The Experts below are selected from a list of 264 Experts worldwide ranked by ideXlab platform
Dianne T. Keough - One of the best experts on this subject based on the ideXlab platform.
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synthesis of branched 9 2 2 phosphonoethoxy ethyl purines as a new class of acyclic nucleoside phosphonates which inhibit plasmodium falciparum hypoxanthine guanine xanthine phosphoribosyltransferase
Bioorganic & Medicinal Chemistry, 2009Co-Authors: Dana Hocková, Dianne T. Keough, Antonin Holý, Milena Masojidkova, John De Jersey, Luke W GuddatAbstract:The malarial parasite Plasmodium falciparum (Pf) lacks the de novo pathway and relies on the Salvage Enzyme, hypoxanthine–guanine–xanthine phosphoribosyltransferase (HGXPRT), for the synthesis of the 6-oxopurine nucleoside monophosphates. Specific acyclic nucleoside phosphonates (ANPs) inhibit PfHGXPRT and possess anti-plasmodial activity. Two series of novel branched ANPs derived from 9-[2-(2-phosphonoethoxy)ethyl]purines were synthesized to investigate their inhibition of PfHGXPRT and human HGPRT. The best inhibitor of PfHGXPRT has a Ki of 1 μM. The data showed that both the position and nature of the hydrophobic substituent change the potency and selectivity of the ANPs.
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inhibition of hypoxanthine guanine phosphoribosyltransferase by acyclic nucleoside phosphonates a new class of antimalarial therapeutics
Journal of Medicinal Chemistry, 2009Co-Authors: Dianne T. Keough, Dana Hocková, Antonin Holý, John De Jersey, Lieve Naesens, Tina S Skinneradams, Luke W GuddatAbstract:The purine Salvage Enzyme hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) is essential for purine nucleotide and hence nucleic acid synthesis in the malaria parasite, Plasmodium falciparum. Acyclic nucleoside phosphonates (ANPs) are analogues of the nucleotide product of the reaction, comprising a purine base joined by a linker to a phosphonate moiety. Ki values for 19 ANPs were determined for Pf HGXPRT and the corresponding human Enzyme, HGPRT. Values for Pf HGXPRT were as low as 100 nM, with selectivity for the parasite Enzyme of up to 58. Structures of human HGPRT in complex with three ANPs are reported. On binding, a large mobile loop in the free Enzyme moves to partly cover the active site. For three ANPs, the IC50 values for Pf grown in cell culture were 1, 14, and 46 μM, while the cytotoxic concentration for the first compound was 489 μM. These results provide a basis for the design of potent and selective ANP inhibitors of Pf HGXPRT as antimalarial drug leads.
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the purine Salvage Enzyme hypoxanthine guanine xanthine phosphoribosyl transferase is a major target antigen for cell mediated immunity to malaria
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Morris O Makobongo, Dianne T. Keough, John De Jersey, George Riding, Chakrit Hirunpetcharat, Peter Willadsen, Michael F GoodAbstract:Although there is good evidence that immunity to the blood stages of malaria parasites can be mediated by different effector components of the adaptive immune system, target antigens for a principal component, effector CD4+ T cells, have never been defined. We generated CD4+ T cell lines to fractions of native antigens from the blood stages of the rodent parasite, Plasmodium yoelii, and identified fraction-specific T cells that had a Th1 phenotype (producing IL-2, IFN-γ, and tumor necrosis factor-α, but not IL-4, after antigenic stimulation). These T cells could inhibit parasite growth in recipient severe combined immunodeficient mice. N-terminal sequencing of the fraction showed identity with hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT). Recombinant HGXPRT from the human malaria parasite, Plasmodium falciparum, activated the T cells in vitro, and immunization of normal mice with recombinant HGXPRT reduced parasite growth rates in all mice after challenge.
