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Stewart Shuman - One of the best experts on this subject based on the ideXlab platform.
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nanomolar inhibitors of trypanosoma brucei RNA Triphosphatase
Mbio, 2016Co-Authors: Paul Smith, Yuko Takagi, Hakim Djaballah, Stewart ShumanAbstract:ABSTRACT Eukaryal taxa differ with respect to the structure and mechanism of the RNA Triphosphatase (RTPase) component of the mRNA capping apparatus. Protozoa, fungi, and certain DNA viruses have a metal-dependent RTPase that belongs to the triphosphate tunnel metalloenzyme (TTM) superfamily. Because the structures, active sites, and chemical mechanisms of the TTM-type RTPases differ from those of mammalian RTPases, the TTM RTPases are potential targets for antiprotozoal, antifungal, and antiviral drug discovery. Here, we employed RNA interference (RNAi) knockdown methods to show that Trypanosoma brucei RTPase Cet1 (TbCet1) is necessary for proliferation of procyclic cells in culture. We then conducted a high-throughput biochemical screen for small-molecule inhibitors of the phosphohydrolase activity of TbCet1. We identified several classes of chemicals—including chlorogenic acids, phenolic glycopyranosides, flavonoids, and other phenolics—that inhibit TbCet1 with nanomolar to low-micromolar 50% inhibitory concentrations (IC 50 s). We confirmed the activity of these compounds, and tested various analogs thereof, by direct manual assays of TbCet1 phosphohydrolase activity. The most potent nanomolar inhibitors included tetracaffeoylquinic acid, 5-galloylgalloylquinic acid, pentagalloylglucose, rosmarinic acid, and miquelianin. TbCet1 inhibitors were less active (or inactive) against the orthologous TTM-type RTPases of mimivirus, baculovirus, and budding yeast (Saccharomyces cerevisiae). Our results affirm that a TTM RTPase is subject to potent inhibition by small molecules, with the caveat that parallel screens against TTM RTPases from multiple different pathogens may be required to fully probe the chemical space of TTM inhibition. IMPORTANCE The stark differences between the structure and mechanism of the RNA Triphosphatase (RTPase) component of the mRNA capping apparatus in pathogenic protozoa, fungi, and viruses and those of their metazoan hosts highlight RTPase as a target for anti-infective drug discovery. Protozoan, fungal, and DNA virus RTPases belong to the triphosphate tunnel metalloenzyme family. This study shows that a protozoan RTPase, TbCet1 from Trypanosoma brucei, is essential for growth of the parasite in culture and identifies, via in vitro screening of chemical libraries, several classes of potent small-molecule inhibitors of TbCet1 phosphohydrolase activity.
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Nanomolar Inhibitors of Trypanosoma brucei RNA Triphosphatase
American Society for Microbiology, 2016Co-Authors: Paul Smith, Yuko Takagi, Hakim Djaballah, Stewart ShumanAbstract:Eukaryal taxa differ with respect to the structure and mechanism of the RNA Triphosphatase (RTPase) component of the mRNA capping apparatus. Protozoa, fungi, and certain DNA viruses have a metal-dependent RTPase that belongs to the triphosphate tunnel metalloenzyme (TTM) superfamily. Because the structures, active sites, and chemical mechanisms of the TTM-type RTPases differ from those of mammalian RTPases, the TTM RTPases are potential targets for antiprotozoal, antifungal, and antiviral drug discovery. Here, we employed RNA interference (RNAi) knockdown methods to show that Trypanosoma brucei RTPase Cet1 (TbCet1) is necessary for proliferation of procyclic cells in culture. We then conducted a high-throughput biochemical screen for small-molecule inhibitors of the phosphohydrolase activity of TbCet1. We identified several classes of chemicals—including chlorogenic acids, phenolic glycopyranosides, flavonoids, and other phenolics—that inhibit TbCet1 with nanomolar to low-micromolar 50% inhibitory concentrations (IC50s). We confirmed the activity of these compounds, and tested various analogs thereof, by direct manual assays of TbCet1 phosphohydrolase activity. The most potent nanomolar inhibitors included tetracaffeoylquinic acid, 5-galloylgalloylquinic acid, pentagalloylglucose, rosmarinic acid, and miquelianin. TbCet1 inhibitors were less active (or inactive) against the orthologous TTM-type RTPases of mimivirus, baculovirus, and budding yeast (Saccharomyces cerevisiae). Our results affirm that a TTM RTPase is subject to potent inhibition by small molecules, with the caveat that parallel screens against TTM RTPases from multiple different pathogens may be required to fully probe the chemical space of TTM inhibition
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Fission yeast RNA Triphosphatase reads an Spt5 CTD code
RNA (New York N.Y.), 2014Co-Authors: Selom K. Doamekpor, Stewart Shuman, Beate Schwer, Ana M. Sanchez, Christopher D. LimaAbstract:mRNA capping enzymes are directed to nascent RNA polymerase II (Pol2) transcripts via interactions with the carboxy-terminal domains (CTDs) of Pol2 and transcription elongation factor Spt5. Fission yeast RNA Triphosphatase binds to the Spt5 CTD, comprising a tandem repeat of nonapeptide motif TPAWNSGSK. Here we report the crystal structure of a Pct1·Spt5-CTD complex, which revealed two CTD docking sites on the Pct1 homodimer that engage TPAWN segments of the motif. Each Spt5 CTD interface, composed of elements from both subunits of the homodimer, is dominated by van der Waals contacts from Pct1 to the tryptophan of the CTD. The bound CTD adopts a distinctive conformation in which the peptide backbone makes a tight U-turn so that the proline stacks over the tryptophan. We show that Pct1 binding to Spt5 CTD is antagonized by threonine phosphorylation. Our results fortify an emerging concept of an “Spt5 CTD code” in which (i) the Spt5 CTD is structurally plastic and can adopt different conformations that are templated by particular cellular Spt5 CTD receptor proteins; and (ii) threonine phosphorylation of the Spt5 CTD repeat inscribes a binary on–off switch that is read by diverse CTD receptors, each in its own distinctive manner.
