the formation of DNA damage after X-irradiation. It is concluded that 3AB and 4AB sensitize HeLaS3 cells for radiation-induced cell killing by inhibiting repair of PLD. Because of the different effects of both inhibitors on repair of PLD and repair of radiation-induced DNA damage (a process known to be affected by inhibition of poly(ADP-ribosyl)ation), it is concluded that the observed inhibition of PLD repair is not caused by inhibition of poly(ADP-ribose)polymerase, and that the inhibitors affect repair of PLD and repair of DNA damage through independent mechanisms.

  20. 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.

  1. 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

  2. Influence of the ADP/ATP ratio, 2-oxoglutarate and divalent ions on Azospirillum brasilense PII protein signalling.

    PubMed

    Gerhardt, Edileusa C M; Araújo, Luíza M; Ribeiro, Ronny R; Chubatsu, Leda S; Scarduelli, Marcelo; Rodrigues, Thiago E; Monteiro, Rose A; Pedrosa, Fábio O; Souza, Emanuel M; Huergo, Luciano F

    2012-06-01

    Proteins belonging to the P(II) family coordinate cellular nitrogen metabolism by direct interaction with a variety of enzymes, transcriptional regulators and transporters. The sensing function of P(II) relies on its ability to bind the nitrogen/carbon signalling molecule 2-oxoglutarate (2-OG). In Proteobacteria, P(II) is further subject to reversible uridylylation according to the intracellular levels of glutamine, which reflect the cellular nitrogen status. A number of P(II) proteins have been shown to bind ADP and ATP in a competitive manner, suggesting that P(II) might act as an energy sensor. Here, we analyse the influence of the ADP/ATP ratio, 2-OG levels and divalent metal ions on in vitro uridylylation of the Azospirillum brasilense P(II) proteins GlnB and GlnZ, and on interaction with their targets AmtB, DraG and DraT. The results support the notion that the cellular concentration of 2-OG is a key factor governing occupation of the GlnB and GlnZ nucleotide binding sites by ATP or ADP, with high 2-OG levels favouring the occupation of P(II) by ATP. Both P(II) uridylylation and interaction with target proteins responded to the ADP/ATP ratio within the expected physiological range, supporting the concept that P(II) proteins might act as cellular energy sensors.

  3. BGP-15, a nicotinic amidoxime derivate protecting heart from ischemia reperfusion injury through modulation of poly(ADP-ribose) polymerase.

    PubMed

    Szabados, E; Literati-Nagy, P; Farkas, B; Sumegi, B

    2000-04-15

    The protective effect of O-(3-piperidino-2-hydroxy-1-propyl)nicotinic amidoxime (BGP-15) against ischemia-reperfusion-induced injury was studied in the Langendorff heart perfusion system. To understand the molecular mechanism of the cardioprotection, the effect of BGP-15 on ischemic-reperfusion-induced reactive oxygen species (ROS) formation, lipid peroxidation single-strand DNA break formation, NAD(+) catabolism, and endogenous ADP-ribosylation reactions were investigated. These studies showed that BGP-15 significantly decreased leakage of lactate dehydrogenase, creatine kinase, and aspartate aminotransferase in reperfused hearts, and reduced the rate of NAD(+) catabolism. In addition, BGP-15 dramatically decreased the ischemia-reperfusion-induced self-ADP-ribosylation of nuclear poly(ADP-ribose) polymerase(PARP) and the mono-ADP-ribosylation of an endoplasmic reticulum chaperone GRP78. These data raise the possibility that BGP-15 may have a direct inhibitory effect on PARP. This hypothesis was tested on isolated enzyme, and kinetic analysis showed a mixed-type (noncompetitive) inhibition with a K(i) = 57 +/- 6 microM. Furthermore, BGP-15 decreased levels of ROS, lipid peroxidation, and single-strand DNA breaks in reperfused hearts. These data suggest that PARP may be an important molecular target of BGP-15 and that BGP-15 decreases ROS levels and cell injury during ischemia-reperfusion in the heart by inhibiting PARP activity.

  4. ADP-induced platelet aggregation after addition of tramadol in vitro in fed and fasted horses plasma.

    PubMed

    Casella, S; Giannetto, C; Giudice, E; Marafioti, S; Fazio, F; Assenza, A; Piccione, G

    2013-04-01

    Adenosine diphosphate (ADP)-induced platelet aggregation in fed and fasted horses after addition of tramadol hydrochloride was evaluated in vitro. On 10 horses citrated blood samples were collected 2h after feeding (fed animals) and 21 h after feeding (fasted animals). Final concentrations of ADP 1 and 0.5 μM, and tramadol hydrochloride (1, 15, 30, 45 and 60 min after the addition of tramadol) were used to determine the maximum degree and initial velocity of platelet aggregation. Repeated measures multifactor analysis of variance (MANOVA) was used to evaluate the effect of feeding/fasting condition, ADP concentration and addition of tramadol. Findings showed statistical differences (P≤0.05) on studied parameters after addition of tramadol to different ADP concentrations in fed and fasted horses. The clinical relevance of these results is that tramadol provides many advantages as a therapeutic option; in fact, it is an inexpensive and a relatively new analgesic in equine veterinary medicine. Further investigations would be appropriate to compare the effects of different opioids but also using different concentrations of tramadol associated with other drugs in order to have substances which can regulate the functional activity of the platelets and to extend the knowledges on equine platelet aggregation. PMID:23031839

  5. Liquid demixing of intrinsically disordered proteins is seeded by poly(ADP-ribose)

    PubMed Central

    Altmeyer, Matthias; Neelsen, Kai J.; Teloni, Federico; Pozdnyakova, Irina; Pellegrino, Stefania; Grøfte, Merete; Rask, Maj-Britt Druedahl; Streicher, Werner; Jungmichel, Stephanie; Nielsen, Michael Lund; Lukas, Jiri

    2015-01-01

    Intrinsically disordered proteins can phase separate from the soluble intracellular space, and tend to aggregate under pathological conditions. The physiological functions and molecular triggers of liquid demixing by phase separation are not well understood. Here we show in vitro and in vivo that the nucleic acid-mimicking biopolymer poly(ADP-ribose) (PAR) nucleates intracellular liquid demixing. PAR levels are markedly induced at sites of DNA damage, and we provide evidence that PAR-seeded liquid demixing results in rapid, yet transient and fully reversible assembly of various intrinsically disordered proteins at DNA break sites. Demixing, which relies on electrostatic interactions between positively charged RGG repeats and negatively charged PAR, is amplified by aggregation-prone prion-like domains, and orchestrates the earliest cellular responses to DNA breakage. We propose that PAR-seeded liquid demixing is a general mechanism to dynamically reorganize the soluble nuclear space with implications for pathological protein aggregation caused by derailed phase separation. PMID:26286827

  6. Substrate binding properties of potato tuber ADP-glucose pyrophosphorylase as determined by isothermal titration calorimetry.

