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Peter Simmonds - One of the best experts on this subject based on the ideXlab platform.
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an adaptive compromise conflicting evolutionary pressures on arthropod borne zika virus Dinucleotide composition in mammalian hosts and mosquito vectors
bioRxiv, 2021Co-Authors: Jelke J Fros, I Visser, Bing Tang, Kexin Yan, Eri Nakayama, Tessa M Visser, C J Koenraadt, Gorben P Pijlman, Andreas Suhrbier, Peter SimmondsAbstract:Most vertebrate RNA viruses show pervasive suppression of CpG and UpA Dinucleotides, closely resembling the Dinucleotide composition of host cell transcriptomes. In contrast, CpG suppression is absent in both invertebrate mRNA and RNA viruses that exclusively infect arthropods. Arthropod-borne (arbo) viruses are transmitted between vertebrate hosts by invertebrate vectors and thus encounter potentially conflicting evolutionary pressures in the different cytoplasmic environments. Using a newly developed Zika virus (ZIKV) model, we have investigated how demands for CpG suppression in vertebrate cells can be reconciled with potentially quite different compositional requirements in invertebrates, and how this affects ZIKV replication and transmission. Mutant viruses with synonymously elevated CpG or UpA Dinucleotide frequencies showed attenuated replication in vertebrate cell lines, which was rescued by knockout of the zinc-finger antiviral protein (ZAP). Conversely, in mosquito cells, ZIKV mutants with elevated CpG Dinucleotide frequencies showed substantially enhanced replication compared to wildtype. Host-driven effects on virus replication attenuation and enhancement were even more apparent in mouse and mosquito models. Infections with CpG-or UpA-high ZIKV mutants in mice did not cause typical ZIKV-induced tissue damage and completely protected mice during subsequent challenge with wildtype virus, which demonstrates their potential as live-attenuated vaccines. In contrast, the CpG-high mutants displayed enhanced replication in Aedes aegypti mosquitoes and a larger proportion of mosquitoes carried infectious virus in their saliva. These findings show that mosquito cells are also capable of discriminating RNA based on Dinucleotide composition. However, the evolutionary pressure on the CpG Dinucleotides of viral genomes in arthropod vectors directly opposes the pressure present in vertebrate host cells, which provides evidence that an adaptive compromise is required for arbovirus transmission. This suggests that the genome composition of arthropod-borne flaviviruses is crucial to maintain the balance between high-level replication in the vertebrate host and persistent replication in the mosquito vector.
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association of zinc finger antiviral protein binding to viral genomic rna with attenuation of replication of echovirus 7
mSphere, 2021Co-Authors: Niluka Goonawardane, Dung Nguyen, Peter SimmondsAbstract:Previous studies have implicated both zinc finger antiviral protein (ZAP) and oligoadenylate synthetase 3 (OAS3)/RNase L in the attenuation of RNA viruses with elevated CpG and UpA Dinucleotides. Mechanisms and interrelationships between these two pathways were investigated using an echovirus 7 (E7) replicon with compositionally modified sequences inserted into the 3' untranslated region. ZAP and OAS3 immunoprecipitation (IP) assays provided complementary data on Dinucleotide composition effects on binding. Elevated frequencies of alternative pyrimidine/purine (CpA and UpG) and reversed (GpC and ApU) Dinucleotides showed no attenuating effect on replication or specific binding to ZAP by IP. However, the bases 3' and 5' of CpG motifs influenced replication and ZAP binding; UCGU enhanced CpG-mediated attenuation and ZAP binding, while A residues shielded CpGs from ZAP recognition. Attenuating effects of elevated frequencies of UpA on replication occurred independently of CpG Dinucleotides and bound noncompetitively with CpG-enriched RNA, consistent with a separate recognition site from CpG. Remarkably, immunoprecipitation with OAS3 antibody reproduced the specific binding to CpG- and UpA-enriched RNA sequences. However, OAS3 and ZAP were coimmunoprecipitated in both ZAP and OAS3 IP and colocalized with E7 and stress granules (SGs) by confocal microscopy analysis of infected cells. ZAP's association with larger cellular complexes may mediate the recruitment of OAS3/RNase L, KHNYN, and other RNA degradation pathways.IMPORTANCE We recently discovered that the OAS3/RNase L antiviral pathway is essential for restriction of CpG- and UpA-enriched viruses, in addition to the requirement for zinc finger antiviral protein (ZAP). The current study provides evidence for the specific Dinucleotide and wider recognition contexts associated with virus recognition and attenuation. It further documents the association of ZAP and OAS3 and association with stress granules and a wider protein interactome that may mediate antiviral effects in different cellular compartments. The study provides a striking reconceptualization of the pathways associated with this aspect of antiviral defense.