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How an mRNA capping enzyme reads distinct RNA polymerase II and Spt5 CTD phosphorylation codes
Genes & Development, 2014Co-Authors: Selom K. Doamekpor, Stewart Shuman, Beate Schwer, Ana M. Sanchez, Christopher D. LimaAbstract:Essential eukaryal mRNA processing events are targeted to nascent transcripts made by RNA polymerase II (Pol2) via physical interactions of the processing machineries with the C-terminal domain (CTD) of Rpb1, the largest subunit of Pol2. The Pol2 CTD consists of tandemly repeated heptapeptides of consensus sequence Y1S2P3T4S5P6S7. The inherently plastic Pol2 CTD structure is sculpted by dynamic phosphorylation and dephoshorylation of the Tyr1, Ser2, Thr4, Ser5, and Ser7 residues and by cis–trans isomerization of the prolines. With up to 128n potential Pol2 CTD primary structures (where n is the number of heptads), the Pol2 CTD provides information about the state of the transcription complex (a CTD code) that is read by diverse Pol2 CTD receptor proteins that control transcription, modify chromatin structure, and catalyze or regulate mRNA capping, splicing, and polyadenylation (Buratowski 2009; Corden 2013; Eick and Geyer 2013; Geronimo et al. 2013). The individual amino acids of the Pol2 CTD heptad and their phosphorylation marks are coding “letters” that have distinct outputs with respect to receptor recognition and impact on cellular gene expression (Schwer et al. 2014). The number of heptad repeats comprising the Pol2 CTD, the minimal number of repeats required for viability, and the prevalence of nonconsensus coding letters tend to increase with progression from unicellular to multicellular eukarya, presumably as a means to orchestrate the actions of increasing numbers of Pol2 CTD receptors. We envision a core Pol2 CTD code that governs events common to most eukarya; for example, the essential function of the Ser5-PO4–Pro6 coding “word” in recruiting mRNA capping enzymes to the Pol2 elongation complex (Schwer and Shuman 2011; Schwer et al. 2012), which is conserved in budding yeast, fission yeast, and mammals, notwithstanding major differences in the genetic and physical organization of the capping apparatus in these taxa (Lima et al. 1999; Changela et al. 2001; Pei et al. 2001; Fabrega et al. 2003; Ghosh et al. 2011). The RNA capping enzymes are the first processing factors to act on the growing Pol2 transcript; indeed, capping can commence after synthesis of only a 19- to 22-mer nascent RNA, the point at which the 5′ triphosphate RNA end is extruded from the RNA-binding pocket of elongating Pol2 and becomes accessible to the capping enzymes (Chiu et al. 2002). The m7GpppN cap is formed by three successive enzymatic reactions: (1) RNA Triphosphatase (TPase) hydrolyzes the RNA 5′ triphosphate end (pppRNA) to a diphosphate (ppRNA) plus inorganic phosphate, (2) GTP:RNA guanylyltransferase (GTase) converts ppRNA to GpppRNA via a covalent GTase-(lysyl-Nζ)–GMP intermediate, and (3) AdoMet:RNA(guanine-N7)-methyltransferase (MTase) converts GpppRNA to m7GpppRNA (Gu and Lima 2005). Whereas it had long been assumed that capping is a constitutive and efficient process for most mRNAs, this view may be too simplistic. The existence of factors that can stimulate capping of specific transcripts and the discovery of enzymes that mediate the decay of partially processed RNA intermediates in the cap synthetic pathway suggest that capping efficiency is a dynamic process (Chiu et al. 2002; Jiao et al. 2010, 2013; Chang et al. 2012). One potential way to regulate capping is by influencing the timely recruitment of the capping enzymes to the Pol2 elongation complex. A conserved theme among eukarya is the direct binding of the GTase component to the Ser5-PO4 form of the Pol2 CTD (Ho and Shuman 1999; Fabrega et al. 2003; Ghosh et al. 2011). In mammals and budding yeast, the TPase components are recruited passively to the Pol2 CTD by virtue of their physical association with the GTase: in cis as a covalently