    PubMed

    Cakir, Bilal; Tuncel, Aytug; Green, Abigail R; Koper, Kaan; Hwang, Seon-Kap; Okita, Thomas W; Kang, ChulHee

    2015-06-01

    Substrate binding properties of the large (LS) and small (SS) subunits of potato tuber ADP-glucose pyrophosphorylase were investigated by using isothermal titration calorimetry. Our results clearly show that the wild type heterotetramer (S(WT)L(WT)) possesses two distinct types of ATP binding sites, whereas the homotetrameric LS and SS variant forms only exhibited properties of one of the two binding sites. The wild type enzyme also exhibited significantly increased affinity to this substrate compared to the homotetrameric enzyme forms. No stable binding was evident for the second substrate, glucose-1-phosphate, in the presence or absence of ATPγS suggesting that interaction of glucose-1-phosphate is dependent on hydrolysis of ATP and supports the Theorell-Chance bi bi reaction mechanism.

  7. 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

  8. Substrate binding properties of potato tuber ADP-glucose pyrophosphorylase as determined by isothermal titration calorimetry.

    PubMed

    Cakir, Bilal; Tuncel, Aytug; Green, Abigail R; Koper, Kaan; Hwang, Seon-Kap; Okita, Thomas W; Kang, ChulHee

    2015-06-01

    Substrate binding properties of the large (LS) and small (SS) subunits of potato tuber ADP-glucose pyrophosphorylase were investigated by using isothermal titration calorimetry. Our results clearly show that the wild type heterotetramer (S(WT)L(WT)) possesses two distinct types of ATP binding sites, whereas the homotetrameric LS and SS variant forms only exhibited properties of one of the two binding sites. The wild type enzyme also exhibited significantly increased affinity to this substrate compared to the homotetrameric enzyme forms. No stable binding was evident for the second substrate, glucose-1-phosphate, in the presence or absence of ATPγS suggesting that interaction of glucose-1-phosphate is dependent on hydrolysis of ATP and supports the Theorell-Chance bi bi reaction mechanism. PMID:25953126

  9. PCR cloning and characterization of multiple ADP-glucose pyrophosphorylase cDNAs from tomato

    NASA Technical Reports Server (NTRS)

    Chen, B. Y.; Janes, H. W.; Gianfagna, T.

    1998-01-01

    Four ADP-glucose pyrophosphorylase (AGP) cDNAs were cloned from tomato fruit and leaves by the PCR techniques. Three of them (agp S1, agp S2, and agp S3) encode the large subunit of AGP, the fourth one (agp B) encodes the small subunit. The deduced amino acid sequences of the cDNAs show very high identities (96-98%) to the corresponding potato AGP isoforms, although there are major differences in tissue expression profiles. All four tomato AGP transcripts were detected in fruit and leaves; the predominant ones in fruit are agp B and agp S1, whereas in leaves they are agp B and agp S3. Genomic southern analysis suggests that the four AGP transcripts are encoded by distinct genes.

  10. Crystal structures of Escherichia coli RecA in complex with MgADP and MnAMP-PNP.

    PubMed

    Xing, Xu; Bell, Charles E

    2004-12-28

    RecA catalyzes the DNA pairing and strand-exchange steps of homologous recombination, an important mechanism for repair of double-stranded DNA breaks. The binding of RecA to DNA is modulated by adenosine nucleotides. ATP increases the affinity of RecA for DNA, while ADP decreases the affinity. Previously, the crystal structures of E. coli RecA and its complex with ADP have been determined to resolutions of 2.3 and 3.0 A, respectively, but the model for the RecA-ADP complex did not include magnesium ion or side chains. Here, we have determined the crystal structures of RecA in complex with MgADP and MnAMP-PNP, a nonhydrolyzable analogue of ATP, at resolutions of 1.9 and 2.1 A, respectively. Both crystals grow in the same conditions and have RecA in a right-handed helical form with a pitch of approximately 82 A. The crystal structures show the detailed interactions of RecA with the nucleotide cofactors, including the metal ion and the gamma phosphate of AMP-PNP. There are very few conformational differences between the structures of RecA bound to ADP and AMP-PNP, which differ from uncomplexed RecA only in a slight opening of the P-loop residues 66-73 upon nucleotide binding. To interpret the functional significance of the structure of the MnAMP-PNP complex, a coprotease assay was used to compare the ability of different nucleotides to promote the active, extended conformation of RecA. Whereas ATPgammaS and ADP-AlF(4) facilitate a robust coprotease activity, ADP and AMP-PNP do not activate RecA at all. We conclude that the crystal structure of the RecA-MnAMP-PNP complex represents a preisomerization state of the RecA protein that exists after ATP has bound but before the conformational transition to the active state. PMID:15610008

  11. 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.

  12. 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

  13. Pyruvate dehydrogenase kinase isoform 2 activity stimulated by speeding up the rate of dissociation of ADP.

    PubMed

    Bao, Haiying; Kasten, Shane A; Yan, Xiaohua; Hiromasa, Yasuaki; Roche, Thomas E

    2004-10-26

    Pyruvate dehydrogenase kinase 2 (PDK2) activity is stimulated by NADH and NADH plus acetyl-CoA via the reduction and reductive acetylation of the lipoyl groups of the dihydrolipoyl acetyltransferase (E2) component. Elevated K(+) and Cl(-) were needed for significant stimulation. Stimulation substantially increased both k(cat) and the K(m) for ATP; the fractional stimulation increased with the level of ATP. With an E2 structure lacking the pyruvate dehydrogenase (E1) binding domain, stimulation of PDK2 was retained, the K(m) for E1 decreased, and the equilibrium dissociation constant for ATP increased but remained much lower than the K(m) for ATP. Stimulation of PDK2 activity greatly reduced the fraction of bound ADP. These results fit an ordered reaction mechanism with ATP binding before E1 and stimulation increasing the rate of dissociation of ADP. Conversion of all of the lipoyl groups in the E2 60mer to the oxidized form (E2(ox)) greatly reduced k(cat) and the K(m) of PDK2 for ATP. Retention over an extended period of time of a low portion of reduced lipoyl groups maintains E2 in a state that supported much higher PDK2 activity than short-term (5 min) reduction of a large portion of lipoyl groups of E2(ox), but reduction of E2(ox) produced a larger fold stimulation. Reduction and to a greater extent reductive acetylation increased PDK2 binding to E2; conversion to E2(ox) did not significantly hinder binding. We suggest that passing even limited reducing equivalents among lipoyl groups maintains E2 lipoyl domains in a conformation that aids kinase function. PMID:15491151