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human cytomegalovirus evades zap detection by suppressing cpg Dinucleotides in the major immediate early 1 gene
PLOS Pathogens, 2020Co-Authors: Yaotang Lin, Peter Simmonds, Stephen Chiweshe, Dominique Mccormick, Anna Raper, Arthur Wickenhagen, Victor Defillipis, Eleanor Gaunt, Sam J Wilson, Finn GreyAbstract:The genomes of RNA and small DNA viruses of vertebrates display significant suppression of CpG Dinucleotide frequencies. Artificially increasing Dinucleotide frequencies results in substantial attenuation of virus replication, suggesting that these compositional changes may facilitate recognition of non-self RNA sequences. Recently, the interferon inducible protein ZAP, was identified as the host factor responsible for sensing CpG in viral RNA, through direct binding and possibly downstream targeting for degradation. Using an arrayed interferon stimulated gene expression library screen, we identified ZAPS, and its associated factor TRIM25, as inhibitors of human cytomegalovirus (HCMV) replication. Exogenous expression of ZAPS and TRIM25 significantly reduced virus replication while knockdown resulted in increased virus replication. HCMV displays a strikingly heterogeneous pattern of CpG representation with specific suppression of CpG motifs within the IE1 major immediate early transcript which is absent in subsequently expressed genes. We demonstrated that suppression of CpG Dinucleotides in the IE1 gene allows evasion of inhibitory effects of ZAP. We show that acute virus replication is mutually exclusive with high levels of cellular ZAP, potentially explaining the higher levels of CpG in viral genes expressed subsequent to IE1 due to the loss of pressure from ZAP in infected cells. Finally, we show that TRIM25 regulates alternative splicing between the ZAP short and long isoforms during HCMV infection and interferon induction, with knockdown of TRIM25 resulting in decreased ZAPS and corresponding increased ZAPL expression. These results demonstrate for the first time that ZAP is a potent host restriction factor against large DNA viruses and that HCMV evades ZAP detection through suppression of CpG Dinucleotides within the major immediate early 1 transcript. Furthermore, TRIM25 is required for efficient upregulation of the interferon inducible short isoform of ZAP through regulation of alternative splicing.
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association of zinc finger antiviral protein zap binding to viral genomic rna with attenuation of replication of echovirus 7
bioRxiv, 2020Co-Authors: Niluka Goonawardane, Dung Nguyen, Peter SimmondsAbstract:Previous studies have implicated both zinc finger antiviral protein (ZAP) and oligoadenylate synthetase 3 (OAS3)/RNASeL in the attenuation of RNA viruses with elevated CpG and UpA Dinucleotides. Mechanisms and inter-relationships between these two pathways were investigated using an echovirus 7 (E7) replicon with compositionally modified sequences inserted into the 39 untranslated region. ZAP and OAS3 immunoprecipitation (IP) assays provided complementary data on Dinucleotide composition effects on binding. Elevated frequencies of alternative pyrimidine/purine (CpA and UpG) and reversed (GpC and ApU) Dinucleotides showed no attenuating effect nor specific binding to ZAP by IP. However, the bases 39 and 59 to CpG motifs influenced replication and ZAP binding; UCGU enhanced CpG-mediated attenuation and ZAP-binding while A residues shielded CpGs from ZAP recognition. Attenuating effects of elevated frequencies of UpA on replication occurred independently of CpG Dinucleotides and bound non-competitively with CpG-enriched RNA consistent with a separate recognition site from CpG. Remarkably, immunoprecipitation with OAS3 antibody reproduced the specific binding to CpG- and UpA-enriched RNA sequences. However, OAS3 and ZAP were coimmunoprecipitated in both ZAP and OAS3 IP, and colocalised with E7 and stress granules (SGs) by confocal microscopy analysis of infected cells. ZAP9s association with larger cellular complexes may mediate the recruitment of OAS3/RNAseL, KHNYN and other RNA degradation pathways.