fused TPase–GTase enzyme, Mce1, in mammals (Ho and Shuman 1999; Changela et al. 2001) or in trans as separately encoded subunits of a TPase•GTase complex in budding yeast (Ho et al. 1999; Gu et al. 2010). The fission yeast Schizosaccharomyces pombe has a distinctive strategy for targeting cap formation to Pol2 transcripts whereby the TPase (Pct1) and GTase (Pce1) enzymes are not associated physically but instead bind independently to the Ser5-phosphorylated Pol2 CTD (Pei et al. 2001). Mammalian and fungal capping enzymes can also gain access to nascent Pol2 transcripts via physical interactions with the transcription elongation factor Spt5 (Wen and Shatkin 1999; Pei and Shuman 2002; Pei et al. 2006; Lidschreiber et al. 2013). Spt5 is a large polypeptide (∼1000–1200 amino acids) composed of multiple domain modules that associates with the Pol2 transcription complex shortly after initiation and can exert negative and positive effects on transcription elongation (Hartzog and Fu 2013). Fission yeast Spt5 has a distinctive C-terminal repeat domain (the “Spt5 CTD”) composed of 18 repeats of a nonapeptide motif (consensus: T1P2A3W4N5S6G7S8K9) that (1) binds the fission yeast RNA capping enzymes Pct1 and Pce1 (Pei and Shuman 2002) and (2) is targeted for threonine phosphorylation by the fission yeast Cdk9 kinase (Pei and Shuman 2002, 2003; Viladevall et al. 2009). Alignment of Spt5 CTD elements in metazoan and fungal species reveals substantive differences, although each contains a series of Thr–Pro or Ser–Pro dipeptide motifs followed by a hydrophobic side chain two residues downstream (Pei and Shuman 2002). The hydrophobic side chain is typically tryptophan in S. pombe; this position is substituted to a tyrosine or histidine in humans. Available genetic evidence points to overlapping roles of the CTDs of fission yeast Pol2 and Spt5 in recruiting the capping enzymes in vivo (Schneider et al. 2010). Unlike the capping enzyme•Pol2 CTD interactions, which stringently depend on the Ser5-PO4 mark, the binding of fission yeast Pct1 and Pce1 to the Spt5 CTD is independent of Thr1 phosphorylation (Pei et al. 2001; Pei and Shuman 2002). The structural principles underpinning capping enzyme•Pol2 CTD interactions were initially elucidated via cocrystallization of Candida albicans GTase (Cgt1) and mammalian GTase (Mce1) bound to Ser5-phosphorylated Pol2 CTD peptide ligands (Fabrega et al. 2003; Ghosh et al. 2011). These two cellular GTases are structurally homologous enzymes composed of two domains: (1) an N-terminal nucleotidyltransferase (NTase) module that contains the guanylate-binding pocket and (2) a C-terminal OB-fold module. A comparison of the Candida and mammalian GTase•Pol2 CTD structures was most revealing because, although the docking sites for Pol2 CTD lie on the surface of the NTase domain in both cases, the docking sites are physically distinct and have little overlap, the atomic contacts to Pol2 CTD are different, and virtually none of the Pol2 CTD-interacting side chains of mammalian GTase are conserved in the Candida enzyme (Ghosh et al. 2011). Thus, capping enzymes from different taxa have evolved different strategies to read the Pol2 CTD code. Here we extend the structural analysis to the GTase enzyme of fission yeast and its interactions with the CTDs of Pol2 and Spt5. We report crystal structures of Pce1 bound to Pol2 CTD and Spt5 CTD ligands. Key findings are that (1) the CTDs of Pol2 and Spt5 interact with completely distinct sites on the NTase and OB domains of the fission yeast GTase, respectively, and (2) whereas the interface of GTase with the Pol2 CTD is dependent on Ser5 phosphorylation, GTase binding to Spt5 CTD is antagonized by Thr1 phosphorylation. We probed by structure-guided mutagenesis the contributions of the GTase•CTD interfaces to CTD binding in vitro and capping enzyme function in vivo. Our findings cohere into a model in which the capping enzyme recognizes and responds to distinct coding cues and phosphorylation marks in the Pol2 and Spt5 CTDs.