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Role of cysteine and lysine residues in human hypoxanthine-guanine phosphoribosyltransferase
Clinical Biochemistry, 1997Co-Authors: Dianne T. Keough, Bryan T. Emmerson, Christine L. Gee, J. De JerseyAbstract:Hypoxanthine-guanine phosphoribosyltransferase (HPRT) is a purine Salvage Enzyme which is widely distributed in human tissues. It catalyses the synthesis of the purine mononucleotides, IMP and GMP. In humans, partial Enzyme deficiency is associated with gout and hyperuricemia while complete Enzyme deficiency results in the Lesch-Nyhan syndrome.
Jue D Wang - One of the best experts on this subject based on the ideXlab platform.
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molecular mechanism of regulation of the purine Salvage Enzyme xprt by the alarmones pppgpp ppgpp and pgpp
Journal of Molecular Biology, 2020Co-Authors: Brent W Anderson, Aili Hao, Kenneth A Satyshur, James L Keck, Jue D WangAbstract:The alarmones pppGpp and ppGpp mediate starvation response and maintain purine homeostasis to protect bacteria. In the bacterial phyla Firmicutes and Bacteroidetes, xanthine phosphoribosyltransferase (XPRT) is a purine Salvage Enzyme that produces the nucleotide XMP from PRPP and xanthine. Combining structural, biochemical, and genetic analyses, we show that pppGpp and ppGpp, as well as a third newly identified alarmone pGpp, all directly interact with XPRT from the Gram-positive bacterium Bacillus subtilis and inhibit XPRT activity by competing with its substrate PRPP. Structural analysis reveals that ppGpp binds the PRPP binding motif within the XPRT active site. This motif is present in another (p)ppGpp target, the purine Salvage Enzyme HPRT, suggesting evolutionary conservation in different Enzymes. However, XPRT oligomeric interaction is distinct from HPRT in that XPRT forms a symmetric dimer with two (p)ppGpp binding sites at the dimer interface. (p)ppGpp's interaction with an XPRT bridging loop across the interface results in XPRT cooperatively binding (p)ppGpp. Also, XPRT displays differential regulation by the alarmones as it is potently inhibited by both ppGpp and pGpp, but only modestly by pppGpp. Lastly, we demonstrate that the alarmones are necessary for protecting GTP homeostasis against excess environmental xanthine in B. subtilis, suggesting that regulation of XPRT is key for regulating the purine Salvage pathway.
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molecular mechanism of regulation of the purine Salvage Enzyme xprt by the alarmones pppgpp ppgpp and pgpp
bioRxiv, 2020Co-Authors: Brent W Anderson, Aili Hao, Kenneth A Satyshur, James L Keck, Jue D WangAbstract:The alarmones pppGpp and ppGpp mediate starvation response and maintain purine homeostasis to protect bacterial species. Xanthine phosphoribosyltransferase (XPRT) is a purine Salvage Enzyme that produces the nucleotide XMP from PRPP and xanthine. Combining structural, biochemical and genetic analyses, we show that pppGpp and ppGpp, as well as a third putative alarmone pGpp, all directly interact with XPRT and inhibit XPRT activity by competing with its substrate PRPP. Structural analysis reveals that ppGpp binds the PRPP binding motif within the XPRT active site. This motif is present in another (p)ppGpp target, the purine Salvage Enzyme HPRT, suggesting evolutionary conservation in different Enzymes. However, XPRT oligomeric interaction is distinct from HPRT in that XPRT forms a symmetric dimer with two (p)ppGpp binding sites at the dimer interface. This results in two distinct regulatory features. First, XPRT cooperatively binds (p)ppGpp with a Hill coefficient of 2. Also, XPRT displays differential regulation by the alarmones as it is potently inhibited by both ppGpp and pGpp, but only modestly by pppGpp. Lastly, we demonstrate that the alarmones are necessary for protecting GTP homeostasis against excess environmental xanthine in Bacillus subtilis, suggesting that regulation of XPRT is key for regulating the purine Salvage pathway.