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cpg and upa Dinucleotides in both coding and non coding regions of echovirus 7 inhibit replication initiation post entry
eLife, 2017Co-Authors: Jelke J Fros, Isabelle Dietrich, Kinda Alshaikhahmed, Tim Casper Passchier, David J A Evans, Peter SimmondsAbstract:Most vertebrate and plant RNA and small DNA viruses suppress genomic CpG and UpA Dinucleotide frequencies, apparently mimicking host mRNA composition. Artificially increasing CpG/UpA Dinucleotides attenuates viruses through an entirely unknown mechanism. Using the echovirus 7 (E7) model in several cell types, we show that the restriction in E7 replication in mutants with increased CpG/UpA Dinucleotides occurred immediately after viral entry, with incoming virions failing to form replication complexes. Sequences of CpG/UpA-high virus stocks showed no evidence of increased mutational errors that would render them replication defective, these viral RNAs were not differentially sequestered in cytoplasmic stress granules nor did they induce a systemic antiviral state. Importantly, restriction was not mediated through effects on translation efficiency since replicons with high CpG/UpA sequences inserted into a non-coding region were similarly replication defective. Host-cells thus possess intrinsic defence pathways that prevent replication of viruses with increased CpG/UpA frequencies independently of codon usage.
Shandar Ahmad - One of the best experts on this subject based on the ideXlab platform.
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Prediction of Dinucleotide-specific RNA-binding sites in proteins.
BMC bioinformatics, 2011Co-Authors: Michael Fernandez, Yutaro Kumagai, Daron M Standley, Akinori Sarai, Kenji Mizuguchi, Shandar AhmadAbstract:Regulation of gene expression, protein synthesis, replication and assembly of many viruses involve RNA-protein interactions. Although some successful computational tools have been reported to recognize RNA binding sites in proteins, the problem of specificity remains poorly investigated. After the nucleotide base composition, the Dinucleotide is the smallest unit of RNA sequence information and many RNA-binding proteins simply bind to regions enriched in one Dinucleotide. Interaction preferences of protein subsequences and Dinucleotides can be inferred from protein-RNA complex structures, enabling a training-based prediction approach. We analyzed basic statistics of amino acid-Dinucleotide contacts in protein-RNA complexes and found their pairing preferences could be identified. Using a standard approach to represent protein subsequences by their evolutionary profile, we trained neural networks to predict multiclass target vectors corresponding to 16 possible contacting Dinucleotide subsequences. In the cross-validation experiments, the accuracies of the optimum network, measured as areas under the curve (AUC) of the receiver operating characteristic (ROC) graphs, were in the range of 65-80%. Dinucleotide-specific contact predictions have also been extended to the prediction of interacting protein and RNA fragment pairs, which shows the applicability of this method to predict targets of RNA-binding proteins. A web server predicting the 16-dimensional contact probability matrix directly from a user-defined protein sequence was implemented and made available at: http://tardis.nibio.go.jp/netasa/srcpred.