  14. Regulation of the Pseudomonas sp. Strain ADP Cyanuric Acid Degradation Operon

    PubMed Central

    García-González, Vicente; Govantes, Fernando; Porrúa, Odil; Santero, Eduardo

    2005-01-01

    Pseudomonas sp. strain ADP is the model strain for studying bacterial degradation of the s-triazine herbicide atrazine. In this work, we focused on the expression of the atzDEF operon, involved in mineralization of the central intermediate of the pathway, cyanuric acid. Expression analysis of atzD-lacZ fusions in Pseudomonas sp. strain ADP and Pseudomonas putida showed that atzDEF is subjected to dual regulation in response to nitrogen limitation and cyanuric acid. The gene adjacent to atzD, orf99 (renamed here atzR), encoding a LysR-like regulator, was found to be required for both responses. Expression of atzR-lacZ was induced by nitrogen limitation and repressed by AtzR. Nitrogen regulation of atzD-lacZ and atzR-lacZ expression was dependent on the alternative σ factor σN and NtrC, suggesting that the cyanuric acid degradation operon may be subject to general nitrogen control. However, while atzR is transcribed from a σN-dependent promoter, atzDEF transcription appears to be driven from a σ70-type promoter. Expression of atzR from a heterologous promoter revealed that although NtrC regulation of atzD-lacZ requires the AtzR protein, it is not the indirect result of NtrC-activated AtzR synthesis. We propose that expression of the cyanuric acid degradation operon atzDEF is controlled by means of a complex regulatory circuit in which AtzR is the main activator. AtzR activity is in turn modulated by the presence of cyanuric acid and by a nitrogen limitation signal transduced by the Ntr system. PMID:15601699

  15. Identification of Inhibitors of Pseudomonas aeruginosa Exotoxin-S ADP-Ribosyltransferase Activity.

    PubMed

    Pinto, Ana Filipa; Ebrahimi, Mahsa; Saleeb, Michael; Forsberg, Åke; Elofsson, Mikael; Schüler, Herwig

    2016-07-01

    The gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen associated with drug resistance complications and, as such, an important object for drug discovery efforts. One attractive target for development of therapeutics is the ADP-ribosyltransferase Exotoxin-S (ExoS), an early effector of the type III secretion system that is delivered into host cells to affect their transcription pattern and cytoskeletal dynamics. The purpose of this study was to formulate a real-time assay of purified recombinant ExoS activity for high-throughput application. We characterized the turnover kinetics of the fluorescent dinucleotide 1,N(6)-etheno-NAD+ as co-substrate for ExoS. Further, we found that the toxin relied on any of five tested isoforms of human 14-3-3 to modify vH-Ras and the Rho-family GTPases Rac1, -2, and -3 and RhoC. We then used 14-3-3β-stimulated ExoS modification of vH-Ras to screen a collection of low-molecular-weight compounds selected to target the poly-ADP ribose polymerase family and identified 3-(4-oxo-3,5,6,7-tetrahydro-4H-cyclopenta[4,5]thieno[2,3-d]pyrimidin-2-yl)propanoic acid as an ExoS inhibitor with micromolar potency. Thus, we present an optimized method to screen for inhibitors of ExoS activity that is amenable to high-throughput format and an intermediate affinity inhibitor that can serve both as assay control and as a starting point for further development. PMID:26850638

  16. Structural Analysis of ADP-Glucose Pyrophosphorylase From the Bacterium Agrobacterium Tumefaciens

    SciTech Connect

    Cupp-Vickery, J.R.; Igarashi, R.Y.; Perez, M.; Poland, M.; Meyer, C.R.

    2009-05-14

    ADP-glucose pyrophosphorylase (ADPGlc PPase) catalyzes the conversion of glucose 1-phosphate and ATP to ADP-glucose and pyrophosphate. As a key step in glucan synthesis, the ADPGlc PPases are highly regulated by allosteric activators and inhibitors in accord with the carbon metabolism pathways of the organism. Crystals of Agrobacterium tumefaciens ADPGlc PPase were obtained using lithium sulfate as a precipitant. A complete anomalous selenomethionyl derivative X-ray diffraction data set was collected with unit cell dimensions a = 85.38 {angstrom}, b = 93.79 {angstrom}, and c = 140.29 {angstrom} ({alpha} = {beta} = {gamma} = 90{sup o}) and space group I{sub 222}. The A. tumefaciens ADPGlc PPase model was refined to 2.1 {angstrom} with an R{sub factor} = 22% and R{sub free} = 26.6%. The model consists of two domains: an N-terminal {alpha}{beta}{alpha} sandwich and a C-terminal parallel {beta}-helix. ATP and glucose 1-phosphate were successfully modeled in the proposed active site, and site-directed mutagenesis of conserved glycines in this region (G20, G21, and G23) resulted in substantial loss of activity. The interface between the N- and the C-terminal domains harbors a strong sulfate-binding site, and kinetic studies revealed that sulfate is a competitive inhibitor for the allosteric activator fructose 6-phosphate. These results suggest that the interface between the N- and C-terminal domains binds the allosteric regulator, and fructose 6-phosphate was modeled into this region. The A. tumefaciens ADPGlc PPase/fructose 6-phosphate structural model along with sequence alignment analysis was used to design mutagenesis experiments to expand the activator specificity to include fructose 1,6-bisphosphate. The H379R and H379K enzymes were found to be activated by fructose 1,6-bisphosphate.