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Crystal structure of vaccinia virus mRNA capping enzyme provides insights into the mechanism and evolution of the capping apparatus
Structure, 2014Co-Authors: O.j.p. Kyrieleis, Stewart Shuman, Jonathan Chang, Marcos De La Peña, Stephen CusackAbstract:Summary Vaccinia virus capping enzyme is a heterodimer of D1 (844 aa) and D12 (287 aa) polypeptides that executes all three steps in m 7 GpppRNA synthesis. The D1 subunit comprises an N-terminal RNA Triphosphatase (TPase)-guanylyltransferase (GTase) module and a C-terminal guanine-N7-methyltransferase (MTase) module. The D12 subunit binds and allosterically stimulates the MTase module. Crystal structures of the complete D1⋅D12 heterodimer disclose the TPase and GTase as members of the triphosphate tunnel metalloenzyme and covalent nucleotidyltransferase superfamilies, respectively, albeit with distinctive active site features. An extensive TPase-GTase interface clamps the GTase nucleotidyltransferase and OB-fold domains in a closed conformation around GTP. Mutagenesis confirms the importance of the TPase-GTase interface for GTase activity. The D1⋅D12 structure complements and rationalizes four decades of biochemical studies of this enzyme, which was the first capping enzyme to be purified and characterized, and provides new insights into the origins of the capping systems of other large DNA viruses.
Radhakrishnan Padmanabhan - One of the best experts on this subject based on the ideXlab platform.
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substitution of ns5 n terminal domain of dengue virus type 2 RNA with type 4 domain caused impaired replication and emergence of adaptive mutants with enhanced fitness
Journal of Biological Chemistry, 2014Co-Authors: Tadahisa Teramoto, Siwaporn Boonyasuppayakorn, Misty Handley, Kyung H Choi, Radhakrishnan PadmanabhanAbstract:Flavivirus NS3 and NS5 are required in viral replication and 5′-capping. NS3 has NS2B-dependent protease, RNA helicase, and 5′-RNA Triphosphatase activities. NS5 has 5′-RNA methyltransferase (MT)/guanylyltransferase (GT) activities within the N-terminal 270 amino acids and the RNA-dependent RNA polymerase (POL) activity within amino acids 271–900. A chimeric NS5 containing the D4MT/D4GT and the D2POL domains in the context of wild-type (WT) D2 RNA was constructed. RNAs synthesized in vitro were transfected into baby hamster kidney cells. The viral replication was analyzed by an indirect immunofluorescence assay to monitor NS1 expression and by quantitative real-time PCR. WT D2 RNA-transfected cells were NS1- positive by day 5, whereas the chimeric RNA-transfected cells became NS1-positive ∼30 days post-transfection in three independent experiments. Sequence analysis covering the entire genome revealed the appearance of a single K74I mutation within the D4MT domain ∼16 days post-transfection in two experiments. In the third, D290N mutation in the conserved NS3 Walker B motif appeared ≥16 days post-transfection. A time course study of serial passages revealed that the 30-day supeRNAtant had gradually evolved to gain replication fitness. Trans-complementation by co-expression of WT D2 NS5 accelerated viral replication of chimeric RNA without changing the K74I mutation. However, the MT and POL activities of NS5 WT D2 and the chimeric NS5 proteins with or without the K74I mutation are similar. Taken together, our results suggest that evolution of the functional interactions involving the chimeric NS5 protein encoded by the viral genome species is essential for gain of viral replication fitness.
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modulation of the nucleoside Triphosphatase RNA helicase and 5 RNA Triphosphatase activities of dengue virus type 2 nonstructural protein 3 ns3 by interaction with ns5 the RNA dependent RNA polymerase
Journal of Biological Chemistry, 2005Co-Authors: Tadahisa Teramoto, Niklaus H Mueller, Jessica Phelan, Krishna H M Murthy, Vannakambadi K Ganesh, Radhakrishnan PadmanabhanAbstract:Abstract Dengue virus type 2 (DEN2), a member of the Flaviviridae family, is a re-emerging human pathogen of global significance. DEN2 nonstructural protein 3 (NS3) has a serine protease domain (NS3-pro) and requires the hydrophilic domain of NS2B (NS2BH) for activation. NS3 is also an RNA-stimulated nucleoside Triphosphatase (NTPase)/RNA helicase and a 5′-RNA Triphosphatase (RTPase). In this study the first biochemical and kinetic properties of full-length NS3 (NS3FL)-associated NTPase, RTPase, and RNA helicase are presented. The NS3FL showed an enhanced RNA helicase activity compared with the NS3-pro-minus NS3, which was further enhanced by the presence of the NS2BH (NS2BH-NS3FL). An active protease catalytic triad is not required for the stimulatory effect, suggesting that the overall folding of the N-terminal protease domain contributes to this enhancement. In DEN2-infected mammalian cells, NS3 and NS5, the viral 5′-RNA methyltransferase/polymerase, exist as a complex. Therefore, the effect of NS5 on the NS3 NTPase activity was examined. The results show that NS5 stimulated the NS3 NTPase and RTPase activities. The NS5 stimulation of NS3 NTPase was dose-dependent until an equimolar ratio was reached. Moreover, the conserved motif, 184RKRK, of NS3 played a crucial role in binding to RNA substrate and modulating the NTPase/RNA helicase and RTPase activities of NS3.