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Prediction of Dinucleotide-specific RNA-binding sites in proteins
BMC Bioinformatics, 2011Co-Authors: Michael Fernandez, Yutaro Kumagai, Daron M Standley, Akinori Sarai, Kenji Mizuguchi, Shandar AhmadAbstract:Abstract Background Regulation of gene expression, protein synthesis, replication and assembly of many viruses involve RNA–protein interactions. Although some successful computational tools have been reported to recognize RNA binding sites in proteins, the problem of specificity remains poorly investigated. After the nucleotide base composition, the Dinucleotide is the smallest unit of RNA sequence information and many RNA-binding proteins simply bind to regions enriched in one Dinucleotide. Interaction preferences of protein subsequences and Dinucleotides can be inferred from protein-RNA complex structures, enabling a training-based prediction approach. Results We analyzed basic statistics of amino acid-Dinucleotide contacts in protein-RNA complexes and found their pairing preferences could be identified. Using a standard approach to represent protein subsequences by their evolutionary profile, we trained neural networks to predict multiclass target vectors corresponding to 16 possible contacting Dinucleotide subsequences. In the cross-validation experiments, the accuracies of the optimum network, measured as areas under the curve (AUC) of the receiver operating characteristic (ROC) graphs, were in the range of 65-80%. Conclusions Dinucleotide-specific contact predictions have also been extended to the prediction of interacting protein and RNA fragment pairs, which shows the applicability of this method to predict targets of RNA-binding proteins. A web server predicting the 16-dimensional contact probability matrix directly from a user-defined protein sequence was implemented and made available at: http://tardis.nibio.go.jp/netasa/srcpred.
M. Teresa Miras-portugal - One of the best experts on this subject based on the ideXlab platform.
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Ca2+ signalling in brain synaptosomes activated by Dinucleotides.
The Journal of membrane biology, 2003Co-Authors: M. Teresa Miras-portugal, Jesús Pintor, Javier GualixAbstract:Diadenosine polyphosphates are a family of Dinucleotides formed by two adenosines joined by a variable number of phosphates. Diadenosine tetraphosphate, Ap4A, diadenosine pentaphosphate Ap5A, and diadenosine hexaphosphate, Ap6A, are stored in synaptic vesicles and are released upon nerve terminal depolarization. At the extracellular level, diadenosine polyphosphates can stimulate presynaptic Dinucleotide receptors. Responses to diadenosine polyphosphates have been described in isolated synaptic terminals (synaptosomes) from several brain areas in different animal species, including man. Dinucleotide receptors are ligand-operated ion channels that allow the influx of cations into the terminals. These cations reach a threshold for N- and P/Q-type voltage-dependent calcium channels, which become activated. The activation of the Dinucleotide receptor together with the activation of these calcium channels triggers the release of neurotransmitters. The ability of Ap5A to promote glutamate, GABA or acetylcholine release has been recently described by the present authors in rat midbrain synaptosomes.
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Ca^2+ Signalling in Brain Synaptosomes Activated by Dinucleotides
The Journal of Membrane Biology, 2003Co-Authors: M. Teresa Miras-portugal, Jesús Pintor, Javier GualixAbstract:Diadenosine polyphosphates are a family of Dinucleotides formed by two adenosines joined by a variable number of phosphates. Diadenosine tetraphosphate, Ap_4A, diadenosine pentaphosphate Ap_5A, and diadenosine hexaphosphate, Ap_6A, are stored in synaptic vesicles and are released upon nerve terminal depolarization. At the extracellular level, diadenosine polyphosphates can stimulate presynaptic Dinucleotide receptors. Responses to diadenosine polyphosphates have been described in isolated synaptic terminals (synaptosomes) from several brain areas in different animal species, including man. Dinucleotide receptors are ligand-operated ion channels that allow the influx of cations into the terminals. These cations reach a threshold for N- and P/Q-type voltage-dependent calcium channels, which become activated. The activation of the Dinucleotide receptor together with the activation of these calcium channels triggers the release of neurotransmitters. The ability of Ap_5A to promote glutamate, GABA or acetylcholine release has been recently described by the present authors in rat midbrain synaptosomes.
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Diinosine polyphosphates, a group of Dinucleotides with antagonistic effects on diadenosine polyphosphate receptor.