  17. 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

  18. Affinity for MgADP and force of unbinding from actin of myosin purified from tonic and phasic smooth muscle

    PubMed Central

    Léguillette, Renaud; Zitouni, Nedjma B.; Govindaraju, Karuthapillai; Fong, Laura M.; Lauzon, Anne-Marie

    2008-01-01

    Smooth muscle is unique in its ability to maintain force at low MgATP consumption. This property, called the latch state, is more prominent in tonic than phasic smooth muscle. Studies performed at the muscle strip level have suggested that myosin from tonic muscle has a greater affinity for MgADP and therefore remains attached to actin longer than myosin from phasic muscle, allowing for cross-bridge dephosphorylation and latch-bridge formation. An alternative hypothesis is that after dephosphorylation, myosin reattaches to actin and maintains force. We investigated these fundamental properties of smooth muscle at the molecular level. We used an in vitro motility assay to measure actin filament velocity (νmax) when propelled by myosin purified from phasic or tonic muscle at increasing [MgADP]. Myosin was 25% thiophosphorylated and 75% unphosphorylated to approximate in vivo conditions. The slope of νmax versus [MgADP] was significantly greater for tonic (−0.51 ± 0.04) than phasic muscle myosin (−0.15 ± 0.04), demonstrating the greater MgADP affinity of myosin from tonic muscle. We then used a laser trap assay to measure the unbinding force from actin of populations of unphosphorylated tonic and phasic muscle myosin. Both myosin types attached to actin, and their unbinding force (0.092 ± 0.022 pN for phasic muscle and 0.084 ± 0.017 pN for tonic muscle) was not statistically different. We conclude that the greater affinity for MgADP of tonic muscle myosin and the reattachment of dephosphorylated myosin to actin may both contribute to the latch state. PMID:18614813

  19. [Influence of ADP-ribose, AMP and adenosine on bioelectric activity of hibernating ground squirrel atrium and papillary muscle].

    PubMed

    Kuz'min, V S; Abramochkin, D V; Sukhova, G S; Rozenshtraukh, L V

    2008-01-01

    The aim of work was to investigate effects of adenosine, AMP and ADP-ribose (1x10(-5)) on bioelectric activity of atrium and papillary muscle of nonhibernating (rat) and hibernating (Yakutian ground squirrel) animals. Action potential (AP) was registered with use of standard microelectrode technique. AP duration (APD) at level of 90% repolarisation in rat atrium in control experiments was 30+/-5 ms, APD at level of 50% repolarisation was 12+/-2 ms. APD at level of 90% repolarisation in rat papillary muscle was 56+/-7 ms, at level of 50% repolarisation was 18+/-2 ms. APD at level of 90% repolarisation in ground squirrel atrium was 77+/-6, APD at level of 50% repolarisation was 38+/-6 ms. APD at level of 90% repolarisation in ground squirrel papillary muscle was 105+/-9 ms, APD at level of 50% repolarisation was 42+/-8 ms. Purine nucleotides and nucleoside, that were tested in work, except ADP-ribose, act as inhibitory factors and decrease APD both in rat and hibernating ground squirrel heart. ADP-ribose decreases APD in papillary muscle of hibernator but did not in its atrium. In ground squirrel atrium AMP and adenosine decrease APD at level of 50% repolarisation by 10+/-3% and 18+/-3% respectively. AMP and adenosine decrease APD at level of 90% repolarisation by 9+/-2% and 11+/-2% respectively. In ground squirrel papillary muscle ADP-ribose, AMP and adenosine decrease APD at level of 50% repolarisation by 26+/-8%, 23+/-8% and 26+/-7%. ADP-ribose, AMP and adenosine decrease APD at level of 90% repolarisation by 12+/-3%, 10+/-3%, 13+/-3%. Thus, decrease of APD in ground squirrel papillary muscle at level of 90% repolarisation during nucleotides and adenosine action was 2-2.5 fold less, than the rat.

  20. 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

  1. 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

  2. The specific, submicromolar-Km ADP-ribose pyrophosphatase purified from human placenta is enzymically indistinguishable from recombinant NUDT9 protein, including a selectivity for Mn2+ as activating cation and increase in Km for ADP-ribose, both elicited by H2O2.

    PubMed

    Carloto, António; Costas, María Jesús; Cameselle, José Carlos; McLennan, Alexander G; Ribeiro, João Meireles

    2006-10-01

    Free ADP-ribose is a putative second messenger and also a potentially toxic compound due to its non-enzymic reactivity towards protein side chains. ADP-ribose hydrolysis is catalysed by NDP-sugar/alcohol pyrophosphatases of differing specificity, including a highly specific, low-K(m) ADP-ribose pyrophosphatase. In humans, a submicromolar-K(m) ADP-ribose pyrophosphatase has been purified from placenta, while recombinant NUDT9 has been described as a similarly specific enzyme with a nudix motif, but with a 10(2)-10(3) higher K(m). Here, a comparative study of both proteins is presented showing that they are in fact enzymically indistinguishable; crucially, they both have submicromolar K(m) for ADP-ribose. This study firmly supports the view that the ADP-ribose pyrophosphatase present in human tissues is a product of the NUDT9 gene. In addition, this study reveals previously unknown properties of both enzyme forms. They display the same, differential properties in the presence of Mg(2+) or Mn(2+) as activating cations with respect to substrate specificity, ADP-ribose saturation kinetics, and inhibition by fluoride. Treatment with H(2)O(2) alters the Mg(2+)/Mn(2+) responses and increases the K(m) values for ADP-ribose, changes that are reversed by DTT. The results are discussed in relation to the proposed roles for ADP-ribose in oxidative/nitrosative stress and for ADP-ribose pyrophosphatase as a protective enzyme whose function is to limit the intracellular accumulation of ADP-ribose.

  3. 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.

  4. Adhesion of platelets to purified solid-phase von Willebrand factor: effects of wall shear rate, ADP, thrombin, and ristocetin.