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expression purification and characterization of the RNA 5 Triphosphatase activity of dengue virus type 2 nonstructural protein 3
Virology, 2002Co-Authors: Greg Bartelma, Radhakrishnan PadmanabhanAbstract:Abstract Dengue virus type 2 (DEN2), a member of the Flaviviridae family of positive-strand RNA viruses, contains a single RNA genome having a type I cap structure at the 5′ end. The viral RNA is translated to produce a single polyprotein precursor that is processed to yield three virion proteins and at least seven nonstructural proteins (NS) in the infected host. NS3 is a multifunctional protein having a serine protease catalytic triad within the N-terminal 180 amino acid residues which requires NS2B as a cofactor for activation of protease activity. The C-terminal portion of this catalytic triad has conserved motifs present in several nucleoside Triphosphatases (NTPases)/RNA helicases. In addition, subtilisin-treated West Nile (WN) virus NS3 from infected cells was reported to have 5′-RNA Triphosphatase activity, suggesting its role in the synthesis of the 5′-cap structure. In this study, full-length DEN2 NS3 was expressed with an N-terminal histidine tag in Escherichia coli and purified in a soluble form. The purified protein has 5′-RNA Triphosphatase activity that cleaves the γ-phosphate moiety of the 5′-triphosphorylated RNA substrate. Biochemical and mutational analyses of the NS3 protein indicate that the nucleoside Triphosphatase and 5′-RNA Triphosphatase activities of NS3 share a common active site.
Stephen Buratowski - One of the best experts on this subject based on the ideXlab platform.
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The Caenorhabditis elegans mRNA 5-Capping Enzyme IN VITRO AND IN VIVO CHARACTERIZATION*
2016Co-Authors: Keith T. Blackwell, Stephen BuratowskiAbstract:Eukaryotic mRNA capping enzymes are bifunctional, carrying both RNA Triphosphatase (RTPase) and guany-lyltransferase (GTase) activities. The Caenorhabditis el-egans CEL-1 capping enzyme consists of an N-terminal region with RTPase activity and a C-terminal region that resembles known GTases, However, CEL-1 has not previously been shown to have GTase activity. Cloning of the cel-1 cDNA shows that the full-length protein has 623 amino acids, including an additional 38 residues at the C termini and 12 residues at the N termini not orig-inally predicted from the genomic sequence. Full-length CEL-1 has RTPase and GTase activities, and the cDNA can functionally replace the capping enzyme genes in Saccharomyces cerevisiae. The CEL-1 RTPase domain is related by sequence to protein-tyrosine phosphatases
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The Caenorhabditis elegans mRNA 5'-capping enzyme. In vitro and in vivo characterization.
Journal of Biological Chemistry, 2003Co-Authors: Toshimitsu Takagi, Amy K. Walker, Chika Sawa, Felix Diehn, Yasutaka Takase, T. Keith Blackwell, Stephen BuratowskiAbstract:Abstract Eukaryotic mRNA capping enzymes are bifunctional, carrying both RNA Triphosphatase (RTPase) and guanylyltransferase (GTase) activities. The Caenorhabditis elegans CEL-1 capping enzyme consists of an N-terminal region with RTPase activity and a C-terminal region that resembles known GTases, However, CEL-1 has not previously been shown to have GTase activity. Cloning of the cel-1 cDNA shows that the full-length protein has 623 amino acids, including an additional 38 residues at the C termini and 12 residues at the N termini not originally predicted from the genomic sequence. Full-length CEL-1 has RTPase and GTase activities, and the cDNA can functionally replace the capping enzyme genes in Saccharomyces cerevisiae. The CEL-1 RTPase domain is related by sequence to protein-tyrosine phosphatases; therefore, mutagenesis of residues predicted to be important for RTPase activity was carried out. CEL-1 uses a mechanism similar to protein-tyrosine phosphatases, except that there was not an absolute requirement for a conserved acidic residue that acts as a proton donor. CEL-1 shows a strong preference for RNA substrates of at least three nucleotides in length. RNA-mediated interference inC. elegans embryos shows that lack of CEL-1 causes development to arrest with a phenotype similar to that seen when RNA polymerase II elongation activity is disrupted. Therefore, capping is essential for gene expression in metazoans.