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evolution of p ppgpp hprt regulation through diversification of an allosteric oligomeric interaction
eLife, 2019Co-Authors: Brent W Anderson, Kenneth A Satyshur, James L Keck, Kuanqing Liu, Christine Wolak, Katarzyna Dubiel, Fukang She, Jue D WangAbstract:The alarmone (p)ppGpp regulates diverse targets, yet its target specificity and evolution remain poorly understood. Here, we elucidate the mechanism by which basal (p)ppGpp inhibits the purine Salvage Enzyme HPRT by sharing a conserved motif with its substrate PRPP. Intriguingly, HPRT regulation by (p)ppGpp varies across organisms and correlates with HPRT oligomeric forms. (p)ppGpp-sensitive HPRT exists as a PRPP-bound dimer or an apo- and (p)ppGpp-bound tetramer, where a dimer-dimer interface triggers allosteric structural rearrangements to enhance (p)ppGpp inhibition. Loss of this oligomeric interface results in weakened (p)ppGpp regulation. Our results reveal an evolutionary principle whereby protein oligomerization allows evolutionary change to accumulate away from a conserved binding pocket to allosterically alter specificity of ligand interaction. This principle also explains how another (p)ppGpp target GMK is variably regulated across species. Since most ligands bind near protein interfaces, we propose that this principle extends to many other protein-ligand interactions.
Andrew D. Hanson - One of the best experts on this subject based on the ideXlab platform.
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Identification of the thiamin Salvage Enzyme thiazole kinase in Arabidopsis and maize
Phytochemistry, 2013Co-Authors: Mohammad Yazdani, Rémi Zallot, Meral Tunc-ozdemir, Valérie De Crécy-lagard, David K. Shintani, Andrew D. HansonAbstract:The breakdown of thiamin (vitamin B1) and its phosphates releases a thiazole moiety, 4-methyl-5-(2-hydroxyethyl)thiazole (THZ), that microorganisms and plants are able to Salvage for re-use in thiamin synthesis. The Salvage process starts with the ATP-dependent phosphorylation of THZ, which in bacteria is mediated by ThiM. The Arabidopsis and maize genomes encode homologs of ThiM (At3g24030 and GRMZM2G094558, respectively). Plasmid-driven expression of either plant homolog restored the ability of THZ to rescue Escherichia coli thiM deletant strains, showing that the plant proteins have ThiM activity in vivo. Enzymatic assays with purified recombinant proteins confirmed the presence of THZ kinase activity. Furthermore, ablating the Arabidopsis At3g24030 gene in a thiazole synthesis mutant severely impaired rescue by THZ. Collectively, these results show that ThiM homologs are the main source of THZ kinase activity in plants and are consequently crucial for thiamin Salvage.
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Characterization of the folate Salvage Enzyme p-aminobenzoylglutamate hydrolase in plants
Phytochemistry, 2007Co-Authors: Gale G. Bozzo, Gilles J. Basset, Valeria Naponelli, Alexandre Noiriel, Jesse F. Gregory, Andrew D. HansonAbstract:Abstract Folates break down in vivo to give pterin and p-aminobenzoylglutamate (pABAGlu) fragments, the latter usually having a polyglutamyl tail. Pilot studies have shown that plants can hydrolyze pABAGlu and its polyglutamates to p-aminobenzoate, a folate biosynthesis precursor. The enzymatic basis of this hydrolysis was further investigated. pABAGlu hydrolase activity was found in all species and organs tested; activity levels implied that the proteins responsible are very rare. The activity was located in cytosol/vacuole and mitochondrial fractions of pea (Pisum sativum L.) leaves, and column chromatography of the activity from Arabidopsis tissues indicated at least three peaks. A major activity peak from Arabidopsis roots was purified 86-fold by a three-column procedure; activity loss during purification exceeded 95%. Size exclusion chromatography gave a molecular mass of ∼200 kDa. Partially purified preparations showed a pH optimum near 7.5, a Km value for pABAGlu of 370 μM, and activity against folic acid. Activity was relatively insensitive to thiol and serine reagents, but was strongly inhibited by 8-hydroxyquinoline-5-sulfonic acid and stimulated by Mn2+, pointing to a metalloEnzyme. The Arabidopsis genome was searched for proteins similar to Pseudomonas carboxypeptidase G, which contains zinc and is the only Enzyme yet confirmed to attack pABAGlu. The sole significant matches were auxin conjugate hydrolase family members and the At4g17830 protein. None was found to have significant pABAGlu hydrolase activity, suggesting that this activity resides in hitherto unrecognized Enzymes. The finding that Arabidopsis has folate-hydrolyzing activity points to an enzymatic component of folate degradation in plants.