    PubMed

    Olson, J D; Zaleski, A; Herrmann, D; Flood, P A

    1989-07-01

    When platelets are stimulated with adenosine diphosphate (ADP), thrombin, or ristocetin, they bind soluble von Willebrand factor (vWF). In contrast, platelets adhere to solid-phase vWF without apparent stimulus. This work characterizes the adhesion of washed human platelets to highly purified solid-phase human vWF. VWF (iodine 125-labeled vWF) was demonstrated to bind in a quantifiable fashion to the internal surfaces of glass capillary tubes, saturating at a surface density of 3.0 mg/ml. The multimeric structure of bound vWF was the same as that of normal vWF. Platelets were washed, labeled with indium 111, and resuspended with washed red blood cells (RBCs) in balanced salt solution containing Ca++, Mg++, and apyrase. The washed platelet RBC suspension was aspirated through capillary tubes to which vWF was adsorbed. Adhesion of platelets to adsorbed vWF was directly dependent on the surface density of vWF. Increasing wall shear rate (100 to 5000 sec-1) produced increasing platelet adhesion to maximum reached at 2500 sec-1. Platelets bound to the solid-phase vWF in an irreversible fashion, and, as demonstrated with scanning electron microscopy, they spread on the surface. When used to stimulate the platelets, ADP, thrombin, and ristocetin all increased the platelet adhesion to solid-phase vWF. ADP- and thrombin-stimulated reactions were inhibited by prior treatment of the platelets with 5'-p-fluorosulfonylbenzoyl adenosine. This inhibitor of ADP binding had no effect on the baseline platelet adhesion reaction (without ADP or thrombin). Adenosine in concentration up to 1 mmol/L failed to inhibit adhesion. The data demonstrate that washed platelets adhere to solid-phase vWF without added agonists, that the reaction is dependent on surface density vWF and wall shear rate, that they bind irreversibly, and that they demonstrate surface spreading. In addition, these platelets can be stimulated to increase their adherence to vWF by using ADP, thrombin, and ristocetin.

  5. Physiological conditions conducive to high cyanophycin content in biomass of Acinetobacter calcoaceticus strain ADP1.

    PubMed

    Elbahloul, Yasser; Krehenbrink, Martin; Reichelt, Rudolf; Steinbüchel, Alexander

    2005-02-01

    The effects of the inorganic medium components, the initial pH, the incubation temperature, the oxygen supply, the carbon-to-nitrogen ratio, and chloramphenicol on the synthesis of cyanophycin (CGP) by Acinetobacter calcoaceticus strain ADP1 were studied in a mineral salts medium containing sodium glutamate and ammonium sulfate as carbon and nitrogen sources, respectively. Variation of all these factors resulted in maximum CGP contents of only about 3.5% (wt/wt) of the cell dry matter (CDM), and phosphate depletion triggered CGP accumulation most substantially. However, addition of arginine to the medium as the sole carbon source for growth promoted CGP accumulation most strikingly. This effect was systematically studied, and an optimized phosphate-limited medium containing 75 mM arginine and 10 mM ammonium sulfate yielded a CGP content of 41.4% (wt/wt) of the CDM at 30 degrees C. The CGP content of the cells was further increased to 46.0% (wt/wt) of the CDM by adding 2.5 microg of chloramphenicol per ml of medium in the accumulation phase. These contents are by far the highest CGP contents of bacterial cells ever reported. CGP was easily isolated from the cells by using an acid extraction method, and this CGP contained about equimolar amounts of aspartic acid and arginine and no detectable lysine; the molecular masses ranged from 21 to 29 kDa, and the average molecular mass was about 25 kDa. Transmission electron micrographs of thin sections of cells revealed large CGP granules that frequently had an irregular shape with protuberances at the surface and often severely deformed the cells. A cphI::OmegaKm mutant of strain ADP1 with a disrupted putative cyanophycinase gene accumulated significantly less CGP than the wild type accumulated, although the cells expressed cyanophycin synthetase at about the same high level. It is possible that the intact CphI protein is involved in the release of CGP primer molecules from initially synthesized CGP. The resulting lower

  6. Expression of plasma membrane receptor genes during megakaryocyte development

    PubMed Central

    Sun, Sijie; Wang, Wenjing; Latchman, Yvette; Gao, Dayong; Aronow, Bruce

    2013-01-01

    Megakaryocyte (MK) development is critically informed by plasma membrane-localized receptors that integrate a multiplicity of environmental cues. Given that the current understanding about receptors and ligands involved in megakaryocytopoiesis is based on single targets, we performed a genome-wide search to identify a plasma membrane receptome for developing MKs. We identified 40 transmembrane receptor genes as being upregulated during MK development. Seven of the 40 receptor-associated genes were selected to validate the dataset. These genes included: interleukin-9 receptor (IL9R), transforming growth factor, β receptor II (TGFBR2), interleukin-4 receptor (IL4R), colony stimulating factor-2 receptor-beta (CSFR2B), adiponectin receptor (ADIPOR2), thrombin receptor (F2R), and interleukin-21 receptor (IL21R). RNA and protein analyses confirmed their expression in primary human MKs. Matched ligands to IL9R, TGFBR2, IL4R, CSFR2B, and ADIPOR2 affected megakaryocytopoiesis. IL9 was unique in its ability to increase the number of MKs formed. In contrast, MK colony formation was inhibited by adiponectin, TGF-β, IL4, and GM-CSF. The thrombin-F2R axis affected platelet function, but not MK development, while IL21 had no apparent detectable effects. ADP-induced platelet aggregation was suppressed by IL9, TGF-β, IL4, and adiponectin. Overall, six of seven of the plasma membrane receptors were confirmed to have functional roles in MK and platelet biology. Also, results show for the first time that adiponectin plays a regulatory role in MK development. Together these data support a strong likelihood that the 40 transmembrane genes identified as being upregulated during MK development will be an important resource to the research community for deciphering the complex repertoire of environmental cues regulating megakaryocytopoiesis and/or platelet function. PMID:23321270

  7. 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.

  8. Biochemical and kinetic characterization of the recombinant ADP-forming acetyl coenzyme A synthetase from the amitochondriate protozoan Entamoeba histolytica.

    PubMed

    Jones, Cheryl P; Ingram-Smith, Cheryl

    2014-12-01

    Entamoeba histolytica, an amitochondriate protozoan parasite that relies on glycolysis as a key pathway for ATP generation, has developed a unique extended PPi-dependent glycolytic pathway in which ADP-forming acetyl-coenzyme A (CoA) synthetase (ACD; acetate:CoA ligase [ADP-forming]; EC 6.2.1.13) converts acetyl-CoA to acetate to produce additional ATP and recycle CoA. We characterized the recombinant E. histolytica ACD and found that the enzyme is bidirectional, allowing it to potentially play a role in ATP production or in utilization of acetate. In the acetate-forming direction, acetyl-CoA was the preferred substrate and propionyl-CoA was used with lower efficiency. In the acetyl-CoA-forming direction, acetate was the preferred substrate, with a lower efficiency observed with propionate. The enzyme can utilize both ADP/ATP and GDP/GTP in the respective directions of the reaction. ATP and PPi were found to inhibit the acetate-forming direction of the reaction, with 50% inhibitory concentrations of 0.81 ± 0.17 mM (mean ± standard deviation) and 0.75 ± 0.20 mM, respectively, which are both in the range of their physiological concentrations. ATP and PPi displayed mixed inhibition versus each of the three substrates, acetyl-CoA, ADP, and phosphate. This is the first example of regulation of ACD enzymatic activity, and possible roles for this regulation are discussed.