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The essential interaction between yeast mRNA capping enzyme subunits is not required for Triphosphatase function in vivo.
Molecular and Cellular Biology, 2000Co-Authors: Yasutaka Takase, Toshimitsu Takagi, Philip B. Komarnitsky, Stephen BuratowskiAbstract:The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: an RNA 5′-Triphosphatase (Cet1) and an mRNA guanylyltransferase (Ceg1). In yeast, the capping enzyme is recruited to the RNA polymerase II (Pol II) transcription complex via an interaction between Ceg1 and the phosphorylated carboxy-terminal domain of the Pol II largest subunit. Previous in vitro experiments showed that the Cet1 carboxy-terminal region (amino acids 265 to 549) carries RNA Triphosphatase activity, while the region containing amino acids 205 to 265 of Cet1 has two functions: it mediates dimerization with Ceg1, but it also allosterically activates Ceg1 guanylyltransferase activity in the context of Pol II binding. Here we characterize several Cet1 mutants in vivo. Mutations or deletions of Cet1 that disrupt interaction with Ceg1 are lethal, showing that this interaction is essential for proper capping enzyme function in vivo. Remarkably, the interaction region of Ceg1 becomes completely dispensable when Ceg1 is substituted by the mouse guanylyltransferase, which does not require allosteric activation by Cet1. Although no interaction between Cet1 and mouse guanylyltransferase is detectable, both proteins are present at yeast promoters in vivo. These results strongly suggest that the primary physiological role of the Ceg1-Cet1 interaction is to allosterically activate Ceg1, rather than to recruit Cet1 to the Pol II complex.
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A Saccharomyces cerevisiae RNA 5'-Triphosphatase related to mRNA capping enzyme.
Nucleic Acids Research, 1999Co-Authors: Christine R. Rodriguez, Toshimitsu Takagi, Eun-jung Cho, Stephen BuratowskiAbstract:The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: the RNA 5'-Triphosphatase (Cet1) and the mRNA guanylyltransferase (Ceg1). Using computer homology searching, a S. cerevisiae gene was identified that encodes a protein resembling the C-terminal region of Cet1. Accordingly, we designated this gene CTL1 (capping enzyme RNATriphosphatase-like 1). CTL1 is not essential for cell viability and no genetic or physical interactions with the capping enzyme genes were observed. The protein is found in both the nucleus and cytoplasm. Recombinant Ctl1 protein releases gamma-phosphate from the 5'-end of RNA to produce a diphosphate terminus. The enzyme is specific for polynucleotide RNA in the presence of magnesium, but becomes specific for nucleotide triphosphates in the presence of manganese. Ctl1 is the second member of the yeast RNA Triphosphatase family, but is probably involved in an RNA processing event other than mRNA capping.
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a protein tyrosine phosphatase like protein from baculovirus has RNA 5 Triphosphatase and diphosphatase activities
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Toshimitsu Takagi, Gregory S Taylor, Takahiro Kusakabe, Harry Charbonneau, Stephen BuratowskiAbstract:The superfamily of protein tyrosine phosphatases (PTPs) includes at least one enzyme with an RNA substrate. We recently showed that the RNA Triphosphatase domain of the Caenorhabditis elegans mRNA capping enzyme is related to the PTP enzyme family by sequence similarity and mechanism. The PTP most similar in sequence to the capping enzyme Triphosphatase is BVP, a dual-specificity PTP encoded by the Autographa californica nuclear polyhedrosis virus. Although BVP previously has been shown to have modest tyrosine and serine/threonine phosphatase activity, we find that it is much more potent as an RNA 5′-phosphatase. BVP sequentially removes γ and β phosphates from the 5′ end of triphosphate-terminated RNA, leaving a 5′-monophosphate end. The activity was specific for polynucleotides; nucleotide triphosphates were not hydrolyzed. A mutant protein in which the active site cysteine was replaced with serine was inactive. Three other dual-specificity PTPs (VH1, VHR, and Cdc14) did not exhibit detectable RNA phosphatase activity. Therefore, capping enzyme and BVP are members of a distinct PTP-like subfamily that can remove phosphates from RNA.
Julien Lescar - One of the best experts on this subject based on the ideXlab platform.