Molecular Pharmacology, 1997Co-Authors: Jesús Pintor, Javier Gualix, M. Teresa Miras-portugalAbstract:A new family of Dinucleotide derivatives, diinosine polyphosphates, has been synthesized through the use of the enzyme 5′ adenylic acid deaminase from Aspergillus sp., starting from the corresponding diadenosine polyphosphates. Functional studies were performed on rat brain synaptic terminals in which a Dinucleotide receptor has been described that is specific for adenine Dinucleotides. The results demonstrated that diinosine polyphosphates did not behave as agonists on the diadenosine polyphosphate receptor (also know as P4 purinoceptor), but they were very efficient as antagonists in abolishing the Ca2+ responses elicited by diadenosine pentaphosphate. The IC50 values for diinosine triphosphate, diinosine tetraphosphate, and diinosine pentaphosphate were 4.90 ± 0.10 μm, 8.33 ± 0.22 μm, and 4.23 ± 0.12 nm, respectively. The diinosine polyphosphates also antagonized the ATP receptors present in synaptic terminals, showing IC50values of 100.08 ± 5.72 μm for diinosine triphosphate, 29.51 ± 1.40 μm for diinosine tetraphosphate and 27.75 ± 1.65 μm for diinosine pentaphosphate. The antagonistic ability of these diinosine nucleotides was studied in comparison with other P1 and P2 purinoceptor antagonists, such as suramin, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid, and 8-cyclopentyl-1,3-dipropylxanthine. These purinergic antagonists did not inhibit the response of the P4 purinoceptor; only the diinosine polyphosphates were able to act as antagonists on the Dinucleotide receptor. Suramin and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid attenuated the responses elicited by ATP, as did the diinosine polyphosphate compounds. The most antagonistic diinosine polyphosphate for the Dinucleotide and ATP receptors was diinosine pentaphosphate, which was 6000 times more selective for the P4 purinoceptor than it was for the ATP receptor.
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Diadenosine polyphosphate-activated inward and outward currents in follicular oocytes of Xenopus laevis
Life Sciences, 1996Co-Authors: Jesús Pintor, M. Teresa Miras-portugal, A U Ziganshin, B F King, Geoffrey BurnstockAbstract:Abstract Ionic currents evoked by α,ω-adenine Dinucleotides (Ap X A; X = 2–6) in follicular oocytes of Xenopus laevis were studied under voltage-clamp conditions. Dinucleotides evoked inward and outward currents in Xenopus oocytes by activating native P 1 and P 2 purinoceptors known to be present on the follicle cell monolayer enveloping oocytes. Inward currents were mediated by a suramin-sen skive P 2 purinoceptor which showed an agonist potency order (at 10μM): Ap 4 A>ATP>Ap 3 A>Ap 5 A, while Ap 2 A and Ap 6 A were inactive. Outward currents were mediated by a novel theophylline-sensitive P 1 purinoceptor which showed an agonist potency order (at 10μM): Ap 2 A>ATP>Ap 4 A=Ap 5 A=Ap 6 A>Ap 3 A. Chromatographic analysis confirmed ectonucleotidase activity at the follicle cell layer of oocytes but at a very low rate of Dinucleotide cleavage, indicating that currents evoked by Dinucleotides resulted from a direct activation of oocyte P 1 and P 2 purinoceptors and not through their breakdown to ATP, ADP and AMP. There was no evidence for specific receptors (i.e., P 4 purinoceptors) for diadenosine polyphosphates in Xenopus oocytes.
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Diadenosine polyphosphate-activated inward and outward currents in follicular oocytes of Xenopus laevis.