  9. 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

  10. 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.

  11. Poly ADP-Ribose Polymerase Inhibition Ameliorates Hind Limb Ischemia Reperfusion Injury in a Murine Model of Type 2 Diabetes

    PubMed Central

    Long, Chandler A.; Boloum, Valy; Albadawi, Hassan; Tsai, Shirling; Yoo, Hyung-Jin; Oklu, Rahmi; Goldman, Mitchell H.; Watkins, Michael T.

    2013-01-01

    Introduction Diabetes is known to increase poly-ADP-ribose-polymerase (PARP) activity and posttranslational poly-ADP-ribosylation of several regulatory proteins involved in inflammation and energy metabolism. These experiments test the hypothesis that PARP inhibition will modulate hind limb ischemia reperfusion (IR) in a mouse model of type-II diabetes; ameliorate the ribosylation and the activity/transnuclear localization of the key glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Methods db/db mice underwent 1.5hrs of hind limb ischemia followed by 1, 7, or 24hrs reperfusion. The treatment group received the PARP inhibitor PJ34 (PJ34) over a 24hrs period; the untreated group received Lactated ringer’s (LR) at the same time points. IR muscles were analyzed for indices of PARP activity, fiber injury, metabolic activity, inflammation, GAPDH activity /intracellular localization and poly-ADP-ribosylation of GAPDH. Results PARP activity was significantly lower in the PJ34 treated groups compared to the LR group at 7 and 24 hours reperfusion. There was significantly less muscle fiber injury in the PJ34 treated group compared to LR treated mice at 24 hrs reperfusion. PJ34 lowered levels of select proinflammatory molecules at 7hrs and 24hrs IR. There were significant increases in metabolic activity only at 24 hours IR in the PJ34 group, which temporally correlated with increase in GAPDH activity, decreased GAPDH poly ADP-ribosylation and nuclear translocation of GAPDH. Conclusions PJ34 reduced PARP activity, GAPDH ribosylation, GAPDH translocation, ameliorated muscle fiber injury, and increased metabolic activity following hind limb IR injury in a murine model of type-II diabetes. PARP inhibition might be a therapeutic strategy following IR in diabetic humans. PMID:23549425

  12. Biochemical and Kinetic Characterization of the Recombinant ADP-Forming Acetyl Coenzyme A Synthetase from the Amitochondriate Protozoan Entamoeba histolytica

    PubMed Central

    Jones, Cheryl P.

    2014-01-01

    Entamoeba histolytica, an amitochondriate protozoan parasite that relies on glycolysis as a key pathway for ATP generation, has developed a unique extended PPi-dependent glycolytic pathway in which ADP-forming acetyl-coenzyme A (CoA) synthetase (ACD; acetate:CoA ligase [ADP-forming]; EC 6.2.1.13) converts acetyl-CoA to acetate to produce additional ATP and recycle CoA. We characterized the recombinant E. histolytica ACD and found that the enzyme is bidirectional, allowing it to potentially play a role in ATP production or in utilization of acetate. In the acetate-forming direction, acetyl-CoA was the preferred substrate and propionyl-CoA was used with lower efficiency. In the acetyl-CoA-forming direction, acetate was the preferred substrate, with a lower efficiency observed with propionate. The enzyme can utilize both ADP/ATP and GDP/GTP in the respective directions of the reaction. ATP and PPi were found to inhibit the acetate-forming direction of the reaction, with 50% inhibitory concentrations of 0.81 ± 0.17 mM (mean ± standard deviation) and 0.75 ± 0.20 mM, respectively, which are both in the range of their physiological concentrations. ATP and PPi displayed mixed inhibition versus each of the three substrates, acetyl-CoA, ADP, and phosphate. This is the first example of regulation of ACD enzymatic activity, and possible roles for this regulation are discussed. PMID:25303954

  13. Structures of yeast mitochondrial ADP/ATP carriers support a domain-based alternating-access transport mechanism.

    PubMed

    Ruprecht, Jonathan J; Hellawell, Alex M; Harding, Marilyn; Crichton, Paul G; McCoy, Airlie J; Kunji, Edmund R S

    2014-01-28

    The mitochondrial ADP/ATP carrier imports ADP from the cytosol and exports ATP from the mitochondrial matrix. The carrier cycles by an unresolved mechanism between the cytoplasmic state, in which the carrier accepts ADP from the cytoplasm, and the matrix state, in which it accepts ATP from the mitochondrial matrix. Here we present the structures of the yeast ADP/ATP carriers Aac2p and Aac3p in the cytoplasmic state. The carriers have three domains and are closed at the matrix side by three interdomain salt-bridge interactions, one of which is braced by a glutamine residue. Glutamine braces are conserved in mitochondrial carriers and contribute to an energy barrier, preventing the conversion to the matrix state unless substrate binding occurs. At the cytoplasmic side a second salt-bridge network forms during the transport cycle, as demonstrated by functional analysis of mutants with charge-reversed networks. Analyses of the domain structures and properties of the interdomain interfaces indicate that interconversion between states involves movement of the even-numbered α-helices across the surfaces of the odd-numbered α-helices by rotation of the domains. The odd-numbered α-helices have an L-shape, with proline or serine residues at the kinks, which functions as a lever-arm, coupling the substrate-induced disruption of the matrix network to the formation of the cytoplasmic network. The simultaneous movement of three domains around a central translocation pathway constitutes a unique mechanism among transport proteins. These findings provide a structural description of transport by mitochondrial carrier proteins, consistent with an alternating-access mechanism.

  14. Structures of Mycobacterium Tuberculosis Folylpolyglutamate Synthase Complexed With ADP And AMPPCD

    SciTech Connect

    Young, P.G.; Smith, C.A.; Metcalf, P.; Baker, E.N.