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the flavivirus ns2b ns3 protease helicase as a target for antiviral drug development
Antiviral Research, 2015Co-Authors: Dahai Luo, Subhash G Vasudevan, Julien LescarAbstract:Abstract The flavivirus NS3 protein is associated with the endoplasmic reticulum membrane via its close interaction with the central hydrophilic region of the NS2B integral membrane protein. The multiple roles played by the NS2B–NS3 protein in the virus life cycle makes it an attractive target for antiviral drug discovery. The N-terminal region of NS3 and its cofactor NS2B constitute the protease that cleaves the viral polyprotein. The NS3 C-terminal domain possesses RNA helicase, nucleoside and RNA Triphosphatase activities and is involved both in viral RNA replication and virus particle formation. In addition, NS2B–NS3 serves as a hub for the assembly of the flavivirus replication complex and also modulates viral pathogenesis and the host immune response. Here, we review biochemical and structural advances on the NS2B–NS3 protein, including the network of interactions it forms with NS5 and NS4B and highlight recent drug development efforts targeting this protein. This article forms part of a symposium in Antiviral Research on flavivirus drug discovery.
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towards the design of antiviral inhibitors against flaviviruses the case for the multifunctional ns3 protein from dengue virus as a target
Antiviral Research, 2008Co-Authors: Julien Lescar, Dahai Luo, Bruno Canard, Aruna Sampath, Siew Pheng Lim, Subhash G VasudevanAbstract:New treatments are urgently needed to combat the increasing number of dengue fever cases in endemic countries as well as amongst a large number of travellers from non-endemic countries. Of the 10 virus encoded proteins, NS3 (non-structural 3) and NS5 carry out all the enzymatic activities needed for polyprotein processing and genome replication, and are considered to be amenable to antiviral inhibition by analogy with successes for similar targets in human immunodeficiency virus and hepatitis C virus. The multifunctional NS3 protein of flavivirus forms a non-covalent complex with the NS2B cofactor and contains the serine-protease activity domain at its N-terminus that is responsible for proteolytic processing of the viral polyprotein and a ATPase/helicase and RNA Triphosphatase at its C-terminal end that are essential for RNA replication. In addition, NS3 seems to be also involved in virus assembly. This review covers the recent biochemical and structural advances on the NS2B-NS3 protease-helicase and presents an outlook for the development of small molecules as antiviral drugs targeting this fascinating multifunctional protein.
Martin Bisaillon - One of the best experts on this subject based on the ideXlab platform.
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the flavivirus ns5 protein is a true RNA guanylyltransferase that catalyzes a two step reaction to form the RNA cap structure
RNA, 2009Co-Authors: Moheshwarnath Issur, Isabelle Bougie, Frederic Picardjean, Brian J Geiss, Simon Despins, Joannie Mayette, Sarah E Hobdey, Martin BisaillonAbstract:The 5'-end of the flavivirus genome harbors a methylated (m7)GpppA(2'OMe) cap structure, which is generated by the virus-encoded RNA Triphosphatase, RNA (guanine-N7) methyltransferase, nucleoside 2'-O-methyltransferase, and RNA guanylyltransferase. The presence of the flavivirus guanylyltransferase activity in NS5 has been suggested by several groups but has not been empirically proven. Here we provide evidence that the N-terminus of the flavivirus NS5 protein is a true RNA guanylyltransferase. We demonstrate that GTP can be used as a substrate by the enzyme to form a covalent GMP-enzyme intermediate via a phosphoamide bond. Mutational studies also confirm the importance of a specific lysine residue in the GTP binding site for the enzymatic activity. We show that the GMP moiety can be transferred to the diphosphate end of an RNA transcript harboring an adenosine as the initiating residue. We also demonstrate that the flavivirus RNA Triphosphatase (NS3 protein) stimulates the RNA guanylyltransferase activity of the NS5 protein. Finally, we show that both enzymes are sufficient and necessary to catalyze the de novo formation of a methylated RNA cap structure in vitro using a triphosphorylated RNA transcript. Our study provides biochemical evidence that flaviviruses encode a complete RNA capping machinery.
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nucleotide analogs and molecular modeling studies reveal key interactions involved in substrate recognition by the yeast RNA Triphosphatase
Nucleic Acids Research, 2009Co-Authors: Moheshwarnath Issur, Isabelle Bougie, Simon Despins, Martin BisaillonAbstract:RNA Triphosphatases (RTPases) are involved in the addition of the distinctive cap structure found at the 5′ ends of eukaryotic mRNAs. Fungi, protozoa and some DNA viruses possess an RTPase that belongs to the triphosphate tunnel metalloenzyme family of enzymes that can also hydrolyze nucleoside triphosphates. Previous crystallization studies revealed that the phosphohydrolase catalytic core is located in a hydrophilic tunnel composed of antiparallel β-strands. However, all past efforts to obtain structural information on the interaction between RTPases and their substrates were unsuccessful. In the present study, we used computational molecular docking to model the binding of a nucleotide substrate into the yeast RTPase active site. In order to confirm the docking model and to gain additional insights into the molecular determinants involved in substrate recognition, we also evaluated both the phosphohydrolysis and the inhibitory potential of an important number of nucleotide analogs. Our study highlights the importance of specific amino acids for the binding of the sugar, base and triphosphate moieties of the nucleotide substrate, and reveals both the structural flexibility and complexity of the active site. These data illustrate the functional features required for the interaction of an RTPase with a ligand and pave the way to the use of nucleotide analogs as potential inhibitors of RTPases of pathogenic importance.