Life sciences, 1996Co-Authors: Jesús Pintor, M. Teresa Miras-portugal, A U Ziganshin, B F King, Geoffrey BurnstockAbstract:Ionic currents evoked by alpha, omega-adenine Dinucleotides (ApXA; X = 2-6) in follicular oocytes of Xenopus laevis were studied under voltage-clamp conditions. Dinucleotides evoked inward and outward currents in Xenopus oocytes by activating native P1 and P2 purinoceptors known to be present on the follicle cell monolayer enveloping oocytes. Inward currents were mediated by a suramin-sensitive P2 purinoceptor which showed an agonist potency order (at 10 microM): Ap4A > ATP > Ap3A > > Ap5A, while Ap2A and Ap6A were inactive. Outward currents were mediated by a novel theophylline-sensitive P1 purinoceptor which showed an agonist potency order (at 10 microM): Ap2A > ATP > > Ap4A = Ap5A = Ap6A > Ap3A. Chromatographic analysis confirmed ectonucleotidase activity at the follicle cell layer of oocytes but at a very low rate of Dinucleotide cleavage, indicating that currents evoked by Dinucleotides resulted from a direct activation of oocyte P1 and P2 purinoceptors and not through their breakdown to ATP, ADP and AMP. There was no evidence for specific receptors (i.e., P4 purinoceptors) for diadenosine polyphosphates in Xenopus oocytes.
Jesús Pintor - One of the best experts on this subject based on the ideXlab platform.
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Ca2+ signalling in brain synaptosomes activated by Dinucleotides.
The Journal of membrane biology, 2003Co-Authors: M. Teresa Miras-portugal, Jesús Pintor, Javier GualixAbstract:Diadenosine polyphosphates are a family of Dinucleotides formed by two adenosines joined by a variable number of phosphates. Diadenosine tetraphosphate, Ap4A, diadenosine pentaphosphate Ap5A, and diadenosine hexaphosphate, Ap6A, are stored in synaptic vesicles and are released upon nerve terminal depolarization. At the extracellular level, diadenosine polyphosphates can stimulate presynaptic Dinucleotide receptors. Responses to diadenosine polyphosphates have been described in isolated synaptic terminals (synaptosomes) from several brain areas in different animal species, including man. Dinucleotide receptors are ligand-operated ion channels that allow the influx of cations into the terminals. These cations reach a threshold for N- and P/Q-type voltage-dependent calcium channels, which become activated. The activation of the Dinucleotide receptor together with the activation of these calcium channels triggers the release of neurotransmitters. The ability of Ap5A to promote glutamate, GABA or acetylcholine release has been recently described by the present authors in rat midbrain synaptosomes.
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Ca^2+ Signalling in Brain Synaptosomes Activated by Dinucleotides
The Journal of Membrane Biology, 2003Co-Authors: M. Teresa Miras-portugal, Jesús Pintor, Javier GualixAbstract:Diadenosine polyphosphates are a family of Dinucleotides formed by two adenosines joined by a variable number of phosphates. Diadenosine tetraphosphate, Ap_4A, diadenosine pentaphosphate Ap_5A, and diadenosine hexaphosphate, Ap_6A, are stored in synaptic vesicles and are released upon nerve terminal depolarization. At the extracellular level, diadenosine polyphosphates can stimulate presynaptic Dinucleotide receptors. Responses to diadenosine polyphosphates have been described in isolated synaptic terminals (synaptosomes) from several brain areas in different animal species, including man. Dinucleotide receptors are ligand-operated ion channels that allow the influx of cations into the terminals. These cations reach a threshold for N- and P/Q-type voltage-dependent calcium channels, which become activated. The activation of the Dinucleotide receptor together with the activation of these calcium channels triggers the release of neurotransmitters. The ability of Ap_5A to promote glutamate, GABA or acetylcholine release has been recently described by the present authors in rat midbrain synaptosomes.
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Diinosine polyphosphates, a group of Dinucleotides with antagonistic effects on diadenosine polyphosphate receptor.