    2009-05-28

    Folate derivatives are essential vitamins for cell growth and replication, primarily because of their central role in reactions of one-carbon metabolism. Folates require polyglutamation to be efficiently retained within the cell and folate-dependent enzymes have a higher affinity for the polyglutamylated forms of this cofactor. Polyglutamylation is dependent on the enzyme folylpolyglutamate synthetase (FPGS), which catalyzes the sequential addition of several glutamates to folate. FPGS is essential for the growth and survival of important bacterial species, including Mycobacterium tuberculosis, and is a potential drug target. Here, the crystal structures of M. tuberculosis FPGS in complex with ADP and AMPPCP are reported at 2.0 and 2.3 angstroms resolution, respectively. The structures reveal a deeply buried nucleotide-binding site, as in the Escherichia coli and Lactobacillus casei FPGS structures, and a long extended groove for the binding of folate substrates. Differences from the E. coli and L. casei FPGS structures are seen in the binding of a key divalent cation, the carbamylation state of an essential lysine side chain and the adoption of an 'open' position by the active-site beta5-alpha6 loop. These changes point to coordinated events that are associated with dihydropteroate/folate binding and the catalysis of the new amide bond with an incoming glutamate residue.

  15. Poly (ADP-Ribose) Polymerase Mediates Diabetes-Induced Retinal Neuropathy

    PubMed Central

    Mohammad, Ghulam; Siddiquei, Mohammad Mairaj

    2013-01-01

    Retinal neuropathy is an early event in the development of diabetic retinopathy. One of the potential enzymes that are activated by oxidative stress in the diabetic retina is poly (ADP-ribose) polymerase (PARP). We investigated the effect of the PARP inhibitor 1,5-isoquinolinediol on the expression of the neurodegeneration mediators and markers in the retinas of diabetic rats. After two weeks of streptozotocin-induced diabetes, rats were treated with 1,5-isoquinolinediol (3 mg/kg/day). After 4 weeks of diabetes, the retinas were harvested and the levels of reactive oxygen species (ROS) were determined fluorometrically and the expressions of PARP, phosporylated-ERK1/2, BDNF, synaptophysin, glutamine synthetase (GS), and caspase-3 were determined by Western blot analysis. Retinal levels of ROS, PARP-1/2, phosphorylated ERK1/2, and cleaved caspase-3 were significantly increased, whereas the expressions of BDNF synaptophysin and GS were significantly decreased in the retinas of diabetic rats, compared to nondiabetic rats. Administration of 1,5-isoquinolinediol did not affect the metabolic status of the diabetic rats, but it significantly attenuated diabetes-induced upregulation of PARP, ROS, ERK1/2 phosphorylation, and cleaved caspase-3 and downregulation of BDNF, synaptophysin, and GS. These findings suggest a beneficial effect of the PARP inhibitor in increasing neurotrophic support and ameliorating early retinal neuropathy induced by diabetes. PMID:24347828

  16. Poly (ADP-ribose) polymerase mediates diabetes-induced retinal neuropathy.

    PubMed

    Mohammad, Ghulam; Siddiquei, Mohammad Mairaj; Abu El-Asrar, Ahmed M

    2013-01-01

    Retinal neuropathy is an early event in the development of diabetic retinopathy. One of the potential enzymes that are activated by oxidative stress in the diabetic retina is poly (ADP-ribose) polymerase (PARP). We investigated the effect of the PARP inhibitor 1,5-isoquinolinediol on the expression of the neurodegeneration mediators and markers in the retinas of diabetic rats. After two weeks of streptozotocin-induced diabetes, rats were treated with 1,5-isoquinolinediol (3 mg/kg/day). After 4 weeks of diabetes, the retinas were harvested and the levels of reactive oxygen species (ROS) were determined fluorometrically and the expressions of PARP, phosporylated-ERK1/2, BDNF, synaptophysin, glutamine synthetase (GS), and caspase-3 were determined by Western blot analysis. Retinal levels of ROS, PARP-1/2, phosphorylated ERK1/2, and cleaved caspase-3 were significantly increased, whereas the expressions of BDNF synaptophysin and GS were significantly decreased in the retinas of diabetic rats, compared to nondiabetic rats. Administration of 1,5-isoquinolinediol did not affect the metabolic status of the diabetic rats, but it significantly attenuated diabetes-induced upregulation of PARP, ROS, ERK1/2 phosphorylation, and cleaved caspase-3 and downregulation of BDNF, synaptophysin, and GS. These findings suggest a beneficial effect of the PARP inhibitor in increasing neurotrophic support and ameliorating early retinal neuropathy induced by diabetes. PMID:24347828

  17. Poly(ADP-ribose) polymerase inhibition reverses vascular dysfunction after {gamma}-irradiation

    SciTech Connect

    Beller, Carsten J. . E-mail: Carsten.Beller@urz.uni-heidelberg.de; Radovits, Tamas; Seres, Leila; Kosse, Jens; Krempien, Robert; Gross, Marie-Luise; Penzel, Roland; Berger, Irina; Huber, Peter E.; Hagl, Siegfried; Szabo, Csaba; Szabo, Gabor

    2006-08-01

    Purpose: The generation of reactive oxygen species during {gamma}-irradiation may induce DNA damage, leading to activation of the nuclear enzyme poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) culminating in endothelial dysfunction. In the present study, we assessed the effect of PARP inhibition on changes in vascular function after acute and short-term irradiation. Methods and Materials: In the acute experiments, aortic rings were exposed to 20 Gy of {gamma}-irradiation. The aortae were harvested after 1 or 7 days. Two additional groups received the ultrapotent PARP inhibitor, INO-1001, for 1 or 7 days after irradiation. The aortic rings were precontracted by phenylephrine and relaxation to acetylcholine and sodium nitroprusside were studied. Results: The vasoconstrictor response to phenylephrine was significantly lower both acutely and 1 and 7 days after irradiation. Vasorelaxation to acetylcholine and sodium nitroprusside was not impaired acutely after irradiation. One and seven days after irradiation, vasorelaxation to acetylcholine and sodium nitroprusside was significantly enhanced. Treatment with INO-1001 reversed vascular dysfunction after irradiation. Conclusion: Vascular dysfunction was observed 1 and 7 days after irradiation, as evidenced by reduced vasoconstriction, coupled with endothelium-dependent and -independent hyperrelaxation. PARP inhibition restored vascular function and may, therefore, be suitable to reverse vascular dysfunction after irradiation.

  18. Poly(ADP-Ribose)Polymerase Activity Controls Plant Growth by Promoting Leaf Cell Number

    PubMed Central

    Schulz, Philipp; Jansseune, Karel; Degenkolbe, Thomas; Méret, Michaël; Claeys, Hannes; Skirycz, Aleksandra; Teige, Markus; Willmitzer, Lothar; Hannah, Matthew A.