Hirofumi Hamada - One of the best experts on this subject based on the ideXlab platform.
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Enhanced growth suppression in esophageal carcinoma cells using adenovirus-mediated fusion gene transfer (uracil phosphoribosyl transferase and herpes simplex virus thymidine kinase)
Cancer Gene Therapy, 2001Co-Authors: Takanori Shimizu, Hideaki Shimada, Takenori Ochiai, Hirofumi HamadaAbstract:Advanced esophageal cancers are highly malignant and frequently resistant to 5-fluorouracil (5-FU). Escherichia coli uracil phosphoribosyltransferase (UP) is a pyrimidine Salvage Enzyme that alters 5-FU metabolism and sensitivity. A recombinant adenovirus encoding the UP gene (AxCA.UP) has been applied in gastric cancer gene therapy to sensitize cancer cells to lower concentrations of 5-FU. We have generated a recombinant adenovirus (AxCA.UT) encoding UP and herpes simplex virus thymidine kinase fusion protein (UT) to examine whether it would enhance the antitumor activity of AxCA.UP treatment. AxCA.UT treatment significantly enhanced the sensitivity of human esophageal cancer cells to and significantly enhanced the growth inhibition effects of UP gene therapy in vitro . Moreover, both 5-FU and ganciclovir showed bystander effects on growth inhibition. In an in vivo study, the therapeutic outcome of AxCA.UT treatment significantly enhanced the antitumor activity of AxCA.UP treatment. These observations suggest that AxCA.UT may be useful in esophageal cancer gene therapy. Cancer Gene Therapy (2001) 8, 512–521
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adenovirus mediated transduction of escherichia coli uracil phosphoribosyltransferase gene sensitizes cancer cells to low concentrations of 5 fluorouracil
Cancer Research, 1998Co-Authors: Fumihiko Kanai, Takayuki Kawakami, Akiko Sadata, Yasushi Shiratori, Hirofumi Hamada, Yoko Yoshida, Makoto Ohashi, Keisuke Tateishi, Torao Tanaka, Masao OmataAbstract:5-fluorouracil (5-FU), although a widely used chemotherapuetic agent, has a limited effect in the treatment of human solid tumors due to their resistance to the cytotoxic effects of 5-FU. Escherichia coli uracil phosphoribosyltransferase (UPRT) is a pyrimidine Salvage Enzyme that catalyzes the synthesis of UMP from uracil and 5-phosphoribosyl-α-1-diphosphate. The present study demonstrates that adenovirus-mediated transduction of E. coli UPRT gene results in marked sensitization of colon, gastric, liver, and pancreas cancer cell lines to low concentration of 5-FU in vitro . The in vitro bystander effect was observed when only 10% of the hepatoma Hep3B cells were infected with UPRT-expressing adenovirus. In addition, 5-FU treatment of human hepatoma or gastric cancer xenografts in nude mice transduced with UPRT was demonstrated to result in significant in vivo antitumor effects. The adenovirus vector transduction of the UPRT gene followed by 5-FU administration is representative of a new chemosensitization strategy for cancer gene therapy.
Brent W Anderson - One of the best experts on this subject based on the ideXlab platform.