Molecular Pharmacology, 1997Co-Authors: Jesús Pintor, Javier Gualix, M. Teresa Miras-portugalAbstract:A new family of Dinucleotide derivatives, diinosine polyphosphates, has been synthesized through the use of the enzyme 5′ adenylic acid deaminase from Aspergillus sp., starting from the corresponding diadenosine polyphosphates. Functional studies were performed on rat brain synaptic terminals in which a Dinucleotide receptor has been described that is specific for adenine Dinucleotides. The results demonstrated that diinosine polyphosphates did not behave as agonists on the diadenosine polyphosphate receptor (also know as P4 purinoceptor), but they were very efficient as antagonists in abolishing the Ca2+ responses elicited by diadenosine pentaphosphate. The IC50 values for diinosine triphosphate, diinosine tetraphosphate, and diinosine pentaphosphate were 4.90 ± 0.10 μm, 8.33 ± 0.22 μm, and 4.23 ± 0.12 nm, respectively. The diinosine polyphosphates also antagonized the ATP receptors present in synaptic terminals, showing IC50values of 100.08 ± 5.72 μm for diinosine triphosphate, 29.51 ± 1.40 μm for diinosine tetraphosphate and 27.75 ± 1.65 μm for diinosine pentaphosphate. The antagonistic ability of these diinosine nucleotides was studied in comparison with other P1 and P2 purinoceptor antagonists, such as suramin, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid, and 8-cyclopentyl-1,3-dipropylxanthine. These purinergic antagonists did not inhibit the response of the P4 purinoceptor; only the diinosine polyphosphates were able to act as antagonists on the Dinucleotide receptor. Suramin and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid attenuated the responses elicited by ATP, as did the diinosine polyphosphate compounds. The most antagonistic diinosine polyphosphate for the Dinucleotide and ATP receptors was diinosine pentaphosphate, which was 6000 times more selective for the P4 purinoceptor than it was for the ATP receptor.
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Diadenosine polyphosphate-activated inward and outward currents in follicular oocytes of Xenopus laevis
Life Sciences, 1996Co-Authors: Jesús Pintor, M. Teresa Miras-portugal, A U Ziganshin, B F King, Geoffrey BurnstockAbstract:Abstract Ionic currents evoked by α,ω-adenine Dinucleotides (Ap X A; X = 2–6) in follicular oocytes of Xenopus laevis were studied under voltage-clamp conditions. Dinucleotides evoked inward and outward currents in Xenopus oocytes by activating native P 1 and P 2 purinoceptors known to be present on the follicle cell monolayer enveloping oocytes. Inward currents were mediated by a suramin-sen skive P 2 purinoceptor which showed an agonist potency order (at 10μM): Ap 4 A>ATP>Ap 3 A>Ap 5 A, while Ap 2 A and Ap 6 A were inactive. Outward currents were mediated by a novel theophylline-sensitive P 1 purinoceptor which showed an agonist potency order (at 10μM): Ap 2 A>ATP>Ap 4 A=Ap 5 A=Ap 6 A>Ap 3 A. Chromatographic analysis confirmed ectonucleotidase activity at the follicle cell layer of oocytes but at a very low rate of Dinucleotide cleavage, indicating that currents evoked by Dinucleotides resulted from a direct activation of oocyte P 1 and P 2 purinoceptors and not through their breakdown to ATP, ADP and AMP. There was no evidence for specific receptors (i.e., P 4 purinoceptors) for diadenosine polyphosphates in Xenopus oocytes.
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Diadenosine polyphosphate-activated inward and outward currents in follicular oocytes of Xenopus laevis.
Life sciences, 1996Co-Authors: Jesús Pintor, M. Teresa Miras-portugal, A U Ziganshin, B F King, Geoffrey BurnstockAbstract:Ionic currents evoked by alpha, omega-adenine Dinucleotides (ApXA; X = 2-6) in follicular oocytes of Xenopus laevis were studied under voltage-clamp conditions. Dinucleotides evoked inward and outward currents in Xenopus oocytes by activating native P1 and P2 purinoceptors known to be present on the follicle cell monolayer enveloping oocytes. Inward currents were mediated by a suramin-sensitive P2 purinoceptor which showed an agonist potency order (at 10 microM): Ap4A > ATP > Ap3A > > Ap5A, while Ap2A and Ap6A were inactive. Outward currents were mediated by a novel theophylline-sensitive P1 purinoceptor which showed an agonist potency order (at 10 microM): Ap2A > ATP > > Ap4A = Ap5A = Ap6A > Ap3A. Chromatographic analysis confirmed ectonucleotidase activity at the follicle cell layer of oocytes but at a very low rate of Dinucleotide cleavage, indicating that currents evoked by Dinucleotides resulted from a direct activation of oocyte P1 and P2 purinoceptors and not through their breakdown to ATP, ADP and AMP. There was no evidence for specific receptors (i.e., P4 purinoceptors) for diadenosine polyphosphates in Xenopus oocytes.