    2014-01-01

    A changing global environment, rising population and increasing demand for biofuels are challenging agriculture and creating a need for technologies to increase biomass production. Here we demonstrate that the inhibition of poly (ADP-ribose) polymerase activity is a promising technology to achieve this under non-stress conditions. Furthermore, we investigate the basis of this growth enhancement via leaf series and kinematic cell analysis as well as single leaf transcriptomics and plant metabolomics under non-stress conditions. These data indicate a regulatory function of PARP within cell growth and potentially development. PARP inhibition enhances growth of Arabidopsis thaliana by enhancing the cell number. Time course single leaf transcriptomics shows that PARP inhibition regulates a small subset of genes which are related to growth promotion, cell cycle and the control of metabolism. This is supported by metabolite analysis showing overall changes in primary and particularly secondary metabolism. Taken together the results indicate a versatile function of PARP beyond its previously reported roles in controlling plant stress tolerance and thus can be a useful target for enhancing biomass production. PMID:24587323

  19. Green Tea Polyphenols Control Dysregulated Glutamate Dehydrogenase in Transgenic Mice by Hijacking the ADP Activation Site

    SciTech Connect

    Li, Changhong; Li, Ming; Chen, Pan; Narayan, Srinivas; Matschinsky, Franz M.; Bennett, Michael J.; Stanley, Charles A.; Smith, Thomas J.

    2012-05-09

    Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and, in animals, is extensively regulated by a number of metabolites. Gain of function mutations in GDH that abrogate GTP inhibition cause the hyperinsulinism/hyperammonemia syndrome (HHS), resulting in increased pancreatic {beta}-cell responsiveness to leucine and susceptibility to hypoglycemia following high protein meals. We have previously shown that two of the polyphenols from green tea (epigallocatechin gallate (EGCG) and epicatechin gallate (ECG)) inhibit GDH in vitro and that EGCG blocks GDH-mediated insulin secretion in wild type rat islets. Using structural and site-directed mutagenesis studies, we demonstrate that ECG binds to the same site as the allosteric regulator, ADP. Perifusion assays using pancreatic islets from transgenic mice expressing a human HHS form of GDH demonstrate that the hyperresponse to glutamine caused by dysregulated GDH is blocked by the addition of EGCG. As observed in HHS patients, these transgenic mice are hypersensitive to amino acid feeding, and this is abrogated by oral administration of EGCG prior to challenge. Finally, the low basal blood glucose level in the HHS mouse model is improved upon chronic administration of EGCG. These results suggest that this common natural product or some derivative thereof may prove useful in controlling this genetic disorder. Of broader clinical implication is that other groups have shown that restriction of glutamine catabolism via these GDH inhibitors can be useful in treating various tumors. This HHS transgenic mouse model offers a highly useful means to test these agents in vivo.

  20. Interplay between Ubiquitin, SUMO, and Poly(ADP-Ribose) in the Cellular Response to Genotoxic Stress

    PubMed Central

    Pellegrino, Stefania; Altmeyer, Matthias

    2016-01-01

    Cells employ a complex network of molecular pathways to cope with endogenous and exogenous genotoxic stress. This multilayered response ensures that genomic lesions are efficiently detected and faithfully repaired in order to safeguard genome integrity. The molecular choreography at sites of DNA damage relies heavily on post-translational modifications (PTMs). Protein modifications with ubiquitin and the small ubiquitin-like modifier SUMO have recently emerged as important regulatory means to coordinate DNA damage signaling and repair. Both ubiquitylation and SUMOylation can lead to extensive chain-like protein modifications, a feature that is shared with yet another DNA damage-induced PTM, the modification of proteins with poly(ADP-ribose) (PAR). Chains of ubiquitin, SUMO, and PAR all contribute to the multi-protein assemblies found at sites of DNA damage and regulate their spatio-temporal dynamics. Here, we review recent advancements in our understanding of how ubiquitin, SUMO, and PAR coordinate the DNA damage response and highlight emerging examples of an intricate interplay between these chain-like modifications during the cellular response to genotoxic stress. PMID:27148359

  1. Conserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins

    PubMed Central

    Atherton, Joseph; Farabella, Irene; Yu, I-Mei; Rosenfeld, Steven S; Houdusse, Anne; Topf, Maya; Moores, Carolyn A

    2014-01-01

    Kinesins are a superfamily of microtubule-based ATP-powered motors, important for multiple, essential cellular functions. How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles—including their nucleotide-free states—at ∼7 Å resolution using cryo-electron microscopy. In both motors, microtubule binding promotes ordered conformations of conserved loops that stimulate ADP release, enhance microtubule affinity and prime the catalytic site for ATP binding. ATP binding causes only small shifts of these nucleotide-coordinating loops but induces large conformational changes elsewhere that allow force generation and neck linker docking towards the microtubule plus end. Family-specific differences across the kinesin–microtubule interface account for the distinctive properties of each motor. Our data thus provide evidence for a conserved ATP-driven mechanism for kinesins and reveal the critical mechanistic contribution of the microtubule interface. DOI: http://dx.doi.org/10.7554/eLife.03680.001 PMID:25209998

  2. Poly(ADP-Ribose) Polymerase Is a Substrate Recognized by Two Metacaspases of Podospora anserina

    PubMed Central

    Strobel, Ingmar

    2013-01-01

    The two metacaspases MCA1 and MCA2 of the fungal aging model organism Podospora anserina (PaMCA1 and PaMCA2, respectively) have previously been demonstrated to be involved in the control of programmed cell death (PCD) and life span. In order to identify specific pathways and components which are controlled by the activity of these enzymes, we set out to characterize them further. Heterologous overexpression in Escherichia coli of the two metacaspase genes resulted in the production of proteins which aggregate and form inclusion bodies from which the active protein has been recovered via refolding in appropriate buffers. The renaturated proteins are characterized by an arginine-specific activity and are active in caspase-like self-maturation leading to the generation of characteristic small protein fragments. Both activities are dependent on the presence of calcium. Incubation of the two metacaspases with recombinant poly(ADP-ribose) polymerase (PARP), a known substrate of mammalian caspases, led to the identification of PARP as a substrate of the two P. anserina proteases. Using double mutants in which P. anserina Parp (PaParp) is overexpressed and PaMca1 is either overexpressed or deleted, we provide evidence for in vivo degradation of PaPA