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molecular mechanism of regulation of the purine Salvage Enzyme xprt by the alarmones pppgpp ppgpp and pgpp
Journal of Molecular Biology, 2020Co-Authors: Brent W Anderson, Aili Hao, Kenneth A Satyshur, James L Keck, Jue D WangAbstract:The alarmones pppGpp and ppGpp mediate starvation response and maintain purine homeostasis to protect bacteria. In the bacterial phyla Firmicutes and Bacteroidetes, xanthine phosphoribosyltransferase (XPRT) is a purine Salvage Enzyme that produces the nucleotide XMP from PRPP and xanthine. Combining structural, biochemical, and genetic analyses, we show that pppGpp and ppGpp, as well as a third newly identified alarmone pGpp, all directly interact with XPRT from the Gram-positive bacterium Bacillus subtilis and inhibit XPRT activity by competing with its substrate PRPP. Structural analysis reveals that ppGpp binds the PRPP binding motif within the XPRT active site. This motif is present in another (p)ppGpp target, the purine Salvage Enzyme HPRT, suggesting evolutionary conservation in different Enzymes. However, XPRT oligomeric interaction is distinct from HPRT in that XPRT forms a symmetric dimer with two (p)ppGpp binding sites at the dimer interface. (p)ppGpp's interaction with an XPRT bridging loop across the interface results in XPRT cooperatively binding (p)ppGpp. Also, XPRT displays differential regulation by the alarmones as it is potently inhibited by both ppGpp and pGpp, but only modestly by pppGpp. Lastly, we demonstrate that the alarmones are necessary for protecting GTP homeostasis against excess environmental xanthine in B. subtilis, suggesting that regulation of XPRT is key for regulating the purine Salvage pathway.
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molecular mechanism of regulation of the purine Salvage Enzyme xprt by the alarmones pppgpp ppgpp and pgpp
bioRxiv, 2020Co-Authors: Brent W Anderson, Aili Hao, Kenneth A Satyshur, James L Keck, Jue D WangAbstract:The alarmones pppGpp and ppGpp mediate starvation response and maintain purine homeostasis to protect bacterial species. Xanthine phosphoribosyltransferase (XPRT) is a purine Salvage Enzyme that produces the nucleotide XMP from PRPP and xanthine. Combining structural, biochemical and genetic analyses, we show that pppGpp and ppGpp, as well as a third putative alarmone pGpp, all directly interact with XPRT and inhibit XPRT activity by competing with its substrate PRPP. Structural analysis reveals that ppGpp binds the PRPP binding motif within the XPRT active site. This motif is present in another (p)ppGpp target, the purine Salvage Enzyme HPRT, suggesting evolutionary conservation in different Enzymes. However, XPRT oligomeric interaction is distinct from HPRT in that XPRT forms a symmetric dimer with two (p)ppGpp binding sites at the dimer interface. This results in two distinct regulatory features. First, XPRT cooperatively binds (p)ppGpp with a Hill coefficient of 2. Also, XPRT displays differential regulation by the alarmones as it is potently inhibited by both ppGpp and pGpp, but only modestly by pppGpp. Lastly, we demonstrate that the alarmones are necessary for protecting GTP homeostasis against excess environmental xanthine in Bacillus subtilis, suggesting that regulation of XPRT is key for regulating the purine Salvage pathway.
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evolution of p ppgpp hprt regulation through diversification of an allosteric oligomeric interaction
eLife, 2019Co-Authors: Brent W Anderson, Kenneth A Satyshur, James L Keck, Kuanqing Liu, Christine Wolak, Katarzyna Dubiel, Fukang She, Jue D WangAbstract:The alarmone (p)ppGpp regulates diverse targets, yet its target specificity and evolution remain poorly understood. Here, we elucidate the mechanism by which basal (p)ppGpp inhibits the purine Salvage Enzyme HPRT by sharing a conserved motif with its substrate PRPP. Intriguingly, HPRT regulation by (p)ppGpp varies across organisms and correlates with HPRT oligomeric forms. (p)ppGpp-sensitive HPRT exists as a PRPP-bound dimer or an apo- and (p)ppGpp-bound tetramer, where a dimer-dimer interface triggers allosteric structural rearrangements to enhance (p)ppGpp inhibition. Loss of this oligomeric interface results in weakened (p)ppGpp regulation. Our results reveal an evolutionary principle whereby protein oligomerization allows evolutionary change to accumulate away from a conserved binding pocket to allosterically alter specificity of ligand interaction. This principle also explains how another (p)ppGpp target GMK is variably regulated across species. Since most ligands bind near protein interfaces, we propose that this principle extends to many other protein-ligand interactions.