Michael Fernandez - One of the best experts on this subject based on the ideXlab platform.
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Prediction of Dinucleotide-specific RNA-binding sites in proteins.
BMC bioinformatics, 2011Co-Authors: Michael Fernandez, Yutaro Kumagai, Daron M Standley, Akinori Sarai, Kenji Mizuguchi, Shandar AhmadAbstract:Regulation of gene expression, protein synthesis, replication and assembly of many viruses involve RNA-protein interactions. Although some successful computational tools have been reported to recognize RNA binding sites in proteins, the problem of specificity remains poorly investigated. After the nucleotide base composition, the Dinucleotide is the smallest unit of RNA sequence information and many RNA-binding proteins simply bind to regions enriched in one Dinucleotide. Interaction preferences of protein subsequences and Dinucleotides can be inferred from protein-RNA complex structures, enabling a training-based prediction approach. We analyzed basic statistics of amino acid-Dinucleotide contacts in protein-RNA complexes and found their pairing preferences could be identified. Using a standard approach to represent protein subsequences by their evolutionary profile, we trained neural networks to predict multiclass target vectors corresponding to 16 possible contacting Dinucleotide subsequences. In the cross-validation experiments, the accuracies of the optimum network, measured as areas under the curve (AUC) of the receiver operating characteristic (ROC) graphs, were in the range of 65-80%. Dinucleotide-specific contact predictions have also been extended to the prediction of interacting protein and RNA fragment pairs, which shows the applicability of this method to predict targets of RNA-binding proteins. A web server predicting the 16-dimensional contact probability matrix directly from a user-defined protein sequence was implemented and made available at: http://tardis.nibio.go.jp/netasa/srcpred.
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Prediction of Dinucleotide-specific RNA-binding sites in proteins
BMC Bioinformatics, 2011Co-Authors: Michael Fernandez, Yutaro Kumagai, Daron M Standley, Akinori Sarai, Kenji Mizuguchi, Shandar AhmadAbstract:Abstract Background Regulation of gene expression, protein synthesis, replication and assembly of many viruses involve RNA–protein interactions. Although some successful computational tools have been reported to recognize RNA binding sites in proteins, the problem of specificity remains poorly investigated. After the nucleotide base composition, the Dinucleotide is the smallest unit of RNA sequence information and many RNA-binding proteins simply bind to regions enriched in one Dinucleotide. Interaction preferences of protein subsequences and Dinucleotides can be inferred from protein-RNA complex structures, enabling a training-based prediction approach. Results We analyzed basic statistics of amino acid-Dinucleotide contacts in protein-RNA complexes and found their pairing preferences could be identified. Using a standard approach to represent protein subsequences by their evolutionary profile, we trained neural networks to predict multiclass target vectors corresponding to 16 possible contacting Dinucleotide subsequences. In the cross-validation experiments, the accuracies of the optimum network, measured as areas under the curve (AUC) of the receiver operating characteristic (ROC) graphs, were in the range of 65-80%. Conclusions Dinucleotide-specific contact predictions have also been extended to the prediction of interacting protein and RNA fragment pairs, which shows the applicability of this method to predict targets of RNA-binding proteins. A web server predicting the 16-dimensional contact probability matrix directly from a user-defined protein sequence was implemented and made available at: http://tardis.nibio.go.jp/netasa/srcpred.