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Inhibition of a metal-dependent viral RNA Triphosphatase by decavanadate.
The Biochemical journal, 2006Co-Authors: Isabelle Bougie, Martin BisaillonAbstract:Paramecium bursaria chlorella virus, a large DNA virus that replicates in unicellular Chlorella-like algae, encodes an RNA Triphosphatase which is involved in the synthesis of the RNA cap structure found at the 5' end of the viral mRNAs. The Chlorella virus RNA Triphosphatase is the smallest member of the metal-dependent RNA Triphosphatases that include enzymes from fungi, DNA viruses, protozoans and microsporidian parasites. In the present study, we investigated the ability of various vanadate oxoanions to inhibit the phosphohydrolase activity of the enzyme. Fluorescence spectroscopy and CD studies were used to directly monitor the binding of decavanadate to the enzyme. Moreover, competition assays show that decavanadate is a potent non-competitive inhibitor of the phosphohydrolase activity, and mutagenesis studies indicate that the binding of decavanadate does not involve amino acids located in the active site of the enzyme. In order to provide additional insight into the relationship between the enzyme structure and decavanadate binding, we correlated the effect of decavanadate binding on protein structure using both CD and guanidinium chloride-induced denaturation as structural indicators. Our data indicated that no significant modification of the overall protein architecture was occurring upon decavanadate binding. However, both fluorescence spectroscopy and CD experiments clearly revealed that the binding of decavanadate to the enzyme significantly decreased the structural stability of the enzyme. Taken together, these studies provide crucial insights into the inhibition of metal-dependent RNA Triphosphatases by decavanadate.
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energetics of RNA binding by the west nile virus RNA Triphosphatase
FEBS Letters, 2006Co-Authors: Ines Benzaghou, Isabelle Bougie, Frederic Picardjean, Martin BisaillonAbstract:The West Nile virus (WNV) RNA genome harbors the characteristic methylated cap structure present at the 5' end of eukaryotic mRNAs. In the present study, we report a detailed study of the binding energetics and thermodynamic parameters involved in the interaction between RNA and the WNV RNA Triphosphatase, an enzyme involved in the synthesis of the RNA cap structure. Fluorescence spectroscopy assays revealed that the initial interaction between RNA and the enzyme is characterized by a high enthalpy of association and that the minimal RNA binding site of NS3 is 13 nucleotides. In order to provide insight into the relationship between the enzyme structure and RNA binding, we also correlated the effect of RNA binding on protein structure using both circular dichroism and denaturation studies as structural indicators. Our data indicate that the protein undergoes structural modifications upon RNA binding, although the interaction does not significantly modify the stability of the protein.
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investigating the role of metal ions in the catalytic mechanism of the yeast RNA Triphosphatase
Journal of Biological Chemistry, 2003Co-Authors: Martin Bisaillon, Isabelle BougieAbstract:The Saccharomyces cerevisiae RNA Triphosphatase (Cet1) requires the presence of metal ion cofactors to catalyze its phosphohydrolase activity, the first step in the formation of the 5′-terminal cap structure of mRNAs. We have used endogenous tryptophan fluorescence studies to elucidate both the nature and the role(s) of the metal ions in the Cet1-mediated phosphohydrolase reaction. The association of Mg2+, Mn2+, and Co2+ ions with the enzyme resulted in a decrease in the intensity of the tryptophan emission spectrum. This decrease was then used to determine the apparent dissociation constants for these ions. Subsequent dual ligand titration experiments demonstrated that the metal ions bind to a common site, for which they compete. The kinetics of real-time metal ion binding to the Cet1 protein were also investigated, and the effects on RNA and nucleotide binding were evaluated. To provide additional insight into the relationship between Cet1 structure and metal ion binding, we correlated the effect of ion binding on protein structure using both circular dichroism and guanidium hydrochloride-induced denaturation as structural indicators. Our data indicate that binding of RNA, nucleotides, and metal ion cofactors does not lead to significant structural modifications of the Cet1 architecture. This suggests a model in which Cet1 possesses a preformed active site, and where major domain rearrangements are not required to form an active catalytic site. Finally, denaturation studies demonstrate that the metal ion cofactors can act by stabilizing the ground state binding of the phosphohydrolase substrate